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Use of porphyrin analyses to study the genetics of heme biosynthesis in the yeast, Saccharomyces cerevisiae

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Use of porphyrin analyses to study the genetics of heme biosynthesis in the yeast, Saccharomyces cerevisiae
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Wolf, Martin C
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English
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ix, 118 leaves : illustrations (1 folded) ; 29 cm

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Subjects / Keywords:
Saccharomyces cerevisiae ( lcsh )
Porphyrins ( lcsh )
Porphyrins ( fast )
Saccharomyces cerevisiae ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 110-118).
General Note:
Submitted in partial fulfillment of the requirements for the degree, Master of Arts, Biology.
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Martin C. Wolf.

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University of Colorado Denver
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Auraria Library
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ocm37898900
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LD1190.L45 1997m .W65 ( lcc )

Full Text
USE OF PORPHYRIN ANALYSES TO STUDY
THE GENETICS OF HEME BIOSYNTHESIS
IN THE YEAST, Saccharomyces cerevisiae
by
Martin C. Wolf
B. A., University of Colorado at Colorado Springs, 1985
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
1997
AL


This thesis for the Master of Arts
degree by
Martin C. Wolf
has been approved
by


Wolf, Martin C. (M.A., Biology)
Use of Porphyrin Analyses to Study the Genetics of Heme Biosynthesis in
the Yeast, Saccharomyces cerevisiae
Thesis directed by Dr. James R. Mattoon
ABSTRACT
Porphyrins are ancient, evolutionarily conserved tetrapyrrolic
macrocycles found in nearly all cells, that serve a variety of functions in their
various forms and derivatives. Besides their central role in the chlorophylls,
pheophytins, and cytochromes of plants and some bacteria, porphyrins play
a vital role in cytochromes, hemoglobin and myoglobin in animals, as well
as in a number of enzymes found in most cells. The specific steps of their
conversion in the biosynthetic pathway leading to heme have gradually
been elucidated and have many similarities in the five kingdoms, but the
genetic mechanisms involved in this pathway and its regulation are still
being determined. The yeast Saccharomyces cerevisiae makes an ideal
eukaryotic model for studying the genetics behind the heme biosynthetic
pathway. In this work methods for analyzing porphyrin accumulation in
small (3-6 g wet weight) batches of yeast cells were developed and refined,
and then applied to three aspects of heme biosynthesis, to study the genes
involved. Methods ranged from whole-cell analyses to extraction and
methylation of porhyrins with spectrophotometric and chromatographic
analyses of them. One aspect of study utilized porphyric mutants with partial
or complete blocks in enzymatic reactions late in the pathway-due either to
mutations in structural genes (HEM) for the enzymes, or in regulatory genes
affecting the pathway. A second aspect utilized porphyric mutants with
defects in RAS-related genes, affecting the regulatory cascade associated
with cAMP and protein kinase. A third aspect utilized porphyric mutants with
defects in cytochrome c oxidase, in the hopes that genetic details related to
the heme a branch of this biosynthetic pathway might be revealed.
This abstract accurately represents the content of the candidate's thesis.
I recommend its publication.
Signed
James R. Mattoon


DEDICATION
To my wife Chris,
and the fruits of our joined nature & nurture,
Tim and Mollie,
without whom this would not have been done


ACKNOWLEDGMENTS
I would like to thank all the professors and instructors in the Biology
Departments at the University of Colorado at both Colorado Springs and
Denver who have inspired and impressed me over the years. I would
especially like to thank Dr. James R. Mattoon who first took me "under his
wing" in the fall of 1985, starting my work with yeast and porphyrins, and
who guided me through many complex mazes. I would like to thank Dr.
Linda K. Dixon for agreeing to chair my committee, and then putting in an
extra mile to see me through at the end. I would like to thank Dr. Sandra L.
Berry-Lowe for joining my committee and providing support and sympathy
with the HPLC. I would also like to thank Annette Beck of the Graduate
School for all her help in the final weeks.


CONTENTS
List of Figures..................................................viii
List of Tables.....................................................ix
Chapter
1. Introduction.....................................................1
1.1 Background..................................................1
1.2 The Porphyrin/Heme Biosynthetic Pathway.....................3
1.3 Porphyrin Research..........................................5
1.4 Saccharomyces cerevisiae Respiratory Background.............7
1.5 Genetics of Heme Biosynthetic Enzymes & Their Regulation...11
1.6 The RAS/cAMP Regulatory System.............................15
1.7 Heme a and Cytochrome c Oxidase............................18
2. Research Goals..................................................20
2.1 Development of Methods.....................................20
2.2 Biological Applications....................................20
3. Methods.........................................................22
3.1 Small-Scale Extraction of Porphyrin Free Acids.............22
3.2 Preparation for Spectrophotometric Analysis of Free Acid
Porphyrin Extracts...........................................28
3.2.1 Operating the DU-7 Spectrophotometer.....................30
3.2.2 Operating the DU-50 Spectrophotometer....................38
3.3 Methyl-Esterification of Free Acid Porphyrin Extracts......42
3.4 TLC-Separation of Porphyrin Methyl Esters..................45
3.4.1 Alternate, Small-Scale TLC Method........................48
3.5 HPLC Procedures............................................51
4. Results and Discussion..........................................64
4.1 The Methods..............................................64
VI


4.1.1 Weight Determinations......................................64
4.1.2 Whole Cell Spectra........................................64
4.1.3 Free Acid Porphyrin Spectra...............................67
4.1.4 Porphyrin Methyl Ester Spectra............................71
4.1.5 Thin-Layer and High-Performance Liquid Chromatography....73
4.2 The Structural Gene Mutants................................75
4.2.1 Uroporphyrinogen Decarboxylase (HEM12) Mutant "P21"......75
4.2.2 Ferrochelatase (HEM 15) Mutant D28/F8.....................79
4.2.3 Ferrochelatase (HEM 15) Mutant "P1".......................81
4.3 The /?AS/cAMP-Related Regulatory Mutants..................83
4.3.1 The ras2 Mutant Group.....................................83
4.3.2 The ras2 sra1 Suppressor Mutants..........................88
4.3.3 The RAS2 Missense Mutant................................. 92
4.3.4 The ras265, High cAMP Mutants.............................94
4.3.5 The cAMP-Permeable HEM Mutant.............................94
4.4 The Cytochrome c Oxidase Mutants...........................102
5. Conclusions.....................................................107
5.1 The Analytical Methods.....................................107
5.2 Porphyrin Patterns in Yeast Mutants........................107
5.3 Mutants with Partial Defects in Heme Biosynthetic Enzymes.... 108
5.4 Mutants with Defects in Regulation of Heme Biosynthesis....108
5.5 Mutants with Defects in Cytochrome c Oxidase...............109
5.6 General Conclusion.........................................109
Bibliography..........................................................110
vii


FIGURES
Figure
1. Porphin, the basic porphyrin macrocycle............................2
2. The heme biosynthetic pathway in yeast.............................2
3. Porphyrins in photodynamic cancer therapy............................6
4. Nuclear-mitochondrial interactions..................................10
5. Whole-cell spectra of D28, grown with & without oxygen.............24
6. The DU-7 keyboard...................................................31
7. Sample scan print-out from the DU-7................................36
8. A blow-up of visible wavelengths from Fig. 7.......................37
9. Sample scan print-out from the DU-50...............................41
10. Typical HPLC chromatogram from the Kipp & Zonen BD41...............58
11. Whole-cell difference spectra: metallo- vs. free porphyrin..........66
12. Overlay of free-acid extracts from proto vs uro strains.............70
13. Overlay of methyl-esters from proto vs uro strains..................72
14. Thin-layer chromatography samples...................................74
15. Overlay whole-cell spectra of "P21", parent, & D28.................78
16. Whole-cell difference spectrum of "P21" minus parent................78
17. Whole-cell spectra of D28/F8 & D28..................................80
18. Whole-cell spectrum of D28/F8 minus D28.............................80
19. Whole-cell spectra of "P1" and parent...............................82
20. Whole-cell difference spectrum of "P1" minus parent................82
21. Overlay whole-cell spectra of "EG", R1a, R12b, & D28..............84
22. Whole-cell difference spectra of "EG", R1a, & R12b minus D28.......85
23. Whole-cell spectra of 112 and 112-699..............................87
24. Whole-cell spectra of S288c and D28...............................89
25. Whole-cell difference spectra of R1a and R12b minus "EG"...........90
26. Whole-cell difference spectra of R1a and R12b minus S288c..........91
27. Whole-cell spectra of 301 and 308.................................93
28. Whole-cell spectra of SC1171, SC1183, and SC167....................95
29. Whole-cell difference spectra of SC1171 & SC1183 minus SC167...96
30. Spectra of free-acid extract from "P1", no cAMP....................98
31. Spectra of free-acid extract from "P1", 2mM cAMP...................99
32. Spectra of methyl-esters from "P1", +/- cAMP.......................100
33. Overlay whole-cell spectra of three COX mutants...................103
34. Whole-cell difference spectra of COX mutants minus control........103
35. HPLC chromatograms showing subsidiary peaks.......................106
viii


TABLES
Table
1. HPLC Conversion Factors........................................58
2. Typical Whole-Cell Difference-Spectra..........................65
3. Typical Absorbance Maxima in Yeast Whole-Cell Spectra..........67
4. Typical Free-Acid Porphyrin Absorbance Maxima..................68
5. Absorbance Maxima for Free-Acid Porphyrin Extracts.............69
6. Absorbance Maxima for Porphyrin Methyl-Ester...................71
7. Yeast Strains Involved in These Studies........................76
8. Summary of Data From All Strains Studied (oversize fold-out)...77
9. Data Summary for the RAS/cAMP Group of Mutants................101
IX


1. INTRODUCTION
1.1 Background
Porphyrins are ancient, evolutionarily conserved tetrapyrrole
macrocycles--four pyrrole rings joined in a larger ring system (Fig. 1)-often
with a metal ion complexed in the center, enabling them (within the
appropriate derivative or polypeptide and membrane contexts) to act as
hydrogen, methyl, or oxygen carriers or transfer agents, or as electron
carriers in transport chains. They may have first served early cells by
protecting them from photooxidation by visible light, as occurs with
porphyrins having a paramagnetic central metal ion, e.g. Fe2+or Cu2+;
porphyrins with other, nonparamagnetic metal ions, e.g. Zn2+, actually
facilitate photooxidation (Camadro & Labbe 1982; Grimal & Labbe-Bois
1980; Margulis 1981; Spikes 1975).
Found at low levels in nearly all cells, these light-sensitive molecules
are precursors or intermediates in the biosynthesis of some of life's most
important compounds, including the vital pigments of most cells: the
chlorophylls, cytochromes and pheophytins in all plants and photosynthetic
bacteria, the hemes in cytochromes of all aerobic cells, the hemes in
hemoglobin and myoglobin in animal cells. Porphyrins also become part of
the ubiquitous enzymes catalase and peroxidase, the corrin ring system of
coenzyme B12, and other enzymes such as nitric oxide synthase, sulfite
oxidase, sulfite reductase, tryptophan pyrrolase and tryptophan oxygenases.
All these compounds are synthesized in cells (in all five kingdoms) from a
single porphyrin intermediate, uroporphyrinogen III, along a similar pathway
(Battersby 1980; Ferreira et al. 1995; Labbe-Bois & Labbe 1978; Margulis
1981; Rawn 1989; Sassa et al. 1975).
1


Fig. 1. Porphin, the basic porphyrin macrocycle, is composed
of four pyrrole rings (A, B, C, D) joined by four methine bridges
(Greek symbols); the macrocycle is a 20-carbon polygon with
four central nitrogens. Side-chains attach at the eight numbered
carbons, while metal or other ions may be complexed in the center.
Glycine
Rg. 2. The heme biosynthetic pathway in yeast involves eight enzymatic steps, catalyzed by both
mitochondrial and cytosolic enzymes. Abbreviations: ALA, 5-aminolevulinate; PBG, porpho-
bilinogen; HMB, hydroxymethylbilane; 'gen, porphyrinogen; 7: heptacarboxylic porphyrin;
6: hexacarboxylic porphyrin; 5: pentacarboxylic porphyrin.
2


1.2 The Porphyrin/Heme Biosynthetic Pathway
In eukaryotes the heme biosynthetic pathway is a complex assembly
line, coordinated between enzymes (and protein regulators) inside
mitochondria and in the cytosol. While the porphyrin ring must have
predated its role in cellular light-gathering arrays that generated ATP and
NADPH for food production in ancestral photosynthetic prokaryotes several
billion years ago, the eukaryotic pathway of heme biosynthesis emerged, in
a give-and-take fashion, from the symbiosis of aerobic mitochondrial
ancestors within their larger, archaebacterial-like hosts. The step most
widely conserved in four of the five kingdoms is that from succinyl CoA to
5-aminolevulinate (ALA), which takes place, in non-plant eukaryotes, in the
mitochondrion (Alberts et al. 1983; Margulis 1981 & 1986); in plants and
some bacteria ALA is made from glutamate, in the chloroplast.
In yeast and most other nonphotosynthetic eukaryotes the amino acid
glycine, formed in the cytosol from the glycolytic intermediate 3-phosphogly-
cerate, enters the mitochondrion and combines with succinyl CoA from the
Krebs cycle, in a condensation reaction catalyzed by ALA synthase (Fig. 2);
ALA synthase requires the cofactor pyridoxal phosphate. There have been
contradictory reports as to whether this is the rate-limiting step in yeast heme
biosynthesis (it now appears the next step is more likely rate-limiting), and
whether or not it is inhibited by heme/protoheme (the consensus now seems
to be not). The ALA which results from the reaction leaves the
mitochondrion and is coupled to another ALA molecule by porphobilinogen
synthase (ALA dehydratase), producing porphobilinogen (PBG), a pyrrole
ring with an acetic acid group on one and a propionic acid group on the
other of its two double-bonded carbons. This step can be inhibited by
protoheme as well as by protoporphyrinogen (proto'gen) and
coproporphyrinogen (copro'gen), or by ferric iron or lead (-lead poisoning
3


results in excretion of excess ALA in the urine). In the next step, four PBGs
are joined as a linear tetrapyrrole, hydroxymethylbilane, by PBG deaminase
and then, after rotation of the D ring by uroporphyrinogen (uro'gen) III
synthase, joined (by the latter enzyme) into a larger, slightly asymmetrical
ring system called uro'gen III. This intermediate, with eight carboxyl groups,
is then serially decarboxylated (at its acetic acid side groups) by uro'gen
decarboxylase to hepta-, hexa-, and penta-carboxylic porphyrinogens, and
then to copro'gen III with four carboxyls; inhibition at this step can be caused
by mercury, copper, manganese, and oxygen (or by halogenated aromatic
hydrocarbons, e g. barbiturates, in animals). Copro'gen III is next acted
upon by copro'gen III oxidase, which requires molecular oxygen as an
electron acceptor, producing proto'gen IX with only two carboxyl groups;
heavy metals such as lead and cadmium inhibit this step. At this point the
pathway in yeast reenters the mitochondrion (occurring at the previous step
in other eukaryotes), as proto'gen IX crosses the membrane and oxygen-
requiring proto'gen IX oxidase catalyzes the next step of aromatization to
protoporphyrin IX. This, as well as the next step, is inhibited by protoheme--
the product of the next step, in which ferrous iron is chelated to the center of
protoporphyrin IX by ferrochelatase; this last step is also inhibited by the
divalent ions of manganese, cadmium, and mercury. Protoheme then may
take one of several paths, depending on the endproduct: if it is en route to
cytochromes b, hemoglobin, or myoglobin, it will not change as a prosthetic
group; if it is to en route to cytochrome cor Cj, its two vinyl side-groups will
interact with sulfhydryl groups on cysteine residues of the apoprotein; if it is
en route to the a cytochromes, it will first be converted to heme a, by the
addition of a long polyunsaturated alkene and an aldehyde group to two of
its carbons. The route to coenzyme B12, the corrins, and siroheme branches
off earlier, from uro'gen III. In photosynthetic organisms the route to the
chlorophylls and pheophytins diverges when protoporphyrin IX is chelated
with magnesium instead of ferrous iron (Amillet et al. 1995; Battersby &
4


McDonald 1976; Battersby et al. 1980; Ferreira et al. 1995; Kanazireva &
Biel 1995; Labbe-Bois & Labbe 1978 & 1990; Rawn 1989; Straka et al.
1981; Tait 1978). More details of the heme pathway will be presented
below, in sections 1.4 and 1.5.
1.3 Porphyrin Research
Porphyrins are currently being used as "tools" in a variety of studies,
as well as being the objects of investigation in other studies. Prathapan et
al. (1993) have been working with synthetic, rigid porphyrin pentamers (one
central porphyrin triple-bonded to four others around it) with high organic
solubility, as models of chlorophyll antennae arrays, utilizing their unique
light absorption and emission properties. Results of work on coordination/
dissociation between metalloporphyrins and cis/trans forms of stilbazoles
(with isomerization occurring under the stimulus of UV irradiation) have led
Iseki et al. (1993) to propose an artificial porphyrin photoswitch to achieve
photoregulation of a chemical reaction on the porphyrin, analogous to the
mechanisms between retinal and rhodopsin in vision, and photomorpho-
genesis in plants. Ricchelli (1995) has reported the use of porphyrins in
biomedical applications for their photosensitizing properties and affinity for
biological membranes. In one example of such applications, Roberts et al.
(1995) monitored age-related differences in the intact human lens by
detecting and measuring triplet excited state lifetimes in a porphyrin during
binding to the lens. In another example, Tsutsui et al. (1975) reported the
use, for diagnostic purposes, of the tendency of certain porphyrin
compounds to localize in tumors and cause fluorescence; the porphyrins
can then be used in light treatment (for their photosensitizing and
photooxidation properties) to destroy the tumor cells. Such "photodynamic
therapy" is now in use (Fig. 3) (Anstett 1996).
Adler et al. (1975) proposed many more uses for porphyrins: as
colorimetric detectors (either clinical or environmental) for carbon monoxide,
5


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6


nitrogen oxides, cyanide, fluorides, strong oxidants, and heavy metals such
as lead, mercury, or copper; as radiation dosimeters for UV or stronger forms
of ionizing radiation; as photodegradative catalytic pigments in plastics or
other potential litter material; as enhancer pigments or films in solar heat
collection devices and filters, as well as in photovoltaic solar cells (in which
the researchers did some successful initial studies); as photoactive
electrodes in fuel cells; and as redox catalysts to increase fuel efficiency or
to decrease emissions from combustion.
Various researchers are studying effects on porphyrin metabolism or
accumulation due to environmental toxins, such as polychlorinated
biphenyls or PCBs (Tsukazaki 1995), the insecticides lindane and DDT
(Attia et al. 1995), and halogenated aromatic hydrocarbons and heavy
metals such as mercury (Akins et al. 1993). The last group has proposed
using porphyrin patterns in certain organs and in excrement as a biomarker
of exposure to toxins by wildlife. The pattern and function of elevated
porphyrin levels in the Harderian glands (in the orbits of most mammals) is
being investigated (Spike et al. 1990 & 1992), as well as circadian changes
in Harderian porphyrins, in conjunction with levels of melatonin and certain
enzymes (Attia et al. 1995). Studying porphyrin accumulation patterns in a
variety of organisms, including yeast, as a means for delineating the details
involved in the genetics and regulation of heme biosynthesis (and
specifically in the human porphyria diseases), is another ongoing area of
current porphyrin research.
1.4 Saccharomyces cerevisiae: Respiratory Background
The yeasts, including Saccharomyces cerevisiae, are facultative
anaerobes; unlike most eukaryotes, which require oxygen for oxidative
respiration, yeasts can flourish quite well without oxygen, as long as glucose
or another glycolytic substrate is available. During such fermentation the
activities of (and biosynthetic pathways leading to) cellular respiration are
7


repressed; once those carbon and energy sources are depleted, or when
they are not available--and if oxygen has become available-then the
pathways leading to a fully functioning oxidative respiratory system become
active (or derepressed), for growth on nonfermentable carbon sources such
as glycerol or ethanol (Alberts et al. 1983; Labbe-Bois & Volland 1977a;
Rawn 1989; Wilkie 1983).
There are two general types of respiration-deficient yeast mutants,
depending on the site of the mutation. Mutations in the relatively small
mitochondrial genome can result in rho mutants (also called "cytoplasmic
petite") which are unable to use nonfermentable carbon sources, requiring
instead glycolytic substrates for growth; rho~{p~) mutants have partially
deleted mitochondrial DNA, whereas p mutants have no mitochondrial
DNA. The respiration deficiency of rho mutants results because yeast
mitochondrial DNA encodes not only some of the proteins and RNAs
involved in expressing its genes-including about twenty-four tRNAs and two
rRNAs-but also some of the very proteins required in aerobic respiration,
including the apoprotein part of cytochrome b, two subunits of ATPase, and
three subunits forming the catalytic core of cytochrome c oxidase (one
subunit of which is bound to cytochromes a and a^) (Evans 1983; Labbe-
Bois 1977; Labbe-Bois & Volland 1977b).
There are also mutations of the yeast nuclear genome that result in
respiration-deficient cells which require glycolytic substrates for growth;
these petite or pet mutants have single gene mutations in nuclear DNA
encoding a variety of proteins destined for the mitochondria, including many
necessary for mitochondrial gene expression, others involved in the
mitochondrial import systems, and a number that are essential in aerobic
respiration, including most of the cytochromes, the enzymes of heme
biosynthesis, the remaining subunits of ATPase, and some of the subunits of
cytochrome c oxidase (Attardi & Schatz 1988; Capaldi 1990; Forsburg &
8


Guarente 1989).
As one would expect for an energy-producing system involving two
genomes, there is a complicated, sensitive communication and feedback
system of gene and enzyme regulation in eukaryotes (including yeast),
many details of which are not yet understood. Growth conditions in yeast,
including oxygen levels and carbon source, can affect heme levels; they
also act on transcriptional regulators of both the nuclear and mitochondrial
genomes, some products of which then in turn affect the other genome or its
products (Fig. 4). For example, oxygen levels affect heme levels in the
mitochondrion, since two of the enzymes late in the heme biosynthetic
pathway are oxygenases; heme then activates certain nuclear regulatory
genes (directly in some cases, and through an intermediary in others*)
which act on nuclear structural genes encoding respiratory proteins
(Forsburg & Guarente 1989):
nuclear reaulatorv aenes action nuclear structural genes
HAP 1,2*,3*.4* + COX 4, 5a (subunits of cyto. c oxidase)
ROX1 - CYC7 (cyto. c isozyme)
- ANB1 (an anaerobically induced gene)
REO 1 - COX 5b (anaerobic subunit of cyto. c oxidase)
Many nuclear-encoded mitochondrial proteins occur in interchangeable
isoforms, which regulate protein functions in a differential manner,
depending on the conditions (or the tissues, in higher eukaryotes); these
isoforms have similar but distinct amino acid sequences, and are encoded in
unlinked nuclear genes. Transcription of certain genes for subunits of heme
biosynthetic enzymes requires activation of upstream activation sites, or
UASs, by specific trans-acting factors, as well as transcriptional factors
bound at the TATA box, for RNA polymerase II to start its work; these factors
themselves are regulated in response to physiological signals (Attardi &
Schatz 1988, Forsburg & Guarente 1989, Mattoon et al. 1990). There are
UASs in the 5' region of CYC1, the nuclear gene for iso-1-cytochrome c,
which affect the efficiency of its transcription as a function of glucose (inhib-
9


Nuclear
Genome

Environmental/Growth
conditions: Oxygen,
Carbon-source
Fig. 4a
enzymes, protein factors,
RNAs, metabolites,
poiypqptides
Mitochondrial
Genome
Fig. 4a. Nuclear-mitochondrial interactions are two-way, coordinating the cellular response
to growth conditions. 4b. A small sampling of the interactions and regulation in the
respiratory system of yeast cells between the nudear and mitochondrial genomes. Solid
arrows represent regulatory actions; dotted arrows represent transport of structural
proteins; + indicates activation; indicates inhibition. Modified from Forsburg & Guarente
(1989).
10


itory) and of heme (activating) (Tzagoloff & Myers 1986). The HAP1 product
(in table above) also binds with heme to an upstream activation site (UAS)
on the CYC1 gene; HAP 2/3 products bind a UAS on CYC1, as well as on
HEM1, thus coordinating transcription of heme and cytochromes c(Mattoon
et al. 1990). The products of the nuclear PET 111 and PET 494 genes, which
are activated by oxygen and inhibited by high glucose levels, are required in
the mitochondrion for translation of cytochrome c oxidase subunits 3 and 1,
respectively. Products of nuclear genes CBP6, CBS1 and CBS2 are
required for translation of mitochondrial-encoded apocytochrome b
(Forsburg & Guarente 1989). More details of this regulatory system will be
discussed below.
1.5 Genetics of the Heme Biosynthetic Enzymes and Their
Regulation
One group of pet respiratory mutants includes the pop and hem
mutants, with mutations that cause partial or complete blocks in reactions
late in the heme biosynthetic pathway, resulting in changes in heme and
therefore cytochrome levels, and/or the accumulation of various porphyrin
intermediates. The pop (porphyrin over-producer) mutants, so designated
by Sherman (Pretlow & Sherman 1967), have been characterized into two
types: mutants with defects in structural genes coding for the biosynthetic
enzymes themselves (genes allelic to the HEM genes); and mutants with
genetic defects altering the regulation of heme biosynthetic enzymes. This
section will discuss the HEM genes and their regulation; the following
sections will consider a particular regulatory system involving the RAS
genes and cAMP (1.6), and the special case of COX gene regulation-
affecting synthesis of the apoprotein subunits of cytochrome c oxidase,
which contains cytochrome aa$and has heme a as a prosthetic group (1.7).
A method using the photooxidative properties of zinc-protoporphyrin
to selectively cull surviving heme-less mutants (which couldn't make, and be
11


killed by, Zn-protoporphyrin) was developed by Labbe-Bois and Grimal in
1980, enabling them to isolate heme mutants in different complementation
groups, and analyze activity of heme biosynthetic enzymes to identify
mutants for most of the steps in this pathway; much progress has been made
since then in genetic analyses of yeast heme biosynthesis (Grimal & Labbe-
Bois 1980, Urban-Grimal & Labbe-Bois 1981).
The first committed enzyme in the heme pathway (see Fig. 2), ALA
synthase, is encoded by HEM1 (allelic to ole3, CYD1, and HEM A) (Labbe-
Bois & Labbe 1990; Labbe-Bois et al. 1980). Evidence over the years has
swung back and forth as to whether this step in yeast heme biosynthesis is
rate-limiting, but currently the next step is generally favored as rate-limiting
(Carvajal et al. 1990, Labbe-Bois & Labbe 1990). Enzyme activity was
found to be lowered on addition of 2% galactose or glucose, chloram-
phenicol, oxygen, or air by Labbe (1971) and on addition of protoheme by
Labbe-Bois & Labbe (1978). On the other hand, Volland & Felix (1984) and
Urban-Grimal et al. (1984, 1986) found inhibition by hemin (with Fe3+) but
no inhibition by protoheme, and Labbe-Bois & Labbe later (1990) report that
neither the enzymes import nor its activity are inhibited by heme. In
photosynthetic bacteria heme apparently does inhibit the enzyme
(Kanazireva & Biel 1995). The yeast HEM1 gene was isolated and cloned
by Arrese et al. (1983). HEM1 is activated at an UAS via the HAP2/3 system,
in response to elevated heme or a shift from glucose to a nonfermentable
carbon source; a consensus sequence for the HAP2/3 products has also
been found on CYCfs UAS 2 and C0X4s UAS. There is apparently
another site close to the HAP2/3 target sequence which keeps HEM1
activated when heme levels are deficient or carbon source changes (Keng &
Guarente 1987). Another gene RHM1 appears to regulate ALA synthase,
perhaps being involved in the enzyme's targeting to the mitochondrial
matrix, or in some other manner controlling its activity (Carvajal et al. 1990).
During aerobic growth on glucose, repression of respiratory metabolism acts
12


on HEM1 through a regulatory gene HEX2(Borralho et al. 1989, Mattoon et
al. 1990b).
The second enzyme in heme biosynthesis, PBG synthase (ALA
dehydratase), is encoded by HEM2 (allelic to HEM 10, ole4, and olerg4)
(Labbe-Bois & Labbe 1990). Now considered the rate-limiting enzyme in
yeast heme biosynthesis, it has low affinity for ALA and lower levels of
activity than ALA synthase; it also correlates the closest with levels of
porphyrins and heme (Labbe-Bois & Labbe 1990). Enzyme activity during
aerobic growth on glucose is inhibited, via the regulatory genes CA7"2and
HEX2 (Borralho et al. 1989, Mattoon et al 1990b); activity decreased with
addition of copro'gen III, proto'gen IX, or protoheme (Labbe-Bois & Labbe
1978). HEM2 has an upstream sequence (a CCAAT box) which may well be
a HAP2/3 binding site (Schlaepfer et al. 1994).
The next "step" in the pathway involves two enzymes; the first is PBG
deaminase (urogen I synthase) and is encoded by HEM3 (allelic to HEM11,
HEMC, ole2, olerg2, and possibly pop6). HEM3 has a HAP2/3/4 binding site
in its promoter region, and expression requires binding of the HAP protein
complex, which in this case does not mediate carbon-source (Keng et al.
1992); although there is no definite evidence, the enzyme may be induced
by PBG (Labbe-Bois & Labbe 1990). The other enzyme at this step,
catalyzing D ring rotation and closure of the linear tetrapyrrole into the larger
ring system of uro'gen III, is uro'gen synthase (uro'gen III cosynthetase); it is
encoded by HEM4, allelic to cyt. In the absence of functional uro'gen
synthase the linear tetrapyrrole (hydroxymethylbilane) spontaneously
cyclizes to make the perfectly symmetrical isomer uro'gen I, which can be
acted on by uro'gen decarboxylase in the next step to produce copro'gen I,
but cannot be further metabolized (Amillet & Labbe-Bois 1995).
The subsequent series of four decarboxylations is catalyzed by
uro'gen decarboxylase, encoded by the HEM 12 gene, allelic to HEM6 and
pop3, and apparently regulated in some manner by the pop1 and pop4 loci.
13


While not allelic, pop4 has a similar phenotype and also lacks cytochrome c
oxidase (Arrese et al. 1982, Labbe-Bois & Labbe 1990). Decarboxylation
takes a certain sequence, starting with the acetic acid on the D ring and
proceeding through rings A, B, and C. Enzyme activity is inhibited by
oxygen, various heavy metals, and by polyhalogenated hydrocarbons
(Labbe-Bois & Labbe 1978, Rytka et al. 1984, Tait 1978).
The next enzyme, copro'gen oxidase, is encoded by HEM13, allelic to
HEMG and linked with HEM12 on chromosome IV in yeast (separated by 10
cMorgans) (Bilinski et al. 1981, Labbe-Bois & Labbe 1990). This enzyme,
cytosolic in yeast, is inside the mitochondrion in mammals and requires
oxygen, though not in plants or perhaps yeast. In yeast there may be one
mechanism using oxygen as an electron acceptor and another operating in
the absence of oxygen, although with the enzyme's very high affinity for
oxygen, small amounts of oxygen diffusing through tubing (as little as 50 nl
of air in a 1-liter culture over 24 hours is adequate) can allow accumulation
or excretion of coproporphyrin in "anaerobic" conditions (Camadro et al.
1986, Labbe-Bois & Labbe 1990, Urban-Grimal & Labbe-Bois 1981).
Glucose repression does not affect levels of this enzyme (Zagorec & Labbe-
Bois 1986). The gene HEM13 is transcriptionally regulated in a negative
manner by both heme and oxygen, via the trans-acting gene product of
CYP1 (allelic to HAP1), a positive regulator of CYC1 ; low heme or oxygen
levels activate HEM 13 expression via HAP1 protein (Labbe-Bois & Labbe
1990, Zagorec et al. 1988).
The next enzymatic step, converting proto'gen IX to protoporphyrin IX,
is catalyzed on the inner mitochondrial membrane by proto'gen oxidase; the
enzyme, encoded by HEM14, requires oxygen as an electron acceptor, and
is at least partially inhibited by heme, or divalent copper or cobalt ions
(Camadro et al. 1982, Labbe-Bois 1990, Tait 1978).
The last step in the pathway also occurs on the inner mitochondrial
membrane in yeast and animals (but inside the chloroplast in plants); it is
14


catalyzed by ferrochelatase (protoheme ferrolyase), which is encoded by
HEM15 (allelic to HEM5 and pop2). Ferrochelatase is inhibited by
heme/protoheme, Mn2+, Cd2+, Hg2+, and N-alkyl-substituted porphyrins, but
is activated by the presence of apocytochromes and oxygen; its activity is
two-four times greater in derepressed cells than in glucose-repressed or
anaerobic cells. It can bind Fe2+ or Zn2+ competitively, but iron inhibits its
zinc-chelatase activity more than vice-versa (Camadro & Labbe 1982,
Ferreira et al. 1995, Labbe-Bois & Labbe 1978, 1990, Lavallee 1987, Tait
1978).
1.6 The RAS/cMAP Regulatory System
The regulatory cascade involving stimulatory guanine nucleotide-
binding proteins (G proteins) acts via the second messenger 3',5'-cyclic
adenosine monophosphate (cAMP) to activate protein kinases, which then
effect widespread phosphorylations of enzymes (either activating or
deactivating them, depending on the particular enzyme) to coordinate vital
changes in response to extracellular signals (such as glucagon or
epinephrine in animals). G proteins may be either stimulators or inhibitors,
but in either case their role is played when bound to guanosine 5'-
triphosphate (GTP); G proteins have a built-in GTPase activity which
hydrolyzes GTP soon after binding to return the G protein to an inactive state
(bound to GDP). If a particular G protein is associated with a positive
regulator, then on binding GTP the complex binds to adenylate cyclase and
stimulates the production of cAMP. A G protein associated with a negative
regulator, on binding GTP, will then bind to adenylate cyclase and inhibit its
production of cAMP. If cAMP results (via a stimulatory G protein), it then
binds to the regulatory subunits of inactive cAMP-dependent protein kinase
(cAPK), causing the release (and activation) of the catalytic subunits, which
then cause the phosphorylation of various enzymes to activate or deactivate
them, as the case may be; meanwhile cAMP phosphodiesterase works to
15


convert cAMP back into AMP to restore the original situation, just as the G
protein's GTPase activity is reverting the G protein to an inactive state. A
classic example in animals occurs with either first messenger epinephrine or
glucagon, whose receptors associate with stimulatory G proteins, causing a
flood of cAMP and hence cAPK, resulting in: the deactivation of glycogen
synthase, the breakdown of glycogen by activated phosphorylases a & b to
release glucose into the bloodstream, the removal of fructose 2,6-
b/sphosphate inhibition on gluconeogenesis, and the activation of lipase to
send fatty acids into p-oxidation for additional energy (Rawn 1989).
Yeast cells have a similar regulatory system involving G proteins,
adenylate cyclase, cAMP, cAMP-dependent protein kinase (with regulatory
and catalytic subunits), and phosphodiesterase. The RAS genes encode G
proteins, and in the normal state their expression in yeast leads (via
increased adenylate cyclase, cAMP and protein kinase) to deactivation of
glycogen synthase, for example, and activation of trehalase-both effects
resulting in increased availability of glucose for continued growth and
progress through the mitotic cell cycle; cAMP levels tend to be elevated
during growth on non-fermentable carbon sources. Prior to meiosis (for
conjugation or budding) yeast cells arrest in the G1 phase of the cell cycle,
apparently through repression of cAMP production and inactivation of
protein kinase; this synchronization of opposite mating types may work
through inactivation of adenylate cyclase or G protein via a pheromone
signal (Matsumoto et al. 1983, Thorner 1982, Uno et al. 1984, Wheals 1987).
RAS genes can easily mutate in one of two positions to eliminate the
protein's GTPase activity, leaving the G protein permanently active, and thus
allowing constitutively stimulated adenylate cyclase to continuously produce
cAMP, resulting in uncontrolled growth-hence the RAS designation as an
oncogene. The yeast genes have "remarkable" homology to mammalian
RAS genes; Kataoka et al. (1984) constructed a missense point mutant,
RAS^3119, similar to transforming alleles of mammalian ras genes. This
16


mutation causes a phenotype similar to overexpression of RAS2, with no
"starvation response:" cells don't accumulate storage carbohydrates such as
glycogen or trehalose, and they fail to sporulate (Tatchell et al. 1985); as
indicated in this study's work with strain 301, they also accumulate
excessive cytochromes (especially c) and porphyrin.
Yeast RAS1 and RAS2 genes are unlinked, and mutations of either
one alone are not lethal, whereas the double mutation is. A disruption of
RAS2 (which eliminates or impairs HAS function), while showing no effect in
growth on glucose, results in a gluconeogenic defect during growth on
nonfermentable carbon sources: poor growth, with accumulation of the
storage carbohydrates glycogen and trehalose; diploids homozygous for
ras2 sporulate on rich media, prematurely, rather than having to undergo
nutrient limitatiion (Tatchell et al. 1985, Wheals 1987). Two such strains
worked with in this study were EG81-22b and 112-699.
Revertants of ras mutations were isolated by Tatchell et al. (1985),
having extragenic suppression of the ras phenotype. Two such sra
(suppressor of ras) strains were worked with in this study: R1a and R12b,
both ras2 sra1 mutants. These revertants have neither the gluconeogenic
defect nor the derepressed sporulation on rich media of ras2 mutants; they
were found to grow even with neither RAS gene functional. The sra1
mutation has since been found to be allelic to bcy1, and involves a mutation
in the regulatory subunit of protein kinase, eliminating the need for binding
of cAMP for activation of the catalytic subunits; hence protein kinase is fully
active in spite of cAMP levels. A similar result occurs for sra1/bcy1
revertants of cyr1 (a mutation of the gene encoding adenylate cyclase) and
of CYR3 and cyr2 mutants (the genes encoding the regulatory and catalytic
subunits, respectively, of protein kinase) (Matsumoto et al. 1984, Thorner
1982, Uno et al. 1982, 1984). All this implies that cAMP, mediated by
upstream components of this regulatory system, is necessary in yeast for
gluconeogenic growth and normal response to nutrient limitation (Tatchell et
17


al. 1985). This study and others investigated the possible role of this cAMP
regulatory system in heme biosynthesis.
1.7 Heme a and Cytochrome c oxidase
Cytochrome c oxidase is a ubiquitous respiratory complex in the
cellular electron transport chain, passing electrons in a series of oxidation-
reductions from cytochrome cto molecular oxygen, and in the process
contributing to the pumping of protons to establish a gradient of proton
backflow that is coupled to the phosphorylation of ADP to make the cellular
energy currency, ATP. In a reflection of evolution, the enzyme's three
bacterial subunits form the catalytic core of this mitochondrial complex in
eukaryotes; these three subunits are encoded in mitochondrial DNA
(COX1/2/3), while the remaining four-to-ten eukaryotic subunits are encoded
in nuclear genes. Subunit 1 contains cytochromes a and 33, with identical
structures but different locations and therefore reduction potentials in the
enzyme complex; cytochrome 3-33 has heme a as a prosthetic group.
Ninety percent of cellular oxygen consumption is due to cytochrome c
oxidase; it is thus easy to understand why substances like cyanide, azide,
and carbon monoxide are poisons, since they bind readily to the heme a
groups, inhibiting the enzyme (Capaldi 1990, Rawn 1989).
Protoheme, the product of ferrochelatase (HEM 15) activity on the
inner mitochondrial membrane, is routed unchanged to different locations
and apoproteins in the production of hemoglobin, myoglobin, and cyto-
chromes b; if destined for cytochrome c it becomes slightly modified by
replacement of vinyl groups at C-2 and C-4 by thiomethyl groups. If, on the
other hand, it is destined for cytochrome a-33, it undergoes more significant
change to first become heme a, with a polyunsaturated alkene at C-2 and an
aldehyde group at C-8 (Rawn 1989). Yeast gene COX10 encodes heme
a:farnesyl-transferase, which farnesylates protoheme, while the product of
COX11 is thought to formylate protoheme, in its transformation to heme a
18


(Glerum & Tzagoloff 1994).
The coordination in biosynthesis, transport, and assembly of the
seven-to-thirteen subunits of cytochrome c oxidase in different eukaryotes is
obviously a complicated affair, many details of which have not been worked
out. It is known that at least two of the nuclear-encoded subunits (COX4 &
5a) require heme (bound to HAP2/3/4 transcription activators) for their
transcription and accumulation, while two other nuclear-encoded genes
(PET 111 & 494) whose products are required for translation of
mitochondrial-encoded subunits 3 and 1, respectively (or 2 and 3, according
to the first paper cited below), are activated by oxygen and inhibited by
glucose; one anaerobic subunit (COX5b) is inhibited by heme bound to
RE01 transcription factor (see Fig. 4 and section 1.4 above). As discussed
in section 1.5 above, heme-bound HAP proteins apparently also activate
upstream promoter sites on several of the HEM genes, so that heme and the
various apocytochrome moieties are coordinated (Attardi & Schatz 1988,
Forsburg & Guarente 1989). One aspect of this study was to analyze levels
of porphyrins and cytochromes in a few cytochrome c oxidase mutants, in
the hopes of finding some insight into the particular heme a sub-branch of
the heme biosynthetic pathway.
19


2. RESEARCH GOALS
2.1 Development of Methods
Determination of which porphyrin intermediates in the heme pathway
accumulate in a given yeast strain, as well as both relative and absolute
quantitative data on that accumulation, can provide valuable information
concerning the role of the gene-product affected by that particular strain's
mutation. The goal of this project was to develop or refine procedures for
analyzing on a small scale the accumulated porphyrins in a batch of yeast
cells, ranging in wet weight from three to six grams. These methods include,
after growth of the culture and whole cell spectrophotometric analysis, the
extraction of free acid porphyrins and their spectrophotometric analysis,
followed by methyl-esterification of the free acid extract for further spectral
analyses, and/or thin-layer or high-performance liquid chromatographic
analyses of the porphyrin components.
2.2 Biological Applications
The methods of porphyrin analysis were applied to studies in three
areas related to the heme biosynthetic pathway. The first area investigated
was porphyric mutants with blocks (ranging from partial to complete) in
enzymatic reactions late in the pathway, resulting in accumulation of various
intermediary porphyrins; these are the pop mutants, mentioned above,
affected either in structural genes (HEM) for heme biosynthetic enzymes, or
in regulatory genes whose products act to control heme biosynthesis in
various ways. The second area investigated was a group of regulatory
mutants, altered in some component of the signaling system involving G-
protein/adenylate cyclase/cAMP/protein kinase, and which also accumulate
porphyrin; these /?AS-related genes may act in response to carbon source
and other physiological variables, and may impact heme biosynthesis. The
20


third area of study involved mutants deficient in the respiratory complex,
cytochrome c oxidase, and which also accumulate porphyrins; because
cytochrome c oxidase contains cytochrome a-a3, incorporating the unique
heme a, it was hoped that details in the biosynthetic pathway specific to the
heme a branch might be elucidated. In each of these three areas of
investigation, the idea was to utilize the porphyrin analytical methods for
providing insights into the particular genetic defect and the possible role of
the corresponding gene in the heme biosynthetic pathway in yeast.
21


3. METHODS
3.1 SMALL-SCALE EXTRACTION OF PORPHYRIN FREE ACIDS
[For up to 5.0 g (wet-weight) cell batches. Modified from "Extraction of
Porphyrin from Yeast" and K. Lee's "Small Scale Extraction of Porphyrins,"
both in Methods Bk. Ill, Section 192 A, which in turn is based on Pretlow &
Sherman (1967).]
Materials: (For 2 strains, through Step 13 of Procedure.)
Access to a working hood.
Chemicals:
15 ml glacial acetic acid
140 ml ethyl acetate
18 ml 2 N. HCI (add 16.6 ml cone. HCI to d.i. water, final vol. 100 ml)
4 ml 10 N. NaOH, in dropper vial (add 40 g NaOH pellets to d.i. water,
final vol. 100 ml)
Glassware:
two 40 ml stoppered glass extraction tubes, aluminum foil-wrapped,
with glass stoppers
one 500 ml suction flask with Buchner funnel and four #2 Whatman
filter papers (two 4.5 cm, two 11.0 cm)
two 30 ml (tall) screw-cap tubes, foil-wrapped, with caps
two 60 ml separatory funnels, pre-greased, and stand for holding
them
two 30 or 40 ml screw-cap tubes with caps (reject any with cracks or
chips on the rims!)
two 20 or 30 ml screw-cap tubes, foil-wrapped, with caps
five 10 ml pipettes and bulb (one per strain; three for chem's)
five Pasteur pipettes and bulb (two per strain, plus one more)
one 125 ml flask for waste solvents
Other:
rubber gloves
marking tape & marker
parafilm
22


vortex mixer
small forceps (for taking tubes out of suction flask)
Thomas Lubriseal (or equivalent)
pH paper (3.0-5.5 range)
long wavelength UV viewer
nitrogen tank with evaporating set-up (rubber tubing and Pasteur
pipettes)
Procedure: Cell Preparation & Harvesting
1) Grow cells in 200 ml of appropriate broth medium in a 500 ml Erlenmeyer
flask in a shaker-incubator at 30 C, 300 rpm, for 48 hours, or as specified by
the protocol for specific strains. The strains should be grown under aerobic
conditions, as growth of yeast anaerobically shuts down respiration and
results in porphyrin accumulation even in normal strains (Kotal et al.) (see
Fig. 5). Based on our data, it is suggested that the following numbers of 500
ml flasks (each with 200 ml of broth) be innoculated:
STRAIN # FLASKS EXPECTED EXPECTED
WET WEIGHT (g) DRY WEIGHT(g)
6288-5A/C1 1 4.4-5.0 1.1-1.5
6288-5A/C1/P21 4 5.0-6.2 1.1-1.9
D28 1 4.7-5.3 1.2-1.6
D28/F8 1 5.0 1.6
AM3-4B/U3 2 35-4.3 ?
AM3-4B/U3/P1 3 3.4-5.8 ?
S288C 1 6.0 1.5
EG81-22b 1 4.9 1.3
R12b 1 4.4 0.8
R1a 1 5.5 1.1
308 1 3.5 0.9
301 3 5.3 0.9
112 1 5.8 1.2
112-699 1 5.4 1.4
SC 167 1 5.3 1.4
SC1171 1 4.7 1.3
SC1183 1 4.4 1.2
JM25 1 5.5 1.4
242-8-5 2 5.0 0.9
155-11-1 2 4.5 0.9
123-20-2 1 4.5 0.9
23


I 111
640nm 580nm 540nm 500nm
Fig. 5. Whole-cell spectra of wild-type strain D28, grown with (dotted) and without
(solid) oxygen. Increased absorbance is clearly apparent in the regions of porphyrin
maxima, especially as seen in the difference spectrum (dashed). Note that cytochromes
are not significantly elevated; note also the 460nm absorbance often seen in difference
spectra of porphyrin accumulators.
24


2) Harvest cells and determine wet weights and dry weights according to
procedure in Yeast Experimental Methods Handbook, p. 17 (#2) (von
Ahlefeldt 1985b). For washing cells and determining wet weights use the
white, teflon 50 ml centrifuge tubes, since the other tubes are made of
plastics that are soluble in the organic solvents used.
3) Prepare 25 mg/ml (dry weight) suspensions for whole-cell spectra, and
run whole-cell spectra on the dual beam spectrophotometer (the "black
box"); see "Yeast Exptl. Methods Hndbk.," p. 18-19.
4) Recentrifuge the remaining suspension in the Sorval RC-2B refrigerated
centrifuge (for 10 min. at 5000 rpm), discard the supernatant, and determine
the wet weight of the pellet after removing as much liquid as possible from
the inner wall of the centrifuge tube.
Extraction: [For up to 5.0 g. wet weight batches; larger amounts will
require larger tubes, etc.] Do the remaining steps in the hood; wear gloves.
Allow four hours minimum through Step 15 (one can take a break after Step
13), and two more hours through Step 19, per pair of strains. Time includes
set-up and clean-up. Cells may be stored now, after Step 4, for up to 60
hours: wrap tubes in foil, cover tops with parafilm and foil, and place in the
coldroom.
Add g.a.a
To pellet add 1.5 ml glacial acetic acid for each remaining
gram wet weight of cells; retain several ml's of it for rinsing tube
walls into extraction tube. Cap tube with parafilm and vortex to
resuspend cells.
5)
Cell pellet
25


6)

Transfer suspension to foil-wrapped 40 ml glass-
stoppered extraction tube. Add three volumes of ethyl
acetate, for each volume of acetic acid used to suspend
Add e.a.
the pellet. Add in 3-4 small aliquots and shake by hand
to prevent clumping of cells. Shake for a total of four
minutes.
\y
Have a suction filtration assembly set up, using a 500 ml
filter flask fitted with tubing, a small Buchner funnel, 2
layers of Whatman #2 filter paper (outer layer is 11.0 cm
diam., folded up around edges; inner layer is 4.5 cm
diam.) for each cell batch. Also have ready 30 ml
(tall) glass screw-cap tubes (flat-bottomed), foil-
wrapped. Pour the cell suspension in slowly, with
vacuum on low, to avoid pulling cells around filter
paper. Filter the extract of each strain separately into a different tube; after
each filtration discard the filter papers and cells, then rinse the funnel with
ethyl acetate.
Removing Metals from Metalloporphyrins: [Do in hood; wear gloves.]
8) Pour filtrate into a 60 ml separatory funnel (pregreased with Thomas
Lubriseal).
9)
Add a volume of 2 N. HCI equal to 1/3 of the filtrate
volume. Shake well. Allow phases to separate. The
acid will displace Zn2+ and other ions, if present, from
the porphyrin.
26


10) Drain the lower (aqueous) phase, containing the porphyrins, into foil-
covered 30 or 40 ml screw-cap tubes. (If tube rims are chipped, there may
be leakage.)
The extraction may be monitored from this point on by checking for the pink
fluorescence characteristic of porphyrins when exposed to long wavelength
UV light.
A' Add NaOH
11) Add 10 N. NaOH in 10-20 drop aliquots, capping and shaking
mixture after each addition (uncapping to release gas
pressure), until a pH of about 3.5 is attained. (Use a Pasteur
pipette and 3.0-5.5 range pH paper to test. As the target pH
gets closer, add only a few drops at a time. Use a ratio of about
10 ml to 45 drops, for 2 N. HCI added in Step 9 to 10 N. NaOH here.) This
step enables the porphyrins to be re-extracted from the aqueous (acid)
phase to the organic (ethyl acetate) phase.
12)
f
n
Add e.a.
To re-extract the free porphyrins, add an equal volume of ethyl
acetate, cap, and shake vigorously for a few minutes. Allow
phases to separate.
13) Using a pipette (and a Pasteur pipette for the final portion) pull off the
upper, organic layer and place in foil-covered tubes. Avoid getting any of
the aqueous phase into the pipette. Cap tube tightly. Extracts may now be
stored in the coldroom, in darkness, overnight.
27


3.2 PREPARATION FOR SPECTROPHOTOMETRIC ANALYSIS
OF FREE ACID PORPHYRIN EXTRACTS
to niti
1)
[Do in hood; wear gloves.]
Set up a hot-water bath at below 70 C (ethyl acetate
boils at 77 C). Fit a Pasteur pipette into a small tube
attached to a nitrogen tank, adjust the valves so that a
fine stream of N2 gas comes out, and play the stream of
gas against the solvent surface, gently agitating it. Lower
the tube into the hot water and hold it there while
agitating the surface, until the volume is less than 2 ml in
the tube. Keep heat low, as needed, by removing the
beaker from the hot plate; excess heat may cause
unwanted decarboxylation of porphyrins.
Minimize the exposure to light during this step;
the evaporation process appears at times to result
in some oxidation or other conversion of
porphyrin, with the appearance of a white
crystalline precipitate.
Transfer the <2 ml volume of porphyrin solution to a screw-cap
or glass-stoppered 12 ml graduated conical testtube, passing it
through a glass wool-fitted Pasteur pipette to filter it; then rinse
the glass wool with a tiny amount of ethyl acetate. Next, using
a Pasteur pipette, add just enough ethyl acetate to raise the
volume to exactly 2.0 ml for each strain; this becomes the "stock". (A white
precipitate forms at times during this final step-usually equally in extracts
from both parental/control strains and porphyrin accumulating strains; this is
28


adequately removed later by centrifugation.) Cap tube, cover with foil, and
put on ice. Extracts may be stored now in coldroom, in darkness, overnight.
3) On the basis of total dry weights of cells extracted, determine relative
dilution factors for each strain, to "equalize" samples for spectra. The
purpose of this adjustment is to eliminate the imbalance caused by having
different weights of starting cell material for each strain to be compared. For
example, given the following strains and dry weights, set the highest dry
weight strain (R1a, 1.95 g) equal to 1.0x dilution factor; divide that weight by
each of the others to get their relative dilution factors (see last column).
(Should the division by the smallest dry weight give a factor larger than 2.0x,
make adjustments on all by multiplying each factor by an appropriate "x"
value less than 1.0, so that the largest factor-for the smallest dry weight--is
no larger than 2.0 ). strain drv weiaht (al rel'tv. dil'n. factor
D28 1.79 1.1X
R1a 1.95 1.0x
112 0.98 2.Ox
112-699 1.09 1.8x
Thus one would use 1.0 ml of the "stock" R1a extract plus 1.0 ml of ethyl
acetate to get a final sample volume of 2.0 ml for performing spectro-
photometric analysis. For the D28 extract one would use 1.1 ml of its "stock"
and 0.9 ml ethyl acetate for a 2 ml spec sample, and so on for each strain.
Prepare these 2 ml spec samples in 5 ml screw-cap vials.
4) Cap each sample tube, vortex, and place in a tabletop centrifuge. (Be
sure that opposite sides are balanced.) Spin at maximum speed for 2-3
minutes. Pour supernatant fluid into a clean, labelled 5 ml screw-cap vial
and put on ice in ice-bucket; keep cover on bucket to avoid exposing the
porphyrins to light.
29


5) Use the DU-7 spectrophotometer (spec) to run spectral analyses on the
strains (see below for operating instructions). The DU-50 spectrophotometer
may also be used; instructions for it follow (section 3.3.2). For the DU-50,
however, if any samples yield an absorbance value much greater than 1.0,
all samples will have to be diluted equally, since the instrument's reliability
decreases as absorbance values exceed 1.0. The H.P. diode array spec
may also be used, but only reads to the nearest 2 nm; instructions can be
found in the manual. This instrument has poor reliability for absorbance
readings below about 0.4 units-values will come out lower than they should
(or at least lower than what the DU-7 would give).
6) After spectrophotometric analyses, evaporate off the ethyl acetate, using
the low-heat hot water bath procedure with a nitrogen stream, under the
hood and in minimal light. Store extracts in capped, foil-covered 5 ml vials,
in the freezer.
7) Clean all glassware (and plasticware) exposed to organic solvents by
rinsing first with water, and then submerging in concentrated acid bath (in
the hood, Rm. 253); wear goggles, apron, and heavy rubber gloves. Follow
with thorough rinsing under tap water, and then the usual wash procedure.
3.2.1 OPERATING THE DU-7 SPECTROPHOTOMETER
1) Plug in instrument thirty minutes in advance to warm up. After a brief
preliminary display, the word "IDLE" will flash across the top of the screen;
when this occurs press keyat top left of keyboard. (See Fig. 6 "DU-
7 Keyboard" ) Next press the appropriate light source keys to turn on lamps;
in all cases for porphyrins we have used both UV and visible: , .
Allow 15-20 minutes for lamps to warm up. The printer is always plugged in
and ready for operation.
30


I
idle uv vis w scan trace

singli scan list dsply (3) [sel]
dual A copy Q)
multi A deleti auto zero start run
Fig. 6: The DU-7 keyboard
7 8 9
4 5 1 6
1 2 3
0 +/-j
CE entej
31


2) Select the appropriate mode of operation, i.e. one of the six keys grouped
at lower left of keyboard. Generally for porphyrin extracts we want to run an
absorbance spectrum: press . (For single, dual, or multi
wavelength modes, see Step 9, below.) The screen will display the
parameter options.
Parameter Options for Scan (Showing choices for a typical porphyrin scan):
Scan #oi (program storage number)
Function [ABS] (absorb, or % transmit.)
Starting 650 (high wavelength of scan)
Ending 350 (low wavelength of scan)
Speed [300] (600 nm/min, 300, or 120)
Upper Limit 1.0 (4.5 max Abs., vert, axis)
Lower Limit 0.0 (-4.5 min Abs., vert, axis)
3) Program your choices for each parameter. For numerical choices, press
appropriate numeric keys, followed by at lower right of keyboard.
If the displayed choice is acceptable, merely press . The cursor
then moves down to the next parameter. For any options displayed inside
[brackets], press [SEL] key (in the center of the keyboard) to change
displayed options; when the desired option appears, press .
NOTE: After the scan, before printing, the wavelength range and
absorbance values can be revised downward (but not upward): wavelength
range can be narrowed from either or both ends, and absorbance limits can
be lowered, for reducing the range and magnifying some detail (See Step
7). It is best to overestimate your expected absorbance now; you can always
lower the upper limit after the scan to enlarge your spectrum-but the upper
limit cannot be raised after running the scan, if the spectrum overshoots the
limit.
4) After warm-up and parameter selection, press , at lower center
of keyboard. This initiates calibration of the instrument; when this is
32


completed, the screen will display a graph with your selected parameters
and, at the top right, the words, "Insert Background Push Run."
NOTE: The DU-7 is a single-beam spectrophotometer, with only one
cuvette compartment; the background (or reference or blank) solution must
be run first, followed by sample(s). Use the same cuvette for ail solutions-or
an optically matched pair. If using UV light, you must use quartz cuvettes.
Use glass or quartz cuvettes when working with organic solvents; plastics
may dissolve. Also, use cuvette caps so that loss of volatile organic solvents
doesn't give increased absorbance readings. Clean cuvettes soon after use
with Windex glass cleaner (and rinse well with deionized water).
5) Lift lid of sample chamber, ensure that the cuvette holder is seated fully
on track and to the right against the chamber wall, and insert the cuvette.
The beam passes through the middle of the cuvette, so the cuvette must be
over half-full, with 1 ml minimum for the narrow cuvettes or 2 ml minimum for
the regular ones. Close the chamber lid, and press . Do not open lid
until the message "Scanning Background" has been replaced by "Insert
Sample Push Run."
6) Remove the cuvette, replace the background solvent with the sample,
and put cuvette back into holder. Close lid and press . Do not open
the lid until reappearance of the message, "Insert Sample Push Run."
NOTE: Using our standard wavelength range of 300 nm (from 650-
350 nm), at a speed of 300 nm/min (i.e. 5 nm/sec-the dual beam spec in
Rm. 253 is run at 1 nm/sec normally), only one sample may be run before
the temporary memory is full. Therefore that scan should be printed (press
key) and then deleted, before running a second sample. On the
other hand, a range of 300 nm, at a speed of 600 nm/min, allows the running
of four samples before the memory is full. All scans are automatically stored
in the memory, and a warning message flashes when it is filled.
33


Manipulating Graph Display, and Printing:
7) After the scan(s), the axes and other aspects of the graph display can be
manipulated for informational purposes or for optimizing the curve's
appearance, prior to printing.
To change any parameters of the display, the cursor's box (white
background) must be moved to that parameter; this is done by pressing dir-
ectional keys in the center square of the keyboard. Then press appro-priate
key(s) for desired value or parameter, followed by . This will alter
only the parameters for the graph on display; subsequent sample-scans will
revert to parameters as originally programmed, in Step 3. The upper limit of
the Absorbance scale cannot be increased after running a scan.
8) The onscreen "Display Mode" has two options: [SINGLE] or [OVERLAY],
An overlay display may be made on up to six scans that are stored in mem-
ory. (Remember, however, that you can only have one scan in memory if
your speed was 300 nm/min; if you wish to make use of this handy overlay
mode for printing, you must have made your scans at 600 nm/min--and you
would have a maximum of four scans in memory. Of course, the other option
would have been to decrease the wavelength range, thus taking up less
memory, allowing more scans). To make an overlay, locate the cursor at
"Display Mode" on the screen; press [SEL] key until [OVERLAY] appears in
the cursor; then press . Move cursor to "Scan #" at the top of the
display, and enter the number of each desired scan, followed by .
(Go to for the scans and their respective numbers as listed in mem-
ory, if you can't remember them). Modify the values of the axes if necessary.
9) Before printing any graph, the cross-hair indicator of the "Scan Trace"
feature should be moved offscreen. This is accomplished by pressing (hold
down) either arrow key at top center of the keyboard ("Scan Trace");
34


otherwise the cross-hair will be printed and may easily be confused with
data points on the graph.
10) To print the displayed graph, press the key. After printing,
move the scan-trace indicator wherever desired on the curve, to read out the
exact absorbance at any given wavelength, or vice-versa. The values
appear in the lower right onscreen, under "Trace." This should be done for
any maxima or peaks, and the resulting data hand-recorded on the print-out.
See Fig.'s 7 and 8 for sample scan print-outs from the DU-7 Spec.
Deleting a Scan (After the message, "Warning-Temporary Memory
Full" appears at top left):
11) Be sure to first print a scan (if you need it) before deleting it! Press
key for screen listing of all stored programs or scans. (Although
background scans use up as much memory as sample scans, only sample
scans will be listed; deleting a sample scan does not delete its background
scan.) Press key. Then press [SEL] key until [SCAN] appears in
the cursor, and press . The scan number will now be in the cursor;
if the number is the correct one, press . If the number is incorrect,
press appropriate numeric keys for the desired scan number as listed; then
press . This deletes the selected scan, leaving room in the
memory for another scan to be run.
NOTE: If the background/reference for the new sample-scan is the
same as for the deleted scan, it is not necessary to re-blank. Press
, and insert the next sample (and push .) However, if the
background/reference for the new scan is different (e g., a difference-
spectrum is to be run on two previously run samples), then you must
recalibrate the instrument: press . Then, when indicated, scan the
new background (or the sample to be subtracted), followed by the sample
itself (or the sample you're subtracting from). Refer to Steps 4-6 above.
35


Fig. 7. Sample scan print-out from the DU-7 spectrophotometer; the sample is a
free-acid porphyrin extract (in ethyl acetate) from yeast strain R12b (vs. ethyl
acetate).
i i r. i
/ i-' S-'i-i
Display Modi
[Single]
r! \rtr-+ i r.r.
I L'i iu '/iwi i
[f
LftsJ
| iji .t
. r- I rr >-
r-Vi


* s:i irj *tm.u A1 .*! i *+? Vi. K? v\t H Kf hhu
-iFfi p?r/i? in

i Ri
\ A fiBS 90LRCE * J nuife. X .nr- l£Q
cqa fl www u -0.0210 Uis/iJJ n V'lr. 350.0 e 4GQC i:r ?*
36


Fig. 8. A blow-up of the visible wavelength portion of the scan in Fig. 7.
bean # s i, bb
Q Jim
J l '-r-F-'
. j , t i
! h *r*v i 51? r*ls*':-'4w
M'JwfW
[Sinqlel
function
Li il* i
Ci am.

4b0 490.0 1 1 i;_r i bto!
CPQ MATT
W v-H ww i IVUU
1.028Z Uis-ij-J Scan
Si iO. 0
to, toitotii
ft < ftftft
to, lotto
! i I H & A
Â¥L \/Â¥wj.
M Ij&AA
n g&gi
r-. i Wi jrt
w I ?-s L:

4S^i \/\
HH-.
i UJw
to, iffii
37


Using Other Modes (besides ):
12) When doing a standard curve (of absorbance vs. concentration) fora
given substance, a series of dilutions is prepared, and these are then read
for absorbance at one or more specified wavelengths. First do a scan of one
of the dilutions, to obtain a spectrum; use the "Scan Trace" cross-hair to
ascertain the exact wavelength(s) of absorbance maxima on the curve.
These are the wavelengths at which you will program the DU-7 to read the
absorbance of your dilution series.
13) After the scan and wavelength determination, press the appropriate
mode keys: , , or --the latter for up to eight
different wavelengths. Then enter the appropriate wavelengths, pressing
after each one. (Don't worry about "Factor" or other parameters or
options available). Then press to calibrate the instrument. When
the top of the screen indicates "Insert Sample Push Run," do so. One of the
"samples" must be the reference/background solvent used in the dilutions;
this absorbance reading must later be subtracted from each sample
absorbance reading-or else used to establish "zero concentration, zero
absorbance" on your graph. The DU-7 won't do this itself.
14) When all samples have been read for absorbance, press for a
print-out of the data. Unfortunately the DU-7 will not create a graph-display
from this data; that you must do manually.
For any problems, or further information, see the Operating Manual for
the Beckman DU-7, in the drawer below the instrument.
3.2.2 OPERATING THE DU-50 SPECTROPHOTOMETER
NOTE: The DU-50 has no "scan-trace" feature for pinpointing
wavelength or absorbance values of peaks or maxima; these have to be
38


interpolated from the graph. Also, absorbance readings should be kept at
or preferably below 1.5 Abs. units.
1) The instrument (in Rm. 252) is generally already plugged in and warmed
up. Turn on the light source by pressing the key in the upper right;
this does not need to warm up. The ultraviolet source is not needed unless
you will use wavelengths below 400 nm; if needed, press the key also,
and let it warm up a minimum of twenty minutes. Make sure the printer is
turned on and is "on-line".
2) For a wavelength scan, press the key to select the scanning
mode. Press appropriate numerical keys for your starting wavelength,
followed by the key; do the same prodedure for the ending
wavelength.
3) Press the key for a speed of 500 nm/min (displayed in the
window), followed by .
4) Set the upper and lower absorbance limits; in general it is convenient to
enter <0.0>, followed by for both limitsthis activates an automatic
ranger that will adjust limits to the sample's absorbance.
5) Press the key to calibrate across the wavelength range.
6) When "Bkg" appears in the display, insert your background solvent
(usually ethyl acetate for free acid prophyrins, or chloroform for methyl ester
porphyrins; use a cuvette lid when scanning samples with volatile organic
solvents) and press the key.
7) When the background scan is done, insert your sample cuvette and press
39


. Data will be automatically fed into the printer for a scan print-out.
(See Fig. 9 for a sample DU-50 print-out).
8) Check the print-out; if any peaks go over 1.5 A, you should dilute the
sample. This instrument's accuracy decreases with higher
concentration/absorbance; with an absorbance of 2.0 or greater, the reading
can be as much as 25% low. Therefore samples should be diluted to be
less than 1.5 A on the pertinent peaks; then repeat the scan (using Steps 7-8
if no one else has used the instrument in the meantime).
9) Once you have a satisfactory scan with all pertinent peaks no greater
than 1.5 A, press to exit from the scan mode.
10) Now use the print-out to calculate the wavelength of each peak (in
particular the Soret peak) and the absorbance at that wavelength.
This instrument has no "scan-trace" feature like the DU-7 has, so you must
do this "manually". One could do a series of single-wavelength readings in
the immediate vicinity of the peak (as estimated from the print-out), but as
there is a range in the reading at any wavelength, generally the calculations
and estimations from the print-out will be as accurate (and as quick) as
running all the single-wavelength readings (and calculating absorbance
averages at each wavelength).
11) If the single-wavelength mode is required or desired at any time, refer to
the plastic-covered "Quick Reference Operator's Guide" for the DU-50,
normally located on top of the instrument.
The H P. Model 8452A "diode array" spectrophotometer may also be
used, as mentioned previously; see Fig.'s 12 & 13 for samples of plotted
scans from this instrument.
40


BECKMAN
DU 50 BFECTRQPHaTDMETER
ABSORBANCE
0.5000 0.4000 0.3000 0.2000 0.1000 0.0000
w
A
V
E
L
E
N
G
T
H
I
N
N
M
Scan Speed: 500 nm/min
Fig. 9. Sample scan print-out from the DU-50 Spectrophotometer; sample is of a free-acid
porphyrin extract (in ethyl acetate) from yeast strain SC1183 (vs. ethyl acetate).
41


3.3 METHYL-ESTERIFICATION OF FREE ACID PORPHYRIN
EXTRACTS
[Modified from "Methylation of Porphyrin Free Acids" in Methods Book III
Section 192 B, and Pretlow's method (1965)].
Materials: (For methylating 2 free acid porphyrin extracts.)
Access to a working hood.
Chemicals:
10 ml 5% (v/v) sulfuric acid/methanol (use 0.5 ml reagent grade-
18.0 M-sulfuric acid added to 9.5 ml HPLC-grade absolute
methanol)
16 ml ethanol-free chloroform (use pentene/amylene-stabilized, or
else wash it in a 60-ml separatory funnel three times with equal
volumes deionized water just before use)
40 ml 1% (v/v) ammonium hydroxide (use 0.4 ml reagent grade-
14.8 M-ammonium hydroxide added to 39.6 ml deionized
water)
24 ml cold deionized water (plus 48 ml additional if washing the
chloroform)
2 pinches anhydrous sodium sulfate
Glassware:
two 20 ml separatory funnels, pre-greased
one 60 ml separatory funnel, pre-greased, and stand for holding it
two 20 ml scew-cap tubes with caps (not needed if ethanol-free
chloroform is on hand)
two 5 ml glass vials with caps
six 10 ml testtubes
two Pasteur pipettes and bulb
one 5 ml pipette and bulb
three 10 ml pipettes
42


Other:
rubber gloves
marking tape & marker
vortex mixer
hot water bath set-up (at 80-85 C) or reflux chamber with hole-plate
for vials
small metal spatula (for sodium sulfate)
Thomas Lubriseal (or equivalent)
long wavelength UV viewer
nitrogen tank with evaporating set-up (rubber tubing and Pasteur
pipettes)
Procedure: [Do in hood; wear gloves ]
1) Redissolve the porphyrin free acids by vortexing in 5 ml of 5% sulfuric
acid/ meth-anol, in a screw-cap vial (with an unchipped rim).
2) Tighten cap securely, wrap the vial in foil, and reflux for 30 minutes in a
hot-water bath at 80-85 C. The bath by the sink in Rm. 252 is adequate, but
should be covered.
3) Let the vials cool (for about ten minutes) to room temperature. Into a 20
ml separatory funnel place 6 ml of ethanol-free or washed chloroform.1 Add
vial contents and shake vigorously, allowing gas to escape between shakes.
4) Add 10 ml of 1% (v/v) ammonium hydroxide in deionized water. Shake
vigorously to get a homogeneous emulsion.
5) Let phases separate fully (check under long wavelength UV light) and
collect the lower (CHCI3) layer.
6) Repeat Steps 4 and 5; for Step 5 this time it may be necessary to allow
phase-separation to continue up to 10 minutes until it is complete when
43


viewed under long wavelength UV.
7) Wash the chloroform solution twice in the separatory funnel with equal
volumes of cold deionized water.
8) To dehydrate the solution, add a pinch or two of anhydrous sodium
sulfate and let sit in a foil-wrapped tube for 15 minutes.
9) Decant into another tube (rinsing the sodium sulfate with half a ml of
chloroform), and evaporate to dryness by directing a fine stream of N2
against the surface. If convenient, now is a good time to filter each sample
for HPLC analysis later (See Steps under "HPLC Procedures"). Before all
the solvent is evaporated, transfer to a very small vial and finish the drying
(rinsing the tube with a bit of CHCI3). Store in a capped, foil-covered vial at
4 C. or less. 1
1Most chloroform (including "HPLC grade") is preserved with ethanol
(0.75-1.2%). The ethanol can convert porphyrin methyl esters into dications
with different spectra and different extinction coefficients; it can also produce
some unwanted ethyl esters, and contribute to the deactivation of the silica
packing in an HPLC column. Pentene or amylene stabilized, ethanol-free
chloroform is available commercially, but if none is on hand, the chloroform
must be washed (in a separatory funnel) three times with equal volumes of
deionized water, just before use.
44


3.4 TLC-SEPARATION OF PORPHYRIN METHYL ESTERS
Materials: (For thin layer separation on 20 x 20 cm plates; for smaller scale,
quicker TLC, using smaller, custom-cut plates, see "Alternate Small-
Scale TLC Method" which follows this section, on page 42.)
Access to a working hood (such as Rm. 255).
Chemicals:
500 ml "eCk" developing solvent (9:1 v/v of 2% ethanolic chloroform/
kerosene) per 5 L chamber (9.0 ml absolute ethanol, 441.0 ml
HPLC-grade chloroform, 50.0 ml deoderized kerosene) [For
large-scale, preparative separations, Sherma & Fried (1991)
recommend using 1:1 v/v of petroleum-ether (40-60 C)/chloro-
form, developing in an ammonia atmosphere by placing a
beaker of 10% ammonia inside the developing chamber.]
washed (or ethanol-free) chloroform for dissolving porphyrin methyl
ester samples (25-500 jul per sample, depending on how much
of the sample is to be separated)
porphyrin methyl ester samples
porphyrin methyl ester marker kit
Glassware:
large (5 L) rectangular glass TLC chamber(s) and lid(s)
1 L flask, graduated cylinders for measuring/mixing developing
solvent
capped bottle for waste solvent when done
Other:
TLC plates (200 fxm thick, plastic-backed MCB "silica 60"-60 A pore
space-that are 20 x 20 cm; or VWR "silica gel 60," 0.20mm
thick, 20 x 20 cm, #EM-5748-7)
oven, for drying plates at 100 C
20 ^l autopipettor & tips, or 5 nl capillary tubes
blow dryer
stapler, thread
TLC plate stand, for holding plate(s) at correct depth in solvent; or use
a holepunch to perforate each (plastic) plate at top, and tie
45


thread to it to suspend the plate at the correct depth & position)
opaque cover for chamber(s)
goggles, gloves
Procedure: (All done inside a working hood, under minimal light; wear
goggles and gloves.)
1) Make up 500 ml of "eCk" developing solvent for each large
chromatographic chamber to be used; keep covered.
2) Pour the developing solvent into the chamber under the hood, place the
metal plate-stand inside (if using it rather than hanging plates by thread),
place the lid on top, and allow about 30 minutes for airspace saturation.
3) Two 20 x 20 cm plates can be developed at a time, per chamber, if the
stand is used; if plates are rolled into cylinders and suspended by thread,
two can also be run together. Warm up the oven, and then dry the TLC
plates for 5-10 minutes at 100 C.
4) Resuspend each sample of dried porphyrin methyl-esters, by adding 50-
200 ^il washed (ethanol free) chloroform and vortexing; the amount to add
depends on the relative apparent (or suspected) concentration of porphyrins
in the sample. Resuspend the methyl ester marker kit (standards) in a
similar manner; these are usually more concentrated than methylated extract
samples, and therefore should be diluted with more CHCl3~say, 100-500 fxl.
Tightly cap until ready to apply; record how much chloroform has been
added to each.
5) Lightly draw an origin line across the plate 2.5 cm up from the bottom;
space the spots about 1.5 cm apart, alternating marker spots with sample
spots, starting no closer to the edge than 2 cm. Spot-apply 5 ^ of each
46


sample, using a 20 [xl autopipettor (set to 5 ixl) or using 5 [.il capillary tubes,
applying in about 1 nl aliquots while letting the spot dry between aliquots;
use a blow dryer (on warm air setting) to dry the spot between aliquots. If
there is some uncertainty as to the amount of porphyrins in the sample, a
range of 5 f.il, 10 jaI, 15 nl spots could be run, alternating with marker spots.
Tightly cap each remaining sample after withdrawing the 5 nl.
6) If rolling the plates, carefully use a holepunch to make a hole at each
upper corner and one at the top in the middle, roll into a vertical cylinder and
carefully staple along "seam" at top and bottom; tie 20 cm thread in each of
opposite holes so the cylinder can hang plumb. (This procedure helps to
minimize the "smile" effect that tends to occur, with solvent moving the
samples farther near the sides as compared to the middle.) Slowly lower the
plate(s) into the chamber so that the penciled origin line is no less than 1 cm
above the surface; if using the rolled plates, tape the threads against the
outside wall of the chamber once plate(s) are in plumb position at correct
depth.
7) Place lid on chamber, cover chamber with an opaque material, and leave
to develop until solvent front reaches about 1 cm from the top (1.25 to 2.0
hours, but start checking after 70 minutes).
8) Remove plate(s) and mark the solvent front with a pencil. Allow plate to
dry in the hood, in darkness, for five minutes. Transfer developing solvent to
an organic waste bottle (unless more TLC runs are planned soon--if so,
keep lid on firmly!).
9) Observe the plate under long wavelength UV light. Only pink fluorescent
spots are porphyrin methyl esters. Circle each such spot gently with a dull
47


pencil, and make a mark for its center of intensity outside the circle. Also
circle/mark the origin spots.
10) Label the marker porphyrins for each marker spot separated; these will
be, in order from bottom upwards: uro-, heptacarboxylic, hexacarboxylic,
pentacarboxylic, copro-, and meso- porphyrin methyl esters. Identify sample
porphyrins by comparing with adjacent marker porphyrins; protoporphyrin in
samples travels slightly farther up the plate than does the meso- marker. If
desired, measure migration distances for each porphyrin spot, and calculate
Rf values.
11) Store the plate(s) in a black plastic bag in the freezer or a coldroom.
Use a fine stream of nitrogen gas to evaporate the suspended samples and
markers to dryness, under minimal light.
12) Using a Photodyne camera with attached hood, photograph the plates
on a UV transmitter.
3.4.1 ALTERNATE, SMALL-SCALE TLC METHOD
Materials: (For quick TLC separation on a custom-sized 6.6 x 10 cm plate,
using a 600 ml beaker for the chamber and 50 ml of developing
solvent per beaker, one plate per beaker; each plate can develop one
sample spot, with a marker spot.)
Access to working fume hood.
Chemicals:
50 ml "eCk" developing solvent per plate, i.e., per sample to be
separated (0.9 ml absolute ethanol, 44.1 ml HPLC-grade
chloroform, 5.0 ml deoderized kerosene)
washed (ethanol-free) chloroform
porphyrin methyl ester samples
porphyrin methyl ester marker kit
48


Glassware:
one 600 ml beaker for each sample or plate to be run
100 ml flask for making up the solvent
graduated cylinder and pipets (with bulb) for measuring chemicals
capped bottle for waste solvent when done
Other:
#2 Whatman filter paper
TLC plates (see above protocol for type of plate; cut one 20 x 20 cm
plate into 6 plates about 6.6 x 10 cm)
oven, for drying plates at 100 C (an HPLC column oven can work,
with plates resting on glass tubing laid over element blocks)
20 (.il auto pipettor & tips
blow dryer
opaque cover
aluminum foil
goggles and gloves
Procedure: [Do inside a working hood, under minimal light; wear goggles
and gloves.]
1) Make up 50 ml "eCk" developing solvent for each sample to be
separated.
2) Fit a wide strip of #2 Whatman filter paper halfway around inside each
600 ml beaker (the paper should extend from the bottom up about two-thirds
to the top). Pour the developing solvent into the beaker(s), and cover snugly
with aluminum foil. Allow 10 minutes for airspace saturation.
3) Using scissors, cut a 20 x 20 cm plate into six 6.6 x 10 cm miniplates.
Warm up the oven and dry the plate(s) at 100 C for 5-10 minutes.
4) Resuspend the porphyrin methyl ester sample(s) and marker kit in
washed (ethanol free) chloroform. (For methylated extracts from strains
having 2-4 g. dry cell weights, try using 100 or 200 nl, depending on the
49


amount of fluorescence; use 500 |xl in the marker kit vial, or less if much has
been used already). Vortex and tightly cap. Record volume of chloroform
added to each.
5) Spot-apply 5 fil samples as in protocol above, onto a penciled origin line
2 cm up from the bottom in this case; place a sample spot at 2.5 cm from the
left side, and a marker spot at 4.0 cm.
6) Slowly lower plate into beaker, making sure the origin is above the
surface; lean plate slightly back against the wall of beaker where there isn't
filter paper. Firmly wrap aluminum foil over the top of the beaker, cover with
opaque material, and leave to develop for about 25 minutes, before solvent
front just reaches the top.
7) Follow procedure in steps 8-12, in above protocol.
50


3.5 HPLC PROCEDURES
High performance liquid chromatography is a much more refined,
sensitive, and reliable method of separating, identifying, and quantifying
porphyrin methyl esters than is thin layer chromatography. With an HPLC
apparatus, the preceeding TLC and TLC-related procedures are not
generally necessary.
While the time period required for an HPLC analysis on one sample is
not too much different from that for a TLC analysis, the relative time period
decreases greatly on the HPLC apparatus as additional samples are added
to an analysis. The minimum time period for a one-sample run (which
includes warm-up & equilibration, a marker run, and flushing & clean-up
afterward) on the HPLC is about 75 minutes. Roughly ten minutes should be
added on for each additional sample that is to be run, assuming that only
one run is required for each sample; more generally at least some sample
runs will have to be repeated to obtain optimal resolution of the peaks, since
the setting of the absorbance range is partly guesswork for any given
sample.
The following procedures apply to the Beckman model 338 HPLC
system (including two 110B pumps, a solvent mixer, a 160 detector with a
mercury lamp and 405 nm filter installed, a 406 analog interface, and a NEC
PC-8300 controller) presently in Rm. 246 (SN: 387770). The columns we
have used for porphyrin analyses are a Waters Microporasil (PN 27477) and
an equivalent Alltech Alphabond silica column (PN 77034, SN 95060702);
column characteristics are 300 x 3.9 mm, packed with irregular 10 urn silica
particles with a 125 A pore size. The mobile-phase solvent is 55% ethyl
acetate and 45% n-heptane (HPLC grade). With our very polar silica
packing and nonpolar mobile phase, thus, we are doing "normal phase" or
adsorption chromatography.
51


Instrument Set-up 1
1) Turn on the power to both the "Solvent Delivery Modules" (pumps A and
B), the mixer, the Detector, the 406 module, and the NEC controller. Power
buttons are on the backs of all instruments, except the NEC which has it on
the right side.
2) Set the LOW LIMIT SET dials on the two pumps all the way
counterclockwise, to reset the pumps; make sure the HIGH LIMIT SET dials
are set to about the beginning of the 4 area.
3) If the NEC window comes up with the start-up screen (BASIC
highlighted), press the left arrow key once to get the cursor to "HPLC", then
press return to get "System Status" screen (TAB. ESC will do the same
thing). Next press TAB for the "Module Access" screen, then press 6 for the
406 analog interface module, resulting in display of the "Run" screen.
4) Once the "Run" screen (RUN in top-left) is displayed, use arrow keys to
move the cursor to the %B field. Test to confirm that the B pump is operating
by entering 100. return, then moving the cursor to the Flow field to enter 0.5.
return. Once the B pump is seen/heard to be operating, enter 0, return in the
%B field to test the functioning of the A pump. (This step assumes that
HPLC-grade ethyl acetate is already in reservoir A, and HPLC-grade n-
heptane is in reservoir B.)
5) After operation of both pumps is confirmed, enter 45, return in the %B
field. This will program a 55:45 ratio of ethyl acetate to n-heptane, as
desired.
6) Move the cursor to the Flow field and enter 15, followed by pressing the.
f3 key (DUR for duration), 4, return. This will program a gradual increase in
52


the flow-rate to 1.5 ml/min over a four minute period.
7) Confirm the program File #1 for a porphyrin run: press shift. f2
(PROGRAM), and once "FILE" appears near the top-right followed by the
cursor, press 1, return. File 1 should look like this (if the file is not present,
use arrow keys to move the cursor, and enter appropriate info, to create this
file):
TIME FUNCTION VALUE DURATION
0.0 RLY ON 1 6.5
0.0 RLY ON 2 0.1
0.0 RLY ON 3 0.02
6.5 END
If you are programming this file, hit return only at the end of each row, and
always enter the TIME info first for each row. To obtain the END function,
press shift. f5 (NEXT), then_f5 (END) again. Relay 1 is to turn on the chart on
the recorder; relay 2 is for an auto-zero; relay 3 makes an injection mark;
after 6.5 minutes the program ends and the chart will stop, but the pumps will
keep running.
When program is displayed, press shift. H (EXIT); when it asks SAVE
CHANGES? enter Y. The window should now show the "Run" screen. Refer
to the NEC System Gold manual, p. 3-116 for programming questions.
8) Let the instruments run (after a flow-rate of 1.5 ml/min has been reached)
for thirty minutes before the first sample run.
9) Check the Minicontroller display for Detector functioning: the display
should read "DET 160" in the upper window, and "D O.K." in the lower
window. If there is some other message (for example, "Power Failure"), turn
the Detector off and check the plugs and electrical connections; if turning it
back on does not produce the "O.K." message, consult the manual for
53


possible problems.
10) Once "D O.K." appears in the lower window, press the right arrow key
(on the Minicontroller) once so that the upper window reads "CHANNEL",
and then press the down arrow key once to obtain the "RANGE" parameter
in the upper window. If the approximate absorbance range of your sample's
components is known, reset the absorbance scale from its default value
(0.100 A) by pressing the appropriate number and decimal keys, followed by
the STO/EX key.
11) The detector needs to warm up for 30 minutes after power up (or 45 min.
if the lowest, most sensitive scales are to be used).
12) Make sure you have enough mobile-phase in the supply flasks for your
runs; if not, or you're not sure, add more to each reservoir. Approx. 65 ml
ethyl acetate and 55 ml n-heptane will be used for a one-sample run (which
includes, again, a marker run and the thirty minutes of pumping before and
after). Add about 10 ml each solvent per additional sample to be run.
Sample Preparation:
13) Prepare the porphyrin methyl ester samples by dissolving them in
ethanol-free chloroform (see Note 1, p. 44, re. chloroform preserved with
ethanol), keeping light exposure to a minimum. For most methylated
extracts, 50-500 nl is an appropriate amount of chloroform. The sample
solution should be slightly pinkish in color; if it is not free of particles
(whether suspended or settled), it should be filtered. Dissolve the marker kit
porphyrins in an appropriate volume of chloroform also; they will not need to
be filtered.
54


14) Sample filtration is done using a 1-2 cc glass syringe with a luer-lock tip,
a 20-23 gauge needle for uptake of sample, and a Fisher Cameo 3N HPLC
nylon filter (0.45 fxm pore size, 3 mm membrane). Draw the sample into the
syringe, then pull slightly farther on the plunger to pull the sample out of the
needle; remove the needle and replace with a filter. Press the plunger
slowly but firmly, capturing the filtrate in a clean vial or microcentrifuge tube.
For quantification purposes, Fisher claims the Cameo 3N filter has a hold-up
volume of "less than 3 iliI".
15) Keep the dissolved sample(s) and marker kit (plus a couple mis each of
both chloroform and prepared mobile-phase) in capped vials or
microcentrifuge tubes, on ice, in a covered ice bucket. Prior to a sample run
it is best to run an injection of porphyrin methyl ester marker kit, to confirm
instrument functioning and permit optimal identifications.
Pre-injection Preparations:
16) Turn on the BD41 recorder by pressing the power button on the top left
of the instrument. Remove the pen cap and insert the long-tip pen into the
upper pen holder.
17) Make sure the chart speed is set by pressing the (L2 button, the mm/sec
button, and the OFF button, if they aren't already depressed.
18) Make sure the 10 mv button for the measuring range (for the long-tip
pen) is pressed, and that the 0 button is up.
19) Turn the zero dial (on the far right) one way or the other to line up the
pen over your zero or base line on the left of the chartpaper, and lower pen.
20) If necessary, reset your A-range on the Minicontroller (see Step 10).
55


Then press the Auto Z key (upper right on Minicontroller). The "offset"
displayed in the window should be around 0.16, but not greater than 0.5; if it
is over 0.5, then the flow cell may need cleaning, or there may be a bubble
lodged in it (see Note1 at end of section, and/or the manuals).
21) On the Minicontroller press the down arrow key twice until "AUTO
RANGE" appears in the upper window; now press the right arrow key to turn
on the auto-range function. With this function on, in the event your sample
concentration is too high for your selected A-range, there will be an
automatic increase of the A-scale by a factor of five for each time the
absorbance approaches 100% on the current scale; the scale will
automatically decrease by a factor of five when the absorbance goes down
to 20% of the expanded scale. Although readings based on this autorange
feature are not accurate, it will at least give you a dilution factor to use for a
new run.
22) Take the 25 nl blunt-tip injection syringe (20 gauge needle), and rinse it
with chloroform by filling it and emptying it into a waste microcentrifuge tube
2-3 times.
23) Then take the injection syringe and fill it with mobile phase (ethyl
acetate/n-heptane). With the injection lever on LOAD, insert the needle
slowly into the port, making sure the needle tip is fully seated (there is slight
resistance the last cm or so).
24) Depress the plunger, injecting a syringe-full of mobile phase into the
valve loop. Remove the syringe and repeat this procedure 2-3 more times,
to fully flush the loop of any residual sample from previous runs. The lever
remains in the LOAD position during this flushing process; some mobile
phase will come out of the short plastic tube that leaves the valve/port area.
56


Marker Kit and Sample Injection:
25) Now take up 5 ^1 of the marker kit solution into the injection syringe and
slowly insert the needle into the port until fully seated.
26) Slowly depress the plunger all the way, and then move the lever to the
INJ position. This will activate the NEC program, starting the chartpaper,
making a small injection mark on the negative side of the zeroline, and auto-
zeroing again. Leave the syringe in the port.
27) Monitor the chromatogram as it goes; approximately 2 cm (~11 squares)
past the injection mark, the solvent front will peak out. This will be a fairly
small peak on A-scalesof 0.1 or more; the smaller the A-range below 0.1,
the larger the solvent front peak. Mesoporphyrin (or in the case of p.m.e.
extracts, protoporphyrin) then peaks out, about 5 mm (2-3 squares) later;
coproporphyrin peaks next, about 7 mm (3.5 squares) later; next is
pentacarboxylic porphyrin 5 mm later (2.5 squares), followed by
hexacarboxylic porphyrin 6mm later (3 squares), then heptacarboxylic
porphyrin about 8 mm later (4 squares), and finally uroporphyrin peaking out
about 10 mm later (5 squares), about 6 minutes after injection (see Fig. 10,
showing a typical chromatogram on the BD41, of a methyl ester marker kit
run).
28) If convenient, label the peaks as they occur, and quantify them. This is
done by multiplying peak-height (as a percent of full-chart scale) times the A-
scale, to obtain the absorbance of the compound; this result is then
multiplied times the HPLC338 conversion factor for that compound (see
Tablel), to obtain the number of picomoles of that compound injected.
29) A full run of an injection containing some uroporphyrin (which is the last
porphyrin to peak out) takes just over six minutes on the Beckman 338
57




(fc./o) -A---, o.ofc/
Z
(6>,75) if£p7A... .o.oc,'
----- o,c-
-------- <^c'
-----------^ftZo (&.) -c'
^£50(7.^) /4--W'
4-0*0 }->
A
|(gp^/lAA^^- A^0.\____________________
Rg. 10. A typical chromatogram from the Kipp & Zonen BD41, showing a run of porphyrin methyl-ester
marker kit (with equimolar amounts of each of six porphyrins) dissolved in chloroform. Note the
injection mark (inj.), the solvent front (S.F.), and the subsequent peaks of the different porphyrins.
Protoporphyrin, when found in a sample, elutes first instead of the standard mesoporphyrin. Values in
parentheses indicate the absorbance on the chart paper scale of 1 to 10; values to the right (A= 0.0799,
e.g.) indicate actual absorbance units, based on the setting for this run (A=0.1).
Table 1. HPLC Conversion Factors
PorDhvrin methvl ester: Conversion Factor:
Uroporphyrin 1750
Hepta-carboxylic porph. 1600
Hexa-carboxylic porph. 1400
Penta-carboxylic porph. 1360
Coproporphyrin 1300
Mesoporphyrin 1370
Protoporphyrin 1020
Table 1. Conversion factors for quantifying porphyrin methyl ester samples dissolved in chloroform, run
through the Beckman model 338 HPLC system, and recorded on the BD41 Kipp & Zonen recorder. For
calculations, based on the absorbance setting and the peak-height on the paper, determine the
absorbance units for each porphyrin in the sample; then multiply each absorbance by its appropriate
conversion factor. Results are picomoles injected. These factors pertain only on the above
instruments, using a mercury lamp and a 405 nm filter in the absorbance detector, with the Alltech
Alphabond silica column.
58


system as currently set-up. After the program shuts off the paper at 6.5
minutes, lift the pen, remove the syringe, and push the injection lever back to
the LOAD position.
30) If the peaks came out too low for good quantification, or if they went
above the absorbance scale as set, it is best to repeat the run on a different
absorbance range (or use a different concentration) so there will be optimal
peak resolution and accuracy; therefore, if necessary, reset the range on the
Minicontroller to an appropriate value, press the Auto Z key on the
Minicontroller, and readjust the zero dial on the recorder so the pen is above
the zeroline. Then repeat the injection procedure (Steps 25-29).
31) Once a satisfactory chromatogram has been obtained for the marker kit,
rinse the syringe with chloroform 4-5 times (as in Step 22), wiping the
outside of the needle with a kimwipe on which some chloroform has been
squirted. Then use the syringe to flush the valve loop with mobile phase
several times (see Steps 23-24).
32) Now compare the relative pinkness (porphyrin concentration) of the
marker kit solution with that of your sample solution, and estimate whether
you will need to change the absorbance range setting for the sample run.
Sometimes all you can do is make a guess; if the scale turns out to have
been too small or large, you'll have to just let the run go on to completion
(6.5 minutes, about 7 cm of paper), and then reset the range and repeat the
run.
33) Run the sample, as in Steps 25-31.
34) Be sure to flush the syringe 4-5 times with chloroform, and the valve
loop 2-3 times with mobile phase, at the end of the run. If there are more
59


samples to run, follow the same steps (25-31).
35) After the last run is finished, lift the pen, depress the maximum chart-
speed button and release the OFF button to feed the paper out for cutting.
Then reset the speed, press down the OFF button, and turn the recorder
power off. Remove the pen cartridge and replace the cap.
36) Turn off the detector, but keep the delivery/pump and other modules
running for 20-30 minutes at the same flowrate to flush the column.
37) During this flushing time, evaporate off the chloroform from the marker
kit and sample(s) under the hood, using a fine stream of nitrogen gas, under
minimal lighting. Cap vials tightly and return marker kit and sample(s) to the
freezer when dried. (If necessary, sample solutions can be kept in the
chloroform in the freezer, or on ice in the coldroom, in tightly capped, foil-
covered vials, for at least five days, with no loss or change.)
38) After flushing the column for 20-30 minutes, in the NEC window go to
the FLOW field, enter 0, f3 (DUR), 4, return, to gradually decrease the
flowrate to zero. After flow has stopped, turn off all modules.
39) If it hasn't been done, thoroughly flush the injection syringe with
chloroform, and wipe the plunger rod with a kimwipe and chloroform. Set
the two parts aside separately, to dry, before replacing the plunger in the
syringe. Insert a blunt 20 gauge needle into the port, seating below the
inner seal, and leave it there while the instrument is not in use.
40) Put any used chloroform and mobile phase into a labelled organic
waste container (Rm. 257-A safety cabinet).
60


NOTES:
1The following steps assume that the mercury lamp is already
installed in the detector, along with the 405 nm filter, that the column is
attached, the pumps are primed, the flow-cell is sufficiently clean that the
absorbance offset displayed on the Minicontroller after zeroing is less than
0.5, and that the system and column are equilibrated with the ethyl
acetate/n-heptane mobile phase. If any of these conditions are lacking, see
below, and/or in the Beckman and System Gold manuals.
For installing the mercury lamp and the 405 nm filter, follow the
instructions in the Beckman 160 Absorbance Detector manual on p. 2-5 in
Section 2.7, and on p. 5-3 in Section 5.3.
To prime the pumps, see the Beckman manual for the 110 B Solvent
Delivery Module, p. 6-3, section 6.22, "Priming the Pump". You'll need a
10 cc luer-lok syringe. We have not bothered with degassing our solvents;
this is apparently not so much of a problem with an organic mobile phase, as
it is with aqueous ones. After switching solvents there may be air bubbles in
the uptake lines, especially if the uptake filters are attached; the bubbles
should be pulled out with the syringe via the same priming port, to avoid
their causing problems in the column or flow cell.
If some other solvent (besides our ethyl acetate/n-heptane mobile
phase) has been used previously, or if it is uncertain, then the system should
be flushed thoroughly with isopropyl alcohol (HPLC-grade 2-propanol) first
(before attaching the column), and then equilibrated with the ethyl acetate/n-
heptane mobile phase. Then the flow cell should also probably be cleaned,
as a change in solvents (or use with certain substances) can cause a build-
up of film (or "gunk") in the flow cell, changing the absorbance readings.
To clean the flow cell, first unscrew the couplings on the inlet and
outlet tubings, leaving the coupling attached to its outlet tubing, and remove
the flow cell from the detector (see p. 5-1 in Section 5.1 of the Beckman 160
Absorbance Detector manual). It is a good idea to first hold the flow cell
61


windows up to a light to get a visual idea of how much cleaning is needed in
the sample cell. Then attach a plastic luer-lok adapter to the outlet coupling,
and pull 20% nitric acid through the flow cell using a 5 cc syringe with a luer
fitting while holding the inlet tubing in the acid solution. Let it sit for fifteen
minutes, then rinse it in a similar manner with d.i. water. If necessary, repeat
the nitric acid flushing again, followed by the rinsing with distilled water after
fifteen minutes. If further cleaning is needed, next repeat the procedure with
1 N. NaOH, followed by water rinsing; and finally repeat it once more with
methanol followed after fifteen minutes with the distilled water. Then
reattach the flow cell and fittings. After such a cleaning series the
absorbance offset on the Minicontroller display (after pressing the Auto Z
key) should be down around 0.163; anything less than 0.3 is apparently
acceptable.
The Alltech column we have has adjustable endcaps, which can be
tightened down (particularly the inlet cap) to eliminate any void that may
build up with use; simply hand-tighten before installing the column-if there
is resistance, there is no void. When attaching the column to the tubing,
slide the ferule up on the tubing, be certain the tubing end is seated all the
way into the endcap, and then tighten the nut down (and the ferule with it)
while holding the tubing in place. Before removing the column for storage, it
should first be equilibrated with isopropyl alcohol; then firmly screw the
plugs (in its box) into the endcaps. Running isopropyl alcohol through the
system will cause the pressure to go up considerably, due to its greater
viscosity, so don't be alarmed when that happens.
An eye should be kept on the pressure level, as indicated in the scale
on the pump (110 B delivery module) marked "PUMP LOAD". It should be
set so the high limit does not exceed 4; but if the pressure starts getting
much above 2 (when not using isopropyl alcohol), this is an indication that
the column end frits (filters) or the column itself needs cleaning. End frits
may be cleaned several times before being replaced by new ones, by
62


immersing them in a 6 N. nitric acid solution and sonicating 10-15 minutes.
Refer to the manuals for any problems, and/or contact Greg Baird, the
Beckman service rep. If any tubing or other components are replaced, be
sure to use the same diameter or size items-don't substitute. In the NEC
System Gold manual, the pertinent sections are 2.12 (on the 338 system, p.
2-122) and 3.8 (on the 406 module, p. 3-106).
63


4 RESULTS AND DISCUSSION
4.1 THE METHODS
In analyzing and comparing the data from over twenty-five
experiments, certain patterns are apparent; the results and these trends will
be presented and described below, following the general chronological
sequence of yeast porphyrin analyses. First to surface are the whole-cell
spectra; next come spectra of the extracted free-acid porphyrins; after the
subsequent methyl-esterification procedure, spectra of porphyrin methyl-
esters may be run, followed by thin-layer chromatography and/or high-
performance liquid chromatography.
4.1.1 WEIGHT DETERMINATION NOTES
In cases where cell yield from a culture is insufficient, it may be
necessary to omit dry-weight determinations to avoid the ensuing cell-loss.
In such cases, for purposes of estimation, the average ratio for porphyrin-
accumulating strains studied here is 4.6, in dividing wet weight by dry
weight. For the associated wild type or parent strains, the average ratio is
4.0, in dividing wet weight by dry.
4.1.2 WHOLE CELL SPECTRA
Based on Vojensky's (1981) findings and on results from these
studies, it is often possible to broadly categorize a strain's dominant
porphyrin make-up based on the regions of the whole-cell spectrum where
the porphyrin peaks occur. The method works fairly well if either
uroporphyrin or protoporphyrin dominate; with certain mixtures or with less
pronounced porphyrin accumulations it is inconclusive.
As colorless porphyrinogens accumulate in a cell they spontaneously
oxidize to the corresponding aromatic porphyrin pigment (fluorescing pink);
64


copro- and uro-porphyrin usually chelate any metal present (typically zinc)
on accumulation, whereas protoporphyrin may or (more often) may not-in
the latter case, remaining a "free" porphyrin. Whole-cell difference spectra of
metalloporphyrins generally show only porphyrin peaks II and III, whereas
free porphyrins may show peak I or both peaks I and IV, in addition to peaks
II and III (see Fig. 11) (Vojensky 1981). Vojensky presented the following
table of porphyrin peak wavelengths (nm):
Table 2: TYPICAL WHOLE-CELL DIFFERENCE-SPECTRA
Porahvrin: Pk. I Pk. II Pk. Ill Pk. IV
Free Proto 640-645 590 540 485-500
Zn Proto - 585-590 540
Zn Uro 575-580 539
Zn Copro - 575-580 539
Based on data from these studies, the table has been condensed
modified as follows:
Predominant PorDhvrin: Pk. I Pk. II Pk. Ill Pk. IV
Proto 635-645 585-590 540-545 (480-500)*
Uro/Copro (620-630)' 575-580 539-540 (500)*
May or may not be present as a peak or a swell in the curve.
In addition to porphyrin peaks, porphyric mutants often show altered
cytochrome levels in their whole-cell spectra, since porphyrins are
intermediates in heme biosynthesis, and hemes in turn are incorporated into
cytochromes, among other things. The following table shows the typical
cytochrome peaks, as well as the interspersed ranges of porphyrin maxima
65


Fig. 11. Whole-cell difference spectra of three porphyrin accumulating mutants
(minus their respective wild-types) are shown. A: Strain 242-8-5 (minus
JM25) shows pronounced peaks II & III in the common two-peak metalloporphyrin
spectrum (while peaks I & IV are still detectable); the strain contains mostly
uroporphyrin, with other decarboxylation products. B: Strain S288c (minus
D28) also shows the double-peak metalloporphyrin spectrum; in this case it is
zinc protoporphyrin. C: Strain D28/F8 (minus D28) shows the more common
free porphyrin four-peak spectrum for protoporphyrin.
66


in a yeast whole-cell spectrum (versus milk, or in a difference-spectrum):
Table 3: TYPICAL ABS. MAXIMA IN YEAST WHOLE-CELL SPECTRA
for porphyrin producing strains, 700-450nm
Cytochrome peaks (nm) Porphyrin peaks (nml
645-620 Pk. I
Cyto. a-as 603 595-575 Pk. II
Cyto. tfx 560
Cyto. c+ci 550 545-540 Pk. Ill
Cyto. £>P 535
Cyto. cP 520
505-480 Pk. IV
Cytochrome maxima shown are for typical wild-type control strains; depending on the
mutation, a porphyrin over-producer may be deficient in one or more cytochromes, as seen by the
absence of those peaks from the whole-cell spectrum; other porphyrin accumulators may have excess
cytochromes. Sometimes a porphyrin maximum may be so high that it masks adjacent cytochrome
peaks--or, vice-versa, a porphyrin maximum gets masked or may be apparent only in a higher minimum
between cytochrome peaks.
Exact location of porphyrin peaks will vary depending on the predominant porphynns
accumulating in that strain (see above).
For application of and details based on the above porphyrin and cytochrome
analyses for the various strains studied, see sections 4.2, 4.3, and 4.4 below.
4.1.3 FREE-ACID PORPHYRIN SPECTRA
The spectrum of a free-acid porphyrin extract (in ethyl acetate)
typically has five maxima: the above four peaks in the visible spectrum, and
the Soret peak found at the upper limit of the visible spectrum-just inside the
near-ultra violet. Whereas the Soret wavelength of a pure porphyrin can be
67


fairly diagnostic, when there is a mixture of accumulated porphyrins in an
extract (as there often is, since uro'gen decarboxylase works on four
sequential porphyrin substrates, and also because various mutations can be
"leaky") it is necessary to look at all five maxima~and even then the
diagnosis is not always clear. To further refine the whole-cell determination
of porphyrin content, the following table may be used for free-acid spectra; it
is based on a table from Vojensky combined with data from Pretlow and this
research (see Table 5):
Table 4: TYPICAL FREE-ACID PORPHYRIN ABS. MAXIMA
(350-650 nm) With Extinction Coefficients
porphyrin in ethvl acetate Soret X (nm) Pk. IV X (nm) Pk. Ill X (nm) Pk. II X (nm) Pk. I X (nm) EmM
Proto 404 500-03* 532-36* 575 630*-33 164
Uro 401 493 536 571-6 628 217
Copro 396-98 495-98* 527-28* 568 622*-26 ?
Any free porphyrin extract is likely to contain more than one porphyrin, depending on what
enzyme has been affected (and how much) by the mutation in that strain; usually the strains here are
predominantly proto- or else predominantly uro- with other decarboxylation porphyrins. Comparison of
an extract's five spectral peak wavelengths with these standards can assist in determining whether
that strain accumulates primarily proto- or primarily uro-; should it contain fair amounts of copro- or
other decarboxy products, differentiation becomes tougher. Soret wavelengths and extinction
coefficients (£mM) for proto- and uro- are from Pretlow (1965); wavelengths for peaks l-IV are from
Vojensky (1981), with asterisked values added from this research.
Data from this research indicate that uro-predominant strains tend to
have the Soret peak between 398-403 nm, with a lower value (398-400) for
those having substantial coproporphyrin. Proto-predominant strains have
the Soret peak between 400-404, with an average of 403 nm for strains with
76% or more protoporphyrin; those with copro and/or other porphyrins tend
to have a lower wavelength. Figure 12 is an overlay of scans of free-acid
68


Strain Soret nm Pk. IV nm Pk. Ill nm Pk. II nm Pk. I nm
D28/F8 403 502-4 536 574-80 630
AM3-4B/U3/P1 404 502-4 536 576 630
EG81-22b 403 502-4 532-7 570-80 631-2
112-699 401 502-5 535-6 572-4 631
112 402 499-505 533 568 630
S288c 402 504 536 574 631
R1a 402 502-4 535-6 573-6 630-1
R12b 402 504 535 569-70 631
SC1171 399 498-9 532 574 626
SC1183 400 498 532 575 625
Proto- 404 500-3 532-6 575 630-3
Uro- 401 493 536 571-6 628
Copro- 396-8 495-8 527-8 568 622-6
6288-5A/C1/P21 398-400 498-9 530-1 566-78 624-5
242-8-5 398 499 528-32 562-9 624-5
155-11-1 403 - 526 562 -
123-20-2 400 - - - -
Table 5. Absorbance maxima for free-acid porphyrin extracts (in ethyl acetate) from the strains in these
studies are shown. Pure porphyrin maxima are also shown (boldprint) for reference, from Table 4. Strains
grouped above the standards are more in line with the proto pattern (except the two SC strains, which are
ambiguous); those grouped below the standards fit more into the uro/copro pattern. Note the overlap
between proto and uro for peaks III and II; the presence of copra with either can make diagnosis difficult.
69


flbsorb ance(RU)
Fig. 12. Overlay spectra of free-acid porphyrin extracts from a proto-dominant strain (D28/F8,
dashed line) and a uro/7/6 strain (6288-5A/C1/P21, solid line), showing the shift to higher
wavelengths for the proto maxima. A: scanned from 350-650nm; B: scanned from 450-650nm,
and blown-up to show the visible wavelength peaks.
70


porphyrins from a proto-predominant strain (D28/F8) and a uro-/copro-
predominant strain (6288-5A/C1/P21), and clearly shows the shift to higher
wavelengths due to the protoporphyrin content of D28/F8. For application of
and details based on the above free-acid porphyrin analyses for the various
strains studied, see sections 4.2, 4.3, and 4.4 below.
4.1.4 PORPHYRIN METHYL-ESTER SPECTRA
Spectra of methyl-esterified extracts (in chloroform) may add further
corroboration to an extract's suspected porphyrin make-up; the following
table gives literature Soret-peak values for pure porphyrins, and experi-
mental values from this study for extracts dominant in proto- or uroporphyrin
(see Table 8):
Table 6: ABS. MAXIMA FOR PORPHYRIN METHYL-ESTER
with Extinction Coefficients (in chloroform)
Dominant porphyrin methvl-ester Soret X (nm) (pure) Experimental extract Soret k £mM
Proto 407-407.5 405-408 171
Uro 405-406 400-404.5 215
Copro 399.5-400 180
Values for pure porphyrins come from Pretlow (1965) and Smith (1975); extinction coefficients
(SmM) are from Smith (1975).
Figure 13 (an overlay of methyl-ester scans of D28/F8 and "P21") clearly
shows the shift to higher wavelengths of the proto-predominant D28/F8,
compared to the uro-/copro-predominant P21-except for the Soret peak in
this case. For application of and details based on the above methyl-ester
71


Rbsorbanca ( RU ) Rbsorbanco CRl>
Fig. 13. Overlay spectra of porphyrin methyl-esters from a proto-dominant strain (D28/F8, solid
line) and a uro/7/6 strain (6288-5A/C1/P21, dashed line), showing the shift to higher
wavelengths for the proto maxima (except for the Soret peak of D28/F8). A: scanned from
350-650nm; B: scanned from 450-650nm, and blown-up to show the visible wavelength peaks.
72


porphyrin analyses for the various strains studied, see sections 4.2, 4.3, and
4.4 below.
4.1.5 THIN-LAYER and HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY
While results from TLC in most cases were basically similar
qualitatively to those from HPLC, if the HPLC is set up for the appropriate
lamp, wavelength filter, column and solvent, it is a far better option both
timewise and for its quantitative and more reliable (& decipherable)
qualitative analyses.
TLC results were generally somewhat problematic, between a
frequent "smile" effect and streaking of spots; TLC does generally permit a
rough qualitative analysis, but even a relative quantitation of the different
porphyrin components in a sample (based on intensity of fluorescence and
sizes of spots) can be unreliable using strictly TLC plates (see Fig. 14).
For application of and details based on porphyrin chromatography
analyses for the various strains studied, see sections 4.2, 4.3, and 4.4 below.
73


A
"eCK solvr
f-
- 'O
o
Hr ~ O
++ o

-WH-t
O-----mJo :
DOSS solv.
4
i ^ o
1 # D
f O
~^r ($)-
mkr
P21
Pit
O-
o-
Fig. 14. Thin-layer chromatography results. A: Both streaking and a "smile" effect can be seen in
these runs; B; The sample shows only slight streaking, and the "smile" effect is minimal; C: a
comparison run of two developing solvents shows fairly good results on the left, with eCK. Plates in A
and B were photographed on a UV transmitter; those in C are photocopies (spots had been circled in
pencil under UV light). Abbreviations: mkr, marker kit standards; eCK, ethanolic chloroform &
kerosene; f, faint; ff, very faint; + signs rate the intensity of bright spots; DOSS solvent contains
benzene, ethyl acetate & methanol.
74


4.2 THE STRUCTURAL GENE MUTANTS
The strains investigated in this category include a pop3 {hem6 or
hem12) mutant, a pop2(hem5 or hem15) mutant, and another hem5
(hem15) mutant which is permeable to cAMP. Table 7, below, lists the yeast
strains used in these studies, along with their genotypes and source. Table
8 summarizes the data of all analyses for all strains studied.
4.2.1 Uroporphyrinogen Decarboxylase (HEM 12) Mutant "P21n
As seen in Fig.'s 15 and 16, showing whole-cell spectra of the wild-
type strain 6288-5A/C1 and the associated mutant 6288-5A/C1/P21, there
are pronounced maxima at about 578 and 541 nm (porphyrin peaks II and
III) in the mutant, with peaks I and IV much less noticeable around 618 and
502 nm-corresponding to the uro-/coproporphyrin predominance pattern
(see Table 2). While some cytochrome peaks are masked in the mutant, it
appears to be deficient in all cytochromes (see Table 3), as seen especially
in the difference-spectrum. Note the maximum located around 465 nm,
which we have not identified, but which often pops up on difference-spectra
for porphyrin accumulators.
Spectra of the free-acid and the methyl-ester samples for P21 yielded
Soret peak values at 389-99 nm and 400 nm, respectively, also correspond-
ing to the uro-/coproporphyrin predominance pattern (see Tables 4 and 6);
free-acid visible peaks l-IV also fall in a pattern indicating a uro-/copro-
mixture (see Table 5).
TLC analysis indicated a greatest percentage of uroporphyrin, with
decreasing amounts of hepta- and hexacarboxylic porphyrins, followed by
much lesser amounts of pentacarboxylic and coproprophyrins. This analysis
was borne out by HPLC results, showing the total porphyrin extract to be
75


Strain Genotype Source
6288-5A/C1 a leu2-3 Ieu2-112 canR
6288-5A/C1/P21 a leu2-3 Ieu2-112 canRhem6-4
D28 a Iys2 gal
D28/F8 a Iys2 hem5-2 this lab
AM3-4B/U3 a ade6 ade10 cam1 cam2 cam3 amp1 ura3 this lab
AM3-4B/U3/P1 a ade6 ade10 cam1 cam2 cam3 amp1 ura3 hem5 this lab
112 a leu2-3-112 ura3-1 can1-100 ade2-1 his3 ICTatchell
112-699 a leu2-3-112 ura3-1 can1-100 ade2-1 his3 ras2-699 (His+) K. Tatchell
S288c a mal(gal2) cup1r M. Carlson
EG81-22b a ras2-530 Ieu2 his4-539 Iys2-801 ura3 K. Tatchell
R12b a ras2-530 sra1-1 K. Tatchell
R1a a ras2-530 sra1-2 ICTatchell
308 a Ieu2 ura3 trp1 his3 lys-RAS2 T. Kataoka
301 a Ieu2 ura3 trp1 his3 lys-RAS2ala18 val19 T. Kataoka
SC 167
SC1171 ras265-2
SC1183 ras265-2
JM25 a ade1 CU Boulder
242-8-5 a canR CU Boulder
155-11-1 a ade1 canR CU Boulder
123-20-2 a ade1 canR CU Boulder
Table 7. Yeast strains involved in these studies.
76


Table 8. Summary of data from all strains studied.
Quantitative data not useful, as methylation occurred prior to determining optimal method of methylation.
Results questionable, considering HPLC and 242-8-5 data.
77


700nm 620 580 540 500 460nm
Fig. 15. Overlaid whole-cell spectra of porphyrin accumulator "P21," its
parent, and wild-type D28.
Fig. 16. Whole-cell difference spectrum of strain "P21" minus its
parent, 6288-5A/C1.
78


40% uro-, 30% hepta-, 15% hexa-, and 5% each of penta- and copro-; total
porphyrin was estimated at 10.0 pmoles per mg dry weight via HPLC, with a
range up to 19.8 pmoles/mg by other methods. Comparisons of free-acid
extracts of P21 and the parental strain averaged 66 times more total
porphyrin in the mutant than in the control.
4.2.2 Ferrochelatase (HEM 15) Mutant D28/F8
As seen in whole-cell spectra (Fig.'s 17 and 18; compare these and
values in Table 8 with Tables 2 and 3) of the mutant D28/F8 and the
corresponding wild-type D28, a typical three- to four-peak pattern indicating
protoporphyrin predominance occurs in the mutant, with peak locations
matching those for protoporphyrin. While cytochromes are fairly masked, the
mutant appears low in a-as and cP. Note in the difference-spectrum that
peak IV at 485 nm has a subsidiary hump as it descends at around 460 nm.
In the free-acid porphyrin spectrum of D28/F8, the Soret peak is at
about 403 nm, also suggesting protoporphyrin, as do the visible maxima
(see Tables 4 and 5). For unknown reasons the methyl-ester spectrum gave
a Soret peak at 396 nm, not fitting either pattern.
The TLC results indicated a predominantly protoporphyrin extract,
with a far lesser amount of coproporphyrin, and still lesser amounts of others
(hexa- and hepta-carboxylic, and uroporphyrin). HPLC anaysis corrobor-
ated the TLC, indicating that 98% of total porphyrin in the mutant was proto-
porphyrin. Calculations from HPLC results indicated a total porphyrin
accumulation of 13.3 pmoles per mg dry weight, with a range from 10.6 to
13.3 pmoles/mg by various methods-approximately 15 times more
porphyrin than in the wild-type D28, based on the free-acid data.
79


700nm 640 610 580 550 520 500 460nm
Fig. 17. Whole-cell spectra of strain D28/F8 and its parent, D28.
700nm 670 640
610 580 550 520
I I
490 460nm
Fig. 18. Whole-cell difference spectrum of strain D28/F8 minus its parent, D28.
80


4.2.3 Ferrochelatase (HEM 15) Mutant "P1"
The cAMP-permeable strains AM3-4B/U3 and associated mutant
AM3-4B/U3/P1 were grown on glycerol and analyzed; whole-cell spectra
(Fig.'s 19 and 20) show the typical three- to four-peak porphyrin pattern of a
protoporphyrin accumulator (see Tables 2 and 3) in the mutant's spectrum,
with peak locations corresponding to protoporphyrin predominance also.
The mutant appears to be low in cytochromes b-, c+ci, and cP. Note in the
difference-spectrum the very broad peak IV, composed of two subsidiary
humps seen in the P1 spectrum at about 500 and 475 nm.
Both free-acid and methyl-ester spectra of P1 had Soret peaks
corresponding to protoporphyrin (404 nm and 408 nm, respectively), as did
the four visible peaks in the free-acid spectrum (see Tables 4-6).
TLC results indicated mainly protoporphyrin in P1, with much smaller
amounts of copro-, pentacarboxylic, and uro- porphyrins. Calculations on
HPLC data indicated nearly 95% protoporphyrin, and 5% uroporphyrin, with
slight traces of others. Total porphyrin via HPLC was 31.0 pmoles/mg dry
weight, ranging from 17.9-46.0 pmoles/mg by other methods; free-acid calc-
ulations yielded 42 times more total porphyrin in P1 than in the wild-type.
With structural gene mutants, the particular type(s) of porphyrins that
accumulate enable a quick determination (or confirmation) of where the
mutation, and hence enzyme block, has occurred. A predominance of
protoporphyrin locates the block at HEM151or ferrochelatase (such as in
strains D28/F8 and AM3-4B/U3/P1), whereas a porphyrin content of mostly
uroporphyrin locates the block at HEM12 for uro'gen decarboxylase; a
mixture of uro with decreasing amounts of porphyrin intermediates (such as
in strain 6299-5A/C1/P21) suggests an incomplete block at HEM 12, in which
81


700nm 670
640
610
580
460nm
550 520 490
Fig. 19. Whole-cell spectra of strain "PI" and its parent, AM3-4B/U3.
Neither strain's medium had added cAMP in this experiment.
700nm 670 640 610 580 550 520 490 460nm
Fig. 20. Whole-cell difference spectrum of strain "PI" minus its parent, AM3-4B/U3.
Neither strain's medium had added cAMP in this esperiment.


the gene mutation has only partially incapacitated the enzyme product. In
each case with the above strains, data from these studies provided confirma-
tion to hunches and/or other results suggesting which gene in heme
biosynthesis was affected. Further information on the common 460 nm
absorbance in whole-cell difference-spectra is not available, except that it
does not show up in porphyrin extracts.
4.3 THE RAS/cAMP-RELATED REGULATORY MUTANTS
These strains have been separated for analysis here into five groups:
the group of ras2 mutants; the group of ras2 mutants having the ras2-
suppressor mutation sra1\ the mis-sense RAS2val19 mutant, having an
amino acid substitution in its RAS2 gene product, and having increased
levels of cAMP; a pair of ras265 mutants having elevated levels of cAMP;
and a cAMP-permeable hem15 (ferrochelatase) mutant (reported on above
in section 4.2.3, with the structural gene mutants).
4.3.1 The ras2 Mutant Group
Strain EG81-22b, a ras2-530 mutant with lower levels of cAMP and
protein kinase, has elevated porphyrin content compared with wild-type
D28, as is apparent in whole-cell spectra (Fig.'s 21 and 22; compare these
and values in Table 8 with Tables 2 and 3). This mutant illustrates the
difficulty that can occur sometimes in trying to determine porphyrin content
from whole-cell, free-acid, and/or methyl-ester spectra, prior to running
chromatographic separations. In Figure 22a this strain shows basically a
two-peak metalloporphyrin spectrum (ignoring the slight bump of peak III
around 500 nm, and the more significant peak just below 460 nm)--suggest-
ing a uro-/copro- content; on the other hand, a different batch of cells (Fig.
22b) shows more pronounced peaks I and IV, in addition to the others-more
in line with the proto- pattern. The wavelengths of the peaks are not any
83


700nm 660 620 580 540 500 460nm
Fig. 21. Overlaid whole-cell spectra of EG81-22b, Rla, R12b and
the wild-type D28. A and B represent two different cell batches.
84


700nm 660 620 580 540 500 460nm
Fig. 22. Whole-cell difference spectra showing D28 sub-
tracted from each of EG81-22b, Rla, and R1 2b. A and B
represent different cell batches, matching A and B, respec-
tively, in Fig. 21.
85


help in the first run (Fig.'s 21a and especially 22a), at 580 and 540 nm~
which could fit either pattern, although they are more suggestive of the uro-
/copro- pattern. In the second run (Fig.'s 21b and especially 22b) the four
peaks are all closer to the proto- pattern. This strain shows decreased levels
of cytochromes c+ct and cP at 550 and 520 nm.
Spectra of its free-acid porphyrins resulted in a Soret average of
402.6 nm; visible peaks I and IV align more with the proto- pattern, while
peaks II and III are inconclusive (see Tables 5 and 8). The methyl-ester
spectra gave a Soret wavelength of 406 nm, which suggests a protoporphy-
rin predominance.
TLC resolved this uncertainty, indicating primarily protoporphyrin, with
varying amounts of coproporphyrin which were altering the wavelengths of
the maxima and the number of peaks in different spectra. HPLC
corroborated the TLC, indicating 76% protoporphyrin, 16% copro-, 5%
hexacarboxylic porphyrin, and insignificant amounts of others. Total
porphyrin content was calculated at an average of 12.9 pmoles per mg dry
weight--18 times that of the wild-type D28, and four times that of the other
control, S288c (see Table 8).
Another mutant, 112-699, has a histidine insertion in its RAS2 protein;
it is a ras2-699 mutant. As seen it the whole-cell spectra (Fig. 23, and Tables
2 and 3), not only did this mutation not increase the porphyrin accumulation
over the control strain 112 (a putative pop mutant, which itself accumulates
porphyrin); the RAS mutant actually has less porphyrin than 112. The
locations of peaks II and III (with a filling-in of the valley at IV, around 490)
don't clearly implicate either proto or uroporphyrins. Note the 460 nm
pigment again, in the difference-spectrum. Both strains are deficient in the c
cytochromes.
The free-acid spectra show the Soret peaks at 401 and 402 nm for
86


700nm 660 620 580 540 500 460nm
Fig. 23. Whole cell spectra of mutants 112 and 11 2-699. A: Both are
overlaid for comparison, with D28. B: Difference spectrum, showing
11 2 minus 11 2-699.
87


112 and the "parent," respectively, which is borderline between uro and
proto patterns; visible peak wavelengths for peaks I and IV point more
toward proto, while peaks II and III are inconclusive; 112's data lean more
toward uro or at least a higher copro component than the RAS mutant (see
Table 5). Quantitations on the data indicated a total porphyrin content of 4.0
pmoles/mg dry weight for 112-699, and 7.4 for 112.
TLC and HPLC separations were not performed on these strains.
The strain S288c was also used as a control for the ras and ras-sra
strains; compared with the control D28 (Fig. 24) it has higher levels of
porphyrin (note the filled-in minima around 580 and 540 nm), with a
relatively lower level of the c cytochromes (550 and 520 nm).
In its free-acid spectrum, S288c has Soret and visible peaks that fit
the proto-predominance pattern, for the most part (see Tables 5 and 8),
whereas its methyl-ester Soret peak is borderline between the two patterns.
TLC and HPLC results showed mainly protoporphyrin (82%, via
HPLC), with a small amount of coproporphyrin (6% via HPLC). Total
porphyrin content was determined to be 3.0 pmoles/mg dry weight-about
four times that of the wild type D28.
4.3.2 The ras2 sra1 Suppressor Mutants
The two ras2 sra1 strains R1a and R12b, with active protein kinase
despite (independent of) low levels of cAMP-due to the suppressor effect of
the sra1 mutation-have very high levels of porphyrins as shown by whole-
cell spectra (Fig.'s 21, 22, 25, 26; compare with Tables 2 and 3). Peaks II
and III are dominant, with a lesser peak I, (see difference spectra, Fig.'s 22,
25), although when subtracting the control S288c (Fig. 26) we see a more
prominent peak I; peak IV is fairly masked by the large 460 nm absorbance
in most spectra. Peaks fit the protoporphyrin wavelength pattern for the most
88


Fig. 24. Whole-cell spectra of control strain S288c. A: Compared in two batches with
wild-type D28. B: Difference spectra from same two batches, of S288c minus D28.
89


I I * I t
TOOnm 660 620 500 540 500 460nm
700nm 660 620 580 540 500 460nm
Fig. 25. Whole-cell difference spectra of strains R1a and R12b minus
EG81-22b. A and B represent two different batches, corresponding to
A and B in Fig.'s 21 and 22.
90


700nm 670 640 610 580 550 520 490 460nm
Fig. 26. Whole-cell difference spectra of strains Rla and R1 2b minus the control S288c.
91