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Hydrolysis glutathione esters

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Title:
Hydrolysis glutathione esters
Creator:
Hogan, Mindi Elisabeth
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Language:
English
Physical Description:
75 leaves : ; 28 cm

Thesis/Dissertation Information

Degree:
Master's ( Master of Science)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Chemistry, CU Denver
Degree Disciplines:
Chemistry

Subjects

Subjects / Keywords:
Glutathione ( lcsh )
Hydrolysis ( lcsh )
Esters ( lcsh )
Esters ( fast )
Glutathione ( fast )
Hydrolysis ( fast )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 71-75).
Thesis:
Department of Chemistry
Statement of Responsibility:
by Mindi Elisabeth Hogan.

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|University of Colorado Denver
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|Auraria Library
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
48713335 ( OCLC )
ocm48713335
Classification:
LD1190.L46 2001m .H63 ( lcc )

Full Text
HYDROLYSIS OF GLUTATHIONE ESTERS
by
Mindi Elisabeth Hogan
B.S., University of Wyoming, 1994
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry
2001


This thesis for the Master of Science
degree by
Mindi Elisabeth Hogan
has been approved
by
Date


Hogan, Mindi Elisabeth (M.S., Chemistiy)
Hydrolysis of Gluathione Esters
Thesis directed by Assistant Professor Ellen J. Levy
ABSTRACT
Glutathione is the most abundant cellular nonprotein thiol, with concentrations
ranging from 0.5 mM to 10 mM. It serves as a transport unit for amino acids, sulfur
and metals; as a reducing agent for proteins, ribonucleotides, and antioxidants; and
possibly as a neurotransmitter. Glutathione also protects cells against damage from
free radicals and xenobiotics. Thus, supplemental glutathione could be used to aid
cellular defenses. While glutathione itself cannot be administered directly, due to
its poor transportation into the cell, glutathione esters can be, as they are more
readily transported across the cellular membrane. In this study, the hydrolysis of
propyl, ethyl, benzyl, isobutyl and n-butyl glutathione monoesters by esterases in
microsomal and cytosolic fractions of rat liver were examined. The esters all showed
some degree of hydrolyzation by the esterases in the microsomal or cytosol
fractions, although the benzyl ester displayed the greatest degree of hydrolyzation.
The enzyme or enzymes responsible for the hydrolysis of the benzyl ester appear to
follow Michaelis-Menten kinetics, having a Km value of 52 mM.
This abstract accurately represents the content of the candidates thesis
recommend its publication.
Signed
hi


DEDICATION
I wish to dedicate this thesis to my husband for his unflagging patience, understanding,
and support throughout this project.


ACKNOWLEDGEMENT
I wish to thank my thesis advisor, Dr. Ellen Levy, and my fellow researchers David
Mills and Doug Tucker, without whom this thesis project would not have been possible.


CONTENTS
Figures.......................................................................viii
Tables...........................................................................x
Chapter
1. Introduction.................................................................1
1.1 Glutathione: Background and Function.........................................2
1.1.1 Free Radicals and Reactive Oxygen Species..................................5
1.1.2 Heavy Metals and Other Xenobiotics.........................................9
1.2 Problems Associated with Glutathione......................................11
1.2.1 Resistance................................................................12
1.3 Supplemental Glutathione....................................................15
1.3.1 Enzyme or Amino Acid Addition.............................................16
1.3.2 Glutathione Addition......................................................18
1.3.3 Esters of Glutathione.....................................................20
1.4 Hydrolysis of Glutathione Esters............................................23
1.5 Purpose.....................................................................25
2. Procedure...................................................................27
2.1 Reagents/Equipment.........................................................27
2.2 Buffer Solutions...........................................................28
2.3 Preparation of Microsomes and Cytosolic Fraction...........................28
2.3.1 Determination of the Protein Content......................................29
2.3.2 Determination of Esterase Activity...................................... 29
vi


2.4 Preparation of the Esters...................................................29
2.5 Preparation of Glutathione Ester Substrate Samples..........................30
2.6 Preparation of the Hydrolysis Samples.......................................30
2.7 Preparation of the Cytosolic Concentration Studies..........................31
2.8 Tietze Assay................................................................31
3. Results......................................................................32
3.1 Tietze Assay Theory.........................................................32
3.2 Glutathione Ester Substrate Studies.........................................33
3.3 Hydrolysis of Glutathione Esters............................................40
3.4 Concentration Studies for Glutathione Benzyl Ester in the Presence of Cytosol.60
3.5 Conclusion..................................................................69
References.......................................................................71
vii


FIGURES
Figure
1.1 Glutathione.................................................................2
1.2 y-glutamylcysteine..........................................................3
1.3 Glutathione Disulfide.......................................................4
1.4 l-methyl-4-phenylpyridinium.................................................7
1.5 Buthionine Sulfoximine.....................................................10
1.6 Thiazolidine...............................................................16
1.7 Glutathione Monoester......................................................22
1.8 Glutathione Diester........................................................22
1.9 Glutathione Monopropyl Ester...............................................25
1.10 Glutathione Monoethyl Ester................................................25
1.11 Glutathione Monobenzyl Ester...............................................25
1.12 Glutathione Monoisobutyl Ester.............................................26
1.13 Glutathione Monobutyl Ester................................................26
3.1 5,5 dithiobis (2-nitrobenzoic acid).......................................32
3.2 Ethyl Ester Concentration Study............................................35
3.3 n-Propyl Ester Concentration Study.........................................36
3.4 n-Butyl Ester Concentration Study..........................................37
3.5 Isobutyl Ester Concentration Study.........................................38
3.6 Benzyl Ester Concentration Study...........................................39
viii


3.7 Hydrolysis of Glutathione Isobutyl Ester...................................43
3.8 Hydrolysis of Glutathione Benzyl Ester.....................................43
3.9 Hydrolysis of Glutathione Benzyl Ester with Microsomes.....................50
3.10 Hydrolysis of Glutathione n-Butyl Ester with Microsomes....................50
3.11 Hydrolysis of Glutathione Isobutyl Ester with Microsomes...................51
3.12 Hydrolysis of Glutathione Ethyl Ester with Microsomes......................51
3.13 Hydrolysis of Glutathione Propyl Ester with Microsomes.....................52
3.14 Hydrolysis of Glutathione Benzyl Ester with Cytosol........................56
3.15 Hydrolysis of Glutathione n-Butyl Ester with Cytosol.......................56
3.16 Hydrolysis of Glutathione Ethyl Ester with Cytosol.........................57
3.17 Hydrolysis of Glutathione Propyl Ester with Cytosol........................57
3.18 Hydrolysis of Glutathione Benzyl Ester Concentration Studies...............65
3.19 Change in Glutathione Benzyl Ester Concentration over Time.................66
3.20 1/V vs 1/[S]...............................................................68
IX


TABLES
Table
3.1 Individual Ethyl Ester Hydrolysis Results..................................35
3.2 Individual n-Propyl Ester Hydrolysis Results...............................36
3.3 Individual n-Butyl Ester Hydrolysis Results................................37
3.4 Individual Isobutyl Ester Hydrolysis Results...............................38
3.5 Individual Benzyl Ester Hydrolysis Results.................................39
3.6 Concentration of Glutathione in Isobutyl Ester Samples.....................41
3.7 Hydrolysis of Glutathione Isobutyl Ester...................................42
3.8 Concentration of Glutathione in Benzyl Ester Samples.......................42
3.9 Hydrolysis of Glutathione Benzyl Ester.....................................42
3.10 Concentration of Glutathione in Ester Samples with Microsomes..............45
3.11 Hydrolysis of Glutathione Esters with Microsomes...........................48
3.12 Concentration of Glutathione in Ester Samples with Cytosol.................53
3.13 Hydrolysis of Glutathione Esters with Cytosol..............................55
3.14 Concentration of Benzyl Ester Concentration Study Samples..................62
3.15 The Hydrolysis of Benzyl Ester Concentration Study Samples.................64
3.16 Change in Concentration of the Esters Over Time............................66
3.17 Velocity for the Benzyl Ester Concentration Study..........................67
3.18 1/[S] and 1/V data.........................................................67
x


1. Introduction
Why do some people seem to stay young and free of disease while others grow old
before their time (Carper, 1995)?
The above question was posed in a 1995 Denver Post article on health. According to
epidemiologist Mara Julius, one of the reasons could be linked to the biologically
produced molecule, glutathione. A study, performed by Julius at the University of
Michigan on senior citizens, found that individuals with lower blood plasma levels of
glutathione were more likely to be suffering from some type of serious ailment than
individuals with higher levels of glutathione (Carper, 1995).
In a similar study, performed by the University of Birmingham in Great Britain,
plasma glutathione levels were examined in 220 people. The subjects were broken into
four distinct groups young, healthy adults; healthy elderly adults; chronically ill,
elderly adults; and elderly, hospitalized adults (Nuttall et al., 1998). The mean
glutathione levels for each group were 0.54 pmol/L, 0.29 pmol/L, 0.24 pmol/L, and 0.17
pmol/L respectively (Nuttall et al., 1998). A discrepancy in the levels of glutathione
between the younger and elderly adults was readily observed. However, a difference
between healthy and ill individuals was also apparent. Lower glutathione levels have
been reported in separate studies on patients suffering from HIV, hepatitis C, and
colitis (Kalebic et al., 1991; Anderson, 1997).
Glutathione does appear to play a role in the health of organisms; but, does it play
an active role or is it merely an indicator of well being? Will glutathione supplements be
1


the answer to aging? These questions can, perhaps, be best answered by taking a closer
look at glutathione itself.
1.1 Glutathione: Background and Function
Glutathione is a ubiquitous tripeptide present in both prokaryotic and eukaryotic
cells (Arrick and Nathan, 1984). In humans, it is found in varying cellular
concentrations from 0.5 mM to 10 mM, depending upon the specific cell type, which
makes it the most abundant cellular nonprotein thiol (Dusre et al., 1989; Yao et al.,
1995). It is also found in plasma, though in significantly lower concentrations only 10-
30 pM (Tew, 1994). Plasma relies on cellular exports, mainly from the fiver, for its
source of glutathione (Meister, 1991).
Glutathione was discovered in 1888 when J. de Rey-Pailhade observed a substance
present within cells that was capable of producing hydrogen sulfide when mixed with
sulfur (Meister, 1989). He called this substance philothion to reflect this particular
reaction. Some 30 years later, the 1929 co-winner of the Nobel Prize for medicine or
physiology F. G. Hopkins (who is, perhaps, better known for his work on Vitamins A and
D) proved that philothion contained the amino acids glutamate and cysteine.
Subsequently, he changed the name of philothion to glutathione to reflect its structure
(Meister, 1989). Refer to Figure 1.1 for the structure of glutathione.
Figure 1.1: Glutathione
H
o
H O
O
+
H3N-CCHCl%C-NH-CC-NHCH^CO
COO
I
CH,
SH
2


However, additional experiments performed by Hopkins indicated that the
compound was, in fact, a tripeptide consisting of L-glutamate, L-cysteine, and glycine
(Meister, 1989). Hence, to elucidate the actual structure, glutathione is sometimes
referred to by its more formal name of y-L-glutamyl-L-cysteinylglycine (Shaw, 1998).
The synthesis of glutathione is a two-step process, involving two different enzymes.
In the initial step, L-cysteine reacts with L-glutamate to form y-glutamylcysteine. Refer
to Figure 1.2 for the structure of y-glutamylcysteine. This reaction is catalyzed by the
aptly named enzyme, y-glutamylcysteine synthetase.
Figure 1.2: y-glutamylcysteine
COOH O H
I II I
H-CCH^CT%C-NH-CCOOH
ch,
SH
This is, incidentally, the feedback inhibition step to regulate the cellular concentration
of glutathione (Tew, 1994). y-glutamylcysteine, in turn, reacts with L-glycine to form
glutathione, facilitated by the enzyme, glutathione synthetase (Shaw, 1998). The
glutathione is either used by the cell or is exported intact to the outside of the cell, thus
providing glutathione for the plasma (Meister, 1983, Science).
The rather simple structure of glutathione enables it to function in many different
biological capacities. Due to the attached thiol group, glutathione acts as a cellular
reducing agent for proteins, antioxidants, and ribonucleotides through the donation of a
hydrogen atom an important function in the maintenance of the redox state within the
cell (Anderson et al., 1985; Meister, 1991). When the redox balance is shifted, due to
3


such problems as various vitamin deficiencies or diabetes, the body becomes more
vulnerable to disease (Maret, 1998). In order to prevent this, the cells ensure the
oxidized form of glutathione, glutathione disulfide, remains at a minimum. The cellular
ratio is about 98% glutathione versus 2% glutathione disulfide. Refer to Figure 1.3 for
the structure of glutathione disulfide.
Figure 1.3: Glutathione Disulfide
COOH
COOH
However, in the endoplasmic reticulum, the amount of glutathione compared to
glutathione disulfide is only about a 75% to 25%, or a 50% to 50% ratio, in order to shift
the redox balance in a direction more conducive to protein folding (Maret, 1998). The
redox maintenance ability of glutathione is important to the erythrocytes, for it
maintains hemoglobin in the Fe+2 state (Lehninger, 1993).
Glutathione is also used as a transport for amino acids, sulfur and metals while also
serving as a storage unit for cysteine (Meister, 1983, Science; Shaw, 1998). Glutathione
4


functions as a cofactor in various intracellular syntheses, such as prostaglandin or
protein synthesis, and in the generation of deoxyribonucleic acids (Chen et al., 1998;
Anderson, 1997; Meister, 1983, Science). There is even speculation that glutathione
may act as a neurotransmitter in a manner similar to glutamic acid (Curry, 1998).
However, glutathione is, perhaps, most notable for its ability to protect the cell against
free radicals, heavy metals, and various xenobiotics (Anderson, 1985).
1.1.1 Free Radicals and Reactive Oxygen Species
Free radicals are quite simply atoms or molecules which possess unpaired electrons.
Naturally, in this state, they are highly reactive. Radical reactions give rise to chain
propagation reactions in which an additional radical is formed during each reaction.
Hence, a single radical can cause multiple damaging reactions.
Free radicals are caused by natural cell electron transfer reactions, such as the
operation of heme proteins, cytochrome P-450, in the endoplasmic reticulum; or by
external sources, such as exposure to radiation (Shaw, 1998; Cooper, 1998). Cytochrome
P-450 is an important catalyst to many hydroxylation reactions, such as the formation of
steroid hormones. It is in this capacity that the protein is able to break down xenobiotic
agents; however, in doing so, it can produce even more biologically reactive species.
One common external source of free radicals is exposure to radiation. Radicals can
also be generated by other xenobiotic agents. One suspected agent is the chemotherapy
agent as-diaminedichloroplatinum (cisplatin). Lipid radicals have been observed after
the addition of cisplatin to liver microsomes (Wang et al., 1996). It has been labeled as
suspected because other experiments involving cisplatin have not produced free
radicals, so the matter is still under investigation.
5


This type of reactivity is especially damaging to a cell, specifically to DNA or lipids.
Attacks on DNA can result in strand breakage and mutation, while attacks on
polyunsaturated lipids break their double bonds. Returning to the glutathione level
study performed by at the University of Birmingham, the same blood samples were also
analyzed for lipid hydroperoxide, to monitor the level of free radicals (Nuttall et al.,
1998). The level of lipid hydroperoxide was lowest in the group of young adults, twice as
high as the result for the young adults in the group of seniors, and four times higher in
the group of ill seniors (Nuttall et al., 1998). Based upon these results, there appears to
be a link between the build-up of free radicals and the corresponding reduction in the
level of glutathione.
Free radicals have also been associated with Parkinsons disease. Terminal
Parkinsons disease patients typically have lower concentration of glutathione and
higher levels of basal malondialdehyde, a chemical which is present as part of lipid
peroxidation. In fact, this molecule was up to 35% higher in the substantia nigra
portion of the brain, the part of the midbrain associated with the control of movement
(Dexter, 1989). Damage to this part of the brain by radicals explains the classic
symptoms of Parkinsons disease poor coordination and uncontrolled tremors.
Parkinsons disease has even been artificially generated through the use of the radical
l-methyl-4-phenylpyridinium (Cooper, 1998). Refer to Figure 1.4 for the structure of 1-
methyl-4-phenylpyridinium.
6


Figure 1.4: l-methyl-4-phenylpyridinium
/f~\ +
wNCH2
Glutathione, together with other antioxidants such as ascorbic acid, uric acid, or 13-
carotene, reacts with various free radicals in a scavenging effort. In fact, glutathione
even reacts with dehydroascorbate to give ascorbic acid and reduced glutathione (Shaw,
1998). When ascorbic acid or glutathione is missing, the other is shortly depleted.
Depletion of Vitamin C is characterized by scurvy, a disease which causes tender joints
and loosening of teeth. Ascorbic acid plays an important role in the reaction to form
connective tissues; hence, the problem with the joints. However, in a study performed
by Martensson and Meister, scurvy in guinea pigs could be prevented by providing
supplemental glutathione (Anderson, 1997).
Glutathione is capable of reacting with many different radicals, including carbon
centered radicals, oxygen-centered radicals or the hydroxyl radicals; the reaction of the
latter with glutathione is much faster than with the previous two types of radicals
(Wardman, 1988). Glutathione reacts more slowly than a radical; however, glutathione
typically has a much greater cellular concentration than the radicals, which ensures
sufficient response. In the effort to remove radicals, glutathione donates a hydrogen
atom and becomes a radical; however, in this state, it demonstrates little reactivity
except towards thiol groups specifically towards a second glutathione radical molecule
(Arrick and Nathan, 1984; Curry, 1998).
7


Hydrogen peroxide and organic hydroperoxides are potentially dangerous sources of
cellular reactive oxygen species (Zhao et al., 1999). Hydrogen peroxide is generated
biologically in liver perisomes, or in mitochondria (Oshino & Chance, 1977; Jain, et al.,
1991). Mitochondria generate adenosine triphosphate (ATP) and in the process convert
oxygen to water; however, occasionally hydrogen peroxide is formed instead. Hydrogen
peroxide, in turn, reacts with iron to produce a hydroxyl radical. Thus, a systemic build-
up of hydrogen peroxide can prove fatal. Because of this, mitochondria contain between
10-20% of the cellular glutathione (Cooper, 1998).
Mitochondria are incapable of producing their own glutathione and must import it
from the cytoplasm; hence, they are particularly sensitive to any reduction in the supply
of glutathione (Cooper, 1998). Glutathione is more rapidly consumed in the
mitochondria of younger animals with a turnaround time approximately one third the
length of an adult organism (Jain et al., 1991). For this reason, glutathione deprivation
in a younger organism can have profound effects, from the formation of cataracts to
mitochondria swelling and consequent destruction (Jain et al., 1991; Meister, 1991).
Mitochondrial damage has been observed in people suffering from Parkinsons disease, a
further indication of the role that radicals play in this disease.
Glutathione reduces hydrogen peroxide to produce water and the oxidized form of
glutathione, glutathione disulfide (GSSG). This reaction is catalyzed by the enzyme
glutathione peroxidase through the following reaction:
2GSH + H2O2 GSSG + 2H2O (Shaw, 1998)
Glutathione disulfide is, in turn, reduced to glutathione by the enzyme glutathione
reductase and nicotinamide adenine dinucleotide phosphate (NADPH).
8


GSSG + NADPH + H+ -> 2GSH + NADP+ (Anderson, 1985)
This is a cellular reaction, so glutathione must continually be imported by the plasma
(Meister, 1983, Science).
In addition to eliminating radicals, glutathione also aids in the subsequent DNA
repair. This was investigated by Revesz and Malaise, in a series of experiments
examining the DNA repair capabilities of normal cells versus mutated cells specifically
cells which have a flaw in the glutathione synthesis system, resulting in reduced
glutathione production (Revesz & Malaise, 1983). In one hour, the normal cells had
repaired most of the DNA strand damage, while in the mutated cells, 30% fewer DNA
breaks were repaired (Revesz & Malaise, 1983).
1.1.2 Heavy Metals and Other Xenobiotics
The presence of excess heavy metals in an organism can cause many detrimental
side effects. One notable example is mercury. Mercury poisoning causes damage to
internal organs, specifically the brain. In the nineteenth century, mercury was used in
the manufacture of hats; hence, the origin of the phrase mad as a hatter (Heath,
1996). Besides tissue damage, metals can also have side reactions such as the reaction of
iron with hydrogen peroxide to produce radicals. The accumulation of iron and
consequential radical generation could, in part, be responsible for Parkinsons disease,
for excess iron has been observed in patients suffering from the disease (Jain et al.,
1991). Studies on glutathione-deficient cells demonstrated their exceptional sensitivity
to the effects of metals, in particular mercury and cadmium ions (Anderson, 1997).
9


In addition to reacting with metals, glutathione also reacts with various xenobiotics,
ranging from air pollutants to drugs. Glutathione reactions are not as efficient in
dismantling various health hazards as systems which target specific toxins; however,
given the theory of natural selection, glutathiones wide-ranging ability to tackle a
number of substances would give it the greatest flexibility in a changing system; and,
thus, provide a selective advantage for subsequent generations (Tew, 1994).
Glutathione levels within cells can be reduced by treating the cells with drugs, such
as acetaminophen (Puri & Meister, 1983). In the case of acetaminophen, cytochrome P-
450 is capable of oxidizing it to a more reactive form. Under low concentrations,
glutathione is able to react with this new form to prevent damage to the cell (Moldeus &
Jernstrom, 1983). In an experiment performed by Dusre et al., to investigate the role
glutathione plays in the resistance to the chemotherapy agent doxorubicin, cellular
glutathione was reduced by first treating the cells with buthionine sulfoximine. Refer to
figure 1.5 for the structure of buthionine sulfoximine.
Figure 1.5: Buthionine Sulfoximine
COOH
I
CH
I
Cli,
H
I
:S"
II
N
I
H CH3
CHCH
10


Buthionine sulfoximine inhibits y-glutamylcysteine synthetase, the enzyme involved
in the first step of glutathione synthetase (Plummer et al., 1981). The cells were then
treated with doxorubicin while a second set of cells was only treated with doxorubicin
(Dusre et al., 1989). There was twice as much activity of the drug in these cells as in the
cells which no longer contained glutathione (Dusre et al., 1989).
To remove or reduce the threat of xenobiotic agents, glutathione reacts in such a
way as to either create products which are more hydrophilic and, thus, easier to remove
from the system, or to reduce the reactivity of the drug. One way of accomplishing this
is to reduce a bifunctional substance to monofunctionality, thus reducing its ability to
damage DNA by forming cross-links with DNA (Tew, 1994). Certain chemotherapy
agents are bifunctional, such as melphalan, a nitrogen mustard, which is used in the
treatment of many different types of cancer including ovarian and breast cancer. (Green
et al., 1984; Tew, 1994; Chaney and Sancar, 1996). Glutathione reactions are facilitated
by the enzyme glutathione-S-transferase, which serves to make the cysteine moiety of
glutathione less basic and, thus, more vulnerable for attack (Tew, 1994). As with
cellular damage induced by radicals, glutathione also assists in the repair of drug-
induced DNA damage, by participating as a cofactor (Yao et al., 1995).
1.2 Problems Associated with Glutathione
While the ability to remove potentially dangerous chemicals from the cell is clearly
advantageous, under certain circumstances, it can work to the detriment of the
organism. One readily identifiable case is the treatment of cancer. Typical cancer
treatments are radiation and chemotherapy. Glutathione is able to reduce the efficacy
11


of both radiation and chemotherapy treatments, as was previously mentioned, by
protecting the tumor cells from attack by these agents.
In the case of the latter treatment, one effect is drug resistance. In an experiment to
examine the resistance of leukemia cells against melphalan, an increase in glutathione
concentration of up to 10 times the normal amount was observed in cells in which
melphalan ceased to be effective at combating leukemia (Calvert et al., 1998). In
ovarian cancer, higher glutathione levels were observed in cancerous cells. This has
also been observed in the cancer drug cisplatin. The platinum contained within the
molecule is attracted to sulfur, thus the chemotherapy agent will attack either DNA
bases or glutathione (Wang, 1996) Of course, by reacting with glutathione, it is no
longer able to combat the tumor. Cells resistant to cisplatin have a higher DNA repair
rate, indicating an increase in glutathione activity (Ozols et al., 1990).
A secondary problem associated with drug resistance does not involve humans, but
instead involves insects. Using the similar glutathione mechanism, insects have become
resistant to pesticides such as dichlorodiphenyltrichloroethane (DDT), an important
concern to areas prone to malarial outbreaks (Meister, 1991).
1.2.1 Resistance
Resistance to chemotherapy drugs is so common that around 85-95% of patients will
be drug-resistant after two years of treatment (Green et al., 1984). Even multiple drug
combinations are susceptible to glutathione reduction (Frankfurt et al., 1991). However,
drug resistance can be reversed by depleting cellular glutathione. Reducing the cellular
level of glutathione, while enabling the chemotherapy agents to attack the cancer, can
also cause additional problems because of the multiple cellular functions of glutathione.
12


For instance, mitochondria can be damaged, proteins can lose structure and enzymes
can cease to function (Minhas & Thornalley, 1995; Meister, 1989). It is clearly
extremely important to balance the benefits of glutathione versus the necessity of
chemotherapy.
Many compounds have the capability to reduce the level of cellular glutathione,
including diamine, diethylmaleate, phorone, or buthionine sulfoximine. Diamine is an
oxidizer capable of forcing glutathione to the oxidized state. However, this only serves
to briefly lower the glutathione concentration because oxidized glutathione is rapidly
converted back to glutathione (Meister, 1991). Diethylmaleate causes up to a 94%
depletion of glutathione in the liver; however, it tends to do more harm than good,
causing detrimental side effects due, in part, to the presence of chemical impurities,
specifically diethylfumarate (Meister, 1991). Phorone causes similar depletion to
diethylmaleate two hours after use, but it too has notable side effects. Chemicals, such
as methylphenyldiazenecarboxylate, that react with the sulfhydryl on glutathione, will
also react indiscriminately with other sulfhydryl molecules inside the cell, resulting in
cell damage and possibly cell death (Plummer et al., 1981).
Buthionine sulfoximine, on the other hand, does not appear to be plagued with side
effects on animal subjects as the aforementioned chemicals are; nor does it appear to
cause convulsions as does the closely related chemical, methionine sulfoximine (Meister,
1991). It is also readily excreted in urine. Only L-buthionine SR-sulfoximine form is
active; the D-buthionine SR-sulfoximine is not. The addition of buthionine sulfoximine
can reduce the glutathione present in tumor cells faster than in normal cells perhaps
due to the fact that certain tumor cells usually only have the level of glutathione
necessary for survival (Kramer et al., 1987). In a study by Kramer et al, depletion of
13


glutathione occurred more rapidly in tumor cells approximately a 90% reduction as
compared to normal cells undergoing the same treatment which could serve to
maintain at least a low level of glutathione in normal cells, thus reducing the likelihood
of cellular damage (Kramer et al., 1987). However, cellular damage will not be entirely
eliminated, for bone marrow has a lower supply of glutathione than normal cells (Chen
et al., 1998). Also, the presence of tumors affects the amount of glutathione in bone
marrow, causing the concentration of glutathione to fall to levels that are only % as high
as normal cells (Baruchel et al., 1995).
In a study performed by ODwyer et al. to investigate the therapeutic advantage to
glutathione depletion with respect to chemotherapy resistance, nine patients with
cancer that was no longer responsive to conventional therapy were treated first with L-
buthionine sulfoximine and then with the chemotherapy drug melphalan (ODwyer et
al., 1992). Four of the patients showed some form of stabilization against the
progression of the cancer. With such promising results, this may appear to be the
perfect response to overcoming the problem of resistance; however, research has shown
that each organ responds differently to buthionine sulfoximine treatment; and, thus,
extensive research would yet be required (Kramer et al., 1987).
In addition to chemotherapy, glutathione reduction can also be useful in other
circumstances. Reducing cellular glutathione would increase the efficacy of radiation
therapy against tumors. Lowering glutathione levels could also be used as a means of
combating malaria. Certain species of parasites, such as the malaria vector
plasmodium have higher cellular concentrations of hydrogen peroxide than the host;
therefore, removing glutathione harms or kills the parasite, while causing minimal
14


damage to the host, which is able to survive the loss of glutathione for longer periods of
time (Meister, 1983, Science).
1.3 Supplemental Glutathione
While glutathione can clearly hinder the treatment of cancer, certain conditions
exist where the addition of supplemental cellular glutathione is advantageous for either
laboratory research, such as the investigation of the effects of glutathione on
chemotherapy resistance; or for practical application, such as for protection of cells
against radiation, peroxides, or xenobiotics. The biological glutathione system is
capable of handling biologically generated reactive oxygen species, but would be readily
overwhelmed with the introduction of significant xenobiotic sources (Oshino & Chance,
1977). However, this can be at least partially rectified by the addition of glutathione to
the deficient cells (Revesz & Malaise, 1983; Hagen et al., 1986). In fact, certain
organisms already use this technique. For instance, certain bacteria have developed
resistance to the drug fosfomycin by producing greater quantities of glutathione (Tew,
1994). Along this same fine, supplemental glutathione could he added to plants to aid
the survival against herbicides, thus destroying the weeds while maintaining the
desired plants (Meister, 1983, Science).
Supplemental glutathione could be used to treat individuals who possess genetic
defects in glutathione production, which can result in brain defects in infants and
anemia in adults (Meister, 1983, Science; Lehninger et al., 1993). Glutathione would
also be of use for treating viral diseases. Experiments by Kalebic et al. show that
glutathione, in concentrations of 15 mM, eliminates viral production for the duration of
the treatment (Kalebic et al., 1991).
15


1.3.1 Enzyme or Amino Acid Addition
Several basic methods exist to increase the level of cellular glutathione. The
production of glutathione could be augmented either by increasing the supply of the
amino acid components of glutathione, or by increasing the enzymes which stimulate
the production of glutathione. Cysteine is normally the rate-limiting amino acid; hence,
amino acid addition has mainly focused on cysteine. R. F. Burk tried unsuccessfully -
to prevent lipid peroxidation in microsomes by directly adding cysteine to the
microsomes (Burk, 1983). The use of certain chemicals capable of reacting internally to
form cysteine, such as N-acetyl-L-cysteine, cysteine esters, y-glutamylcysteine or L-2-
oxothiazolidine-4-carboxylate (thiazolidine) to increase glutathione has met with greater
success (Minhas & Thornalley, 1995; Williamson et al., 1982). Refer to Figure 1.6 for
the structure of thiazolidine.
Figure 1.6: Thiazolidine
S-------
H
N-acetyl-L-cysteine has successfully suppressed reverse transcriptase in HIV. In
experiments performed by Kalebic et al, the activity of reverse transcriptase could be
reduced to less than 10% by the addition of 15 mM N-acetyl-L-cysteine (Kalebic et al.,
1991).
Thiazolidine can be used to create S-carboxy-L-cysteine, which degrades to cysteine
via 5-oxo-L-prolinase, a naturally occurring enzyme (Williamson, 1982; Meister, 1983).
16


H + 2H20 + ATP - L-cysteine + C02 + ADP +P; (Meister, 1989)
In a study performed by Baruchel et al., the addition of thiazolidine to the kidneys,
liver, and bone marrow, increased the concentration of glutathione by approximately
44% in the kidneys, by 65% in the liver, but only by 15% in bone marrow (Baruchel et
al., 1995). More importantly, thiazolidine increased the amount of glutathione within
normal cells, but without a corresponding increase in the tumor cells (Chen et al., 1998).
Consequently, buthionine sulfoximine could be used to remove glutathione; then, the
glutathione could be selectively returned to normal cells to protect them against further
chemotherapy or radiation therapy. Thus, the tumor cells would be preferentially
selected.
Even with all the apparent benefits of these methods of glutathione addition,
several problems still arise. For instance, the administration of cysteine does not
guarantee its use in the formation of glutathione, as many other substances also require
cysteine for synthesis. Also, the addition of cysteine is subject to the glutathione
feedback inhibition system, y-glutamylcysteine synthetase regulates the concentration
of glutathione in the body and prevents high concentrations of glutathione from being
formed (Anderson et al., 1985; Wellner and Anderson, 1984). Cysteine is capable of
intracellular degradation, rendering the addition somewhat moot (Arrick and Nathan,
1984). It can be oxidized to cystine internally, which would require a chemical reaction
to disassemble prior to glutathione synthesis. Most importantly, an excess of cysteine
can cause neural damage (Meister, 1989; Williamson et al., 1982). In the central
17


nervous system, cysteine partially prevents the uptake of the neurotransmitter
glutamate, when present in the extracellular space (Cooper, 1998). The damage caused
by cysteine is even more pronounced in young animals, leading to lesions in the brain
similar to those produced by acidic amino acids (Karlsen et al., 1981). In a study
performed by R. L. Karlsen et. al, all rats injected with a 1.2 mg/g body weight solution
of cysteine developed brain lesions of one type or another; and, consequently, there was
only a 50% survival rate over the first two days (Karlsen et al., 1981).
1.3.2 Glutathione Addition
A second way to increase glutathione is to directly add glutathione. The addition of
glutathione would circumvent the glutathione feedback inhibition system and would not
require ATP to create (Minhas & Thornalley, 1995). The aforementioned R. F. Burk
also tried to prevent lipid peroxidation through the direct administration of glutathione
with more positive results than with the addition of cysteine (Burk, 1983). Glutathione
did not prevent the attack on the lipid; however, it was able to delay it by almost four
times the normal reaction time, indicating that glutathione serves an important role in
prevention (Burk, 1983).
Unfortunately, glutathione is not readily transported into the cells. Thus, the
glutathione supplements mentioned in the Denver Post article would be useless, as the
article itself laments (Carper, 1995). Glutathione appears to be exported out of cells to
aid in the defense of the cellular membrane against xenobiotic or naturally occurring
reactive agents (Meister, 1991). However, it does not appear as if significant portions of
intact glutathione are imported into cells. Glutathione is first transported outside the
cell where it can react with cystine to form y-glutamylcystine. This latter molecule can
18


then move across the cell membrane, where it is subsequently broken apart to form
cysteine and y-glutamylcysteine. However, certain cells such as those in the kidney and
small intestine are equipped with sodium ion transports which enable them to take up
intact glutathione (Hagen et al., 1986).
There is also evidence that glutathione can be transferred between cells via gap
junctions proteins on the cell membrane which connect to adjacent cells and allow
substances smaller than about 1.5 nm through (Campbell, 1990; Chen et al., 1998).
Cancer cells do not appear to have fewer gap junctions to serve as transport, so it might
be possible to selectively add glutathione to normal cells while providing a minimal
supply to cancer cells (Chen et al., 1998). Nonetheless, this would not solve the
transport problem when glutathione is added in the bloodstream. A transport system
does exist to export glutathione to the plasma which will transport glutathione across
the cell membrane, but it only appears to function when the concentration of
glutathione outside the cell is many times more concentrated than the concentration of
glutathione within the cell membrane (Garcia-Ruiz et al., 1992). In studies where the
degradation of glutathione was inhibited by the chemical acivicin, transport of whole
glutathione across the cell was observed. However, if the breakdown of glutathione
were not inhibited, this may not be a popular mechanism. The entire discussion of
transportation of glutathione throughout the system is still a matter of debate. An
easier way of transporting glutathione into cells has emerged through the esterification
of glutathione.
19


1.3.3 Esters of Glutathione
As with cysteine, modified forms of glutathione clearly need to be employed. Esters
of glutathione are more effective than glutathione for increasing cellular glutathione
levels as they are more readily transported across the cell membrane. Once inside the
cell, the esters are converted to glutathione via intracellular esterases. The addition of
the ester reduces the number of charges on the glutathione by eliminating the negative
charge on the glycine, making the molecule more hydrophobic and, thus, easier to
transport through the cellular membrane (Meister, 1989). The ease of ester transport
has been demonstrated in other studies, through the rupture of lysosomes after the
introduction of amino acid esters (Goldman and Naider, 1974). The esters are readily
transported inside the lysosomes where they are hydrolyzed; however, the amino acid
constituents are not transported as rapidly through the membrane. Water enters the
lysosome via osmotic flow, resulting in the eventual bursting of the lysosome. Studies in
the effectiveness of glutathione esters versus glutathione for different cell types show an
increase in intracellular glutathione levels for the kidney, liver, spleen, pancreas, and
heart that is, in some cases, more than three times as great for the glutathione esters
versus unmodified glutathione (Anderson et al., 1985).
In a study performed by Anderson et al, the effectiveness of glutathione esters
versus glutathione was evaluated by treating mice first with either glutathione or
glutathione ester and then with the cancer drug cis-diaminedichloroplatinum, referred
to by the drug name of cisplatin (Anderson etal., 1990). Cisplatin can cause 100%
fatality in mice at a dose of 70 pmol/kg; however, mice with compromised glutathione
production (by pretreatment with buthionine sulfoximine) have a 100% mortality rate at
a dose of only 45 pmol/kg. Roughly 100% protection against cisplatin damage is
20


achieved with 5 mmol/kg of glutathione esters, while glutathione itself requires a
concentration twice as high to give equivalent protection for a 45 pmol/kg dose of
cisplatin. Part of the functioning of the glutathione esters could also be due in part to
the ability of the esters themselves to affect the xenobiotic agents (Meister, 1991).
Glutathione esters have been given in the form of injections, though even oral dosing of
glutathione monoester caused the levels of glutathione to rise (Anderson et al., 1994).
Glutathione esters also protect cells treated with buthionine sulfoximine from
mitochondria damage as a result of glutathione depletion (Anderson et al, 1994, Meister,
1989). Glutathione under similar conditions is unable to do this. In a second study
involving rats, glutathione did not increase the mitochondrial glutathione or the cortex
glutathione, whereas both increased with the addition of glutathione esters. This could
be of significant import when trying to prevent peroxide damage to mitochondria
(Meister, 1991).
The effectiveness of glutathione esters to protect against drugs has also been
explored. In one study, mice were given a lethal dose of acetaminophen (Puri and
Meister, 1983). A group of these mice was also given glutathione esters. All the mice
treated with glutathione ester lived, while the ones not treated died shortly thereafter
(Puri and Meister, 1983).
Glutathione esters have been used to suppress reverse transcriptase. In the same
experiment in which the effectiveness of N-acetyl-L-cysteine against reverse
transcriptase was investigated, glutathione esters were able to prevent reverse
transcriptase in HIV from functioning by restricting protein synthesis in the virus with
a reduction in activity directly related to the length of the treatment (Kalebic et al.,
1991).
21


Glutathione esters also appear to protect against radiation damage. Wellner et al.
tested this theory by removing cellular glutathione with buthionine sulfoximine,
treating the cells with either glutathione esters or glutathione and then irradiating the
cells (Wellner & Anderson, 1984). The control cells were no longer viable, while 42% of
the glutathione-treated cells were still viable. The cells treated with glutathione esters
were 86% viable, proving the effectiveness of glutathione esters (Wellner & Anderson,
1984). In fact, glutathione esters could be administered after irradiation and still
protect the cells to an extent where there was a survival rate of up to 60% (Wellner &
Anderson, 1984).
Both mono and diesters of glutathione have been created. (For the structure of the
monoester and the diester of glutathione, refer to figures 1.7 and 1.8 respectively.)
Figure 1.7: Glutathione Monoester
H
O
O
O
+
H3NCc%CH^CNHCHCNHC%- COR
COO
I
Ct^SH
Figure 1.8: Glutathione Diester
H
O
O
O
H3NCCt%CI%CNHCH-C-NHCH^C-O-R
COOR CH.SH
22


Ethyl ester is generally preferred for animal studies, since the ethanol which is
produced after esterification has less harmful effects on the cells than other alcohols.
Diesters are hydrolyzed more quickly to the monoester than the monoester is to
glutathione (Minhas & Thornalley, 1995; Anderson, 1997). The diesters have the
advantage of rapid transport into the cells due to the elimination of the negative charge
on both the glycine and the glutamate residues. However, in mice and rats, but
interestingly enough not other rodents, the esters are broken down in the plasma to
monoesters due to the presence of glutathione diester a-esterase (Levy et al., 1994).
Hence, in rodents, use of the diester is equivalent to the use of the monoester (Levy et
al, 1993). A cell treated with the diester will have almost three times the amount of
glutathione than a similar cell treated with the monoester (Anderson, 1997).
1.4 Hydrolysis of Glutathione Esters
The glutathione esters do appear to be more effective in increasing cellular
glutathione levels than glutathione. However, for the glutathione esters to be converted
to glutathione, hydrolysis must occur by internal esterases because the rate of
uncatalyzed hydrolysis of esters is too slow at physiological pH and temperature to be of
any medical use. Many non-specific esterases are available in the cell which would be
able to attack the ester site on the glutathione (Maki et al., 1991).
Cells possess many different esterases capable of breaking apart a wide variety of
hydrocarbon or aromatic esters. (Restricting the definition of the enzyme capable of
reducing glutathione esters to glutathione to esterase could be too confining as amidases
are known to catalyze xenophobic esters as well as their more normal targets, amides
(Heymann, 1980).) The number of esterases present is still under investigation. At
23


least six have been identified in rat liver. There is great difficulty in analyzing
esterases; a newly discovered esterase could turn out to be, in fact, a previously
discovered esterase. In addition, the concentrations of esterases can vary based upon
the sex of the individual or upon the species (Maki et al., 1991). In many cases, the
newly discovered enzyme may, in fact, be an isoenzyme of a previously known enzyme.
The carboxylesterases in many cases serve the same function as glutathione; that is
to say, they too dismantle xenobiotic entities (Walker, 1994). Esterases serve to detoxify
the liver system by dismantling xenophobic organisms into less destructive entities
(Heymann, 1980). A family of nonspecific isoenzymes simply entitled B-esterases is
capable of hydrolyzing both aliphatic and aromatic esters. (Heymann, 1980).
Incidentally, these enzymes are also present in the microsomal fraction (Heymann,
1980).
Tumor cells and normal cells differ with respect to esterases and with respect to the
glutathione concentrations. It might, therefore, be possible to differentiate between
tumors and normal tissue. That is to say, cellular glutathione could be depleted and
then treated with glutathione esters which are designed to be hydrolyzed only by
esterases found in normal tissue, thus protecting normal cells against chemotherapy
agents. If glutathione esters could be developed that would be preferentially converted
by normal cells as opposed to the cancer cells, a means of protecting normal cells from
the chemotherapy agents could be developed. However, this would clearly require a
greater understanding into the esterases involved in the hydrolysis of glutathione
esters.
24


1.5 Purpose
In order to elucidate the aforementioned problem, the hydrolysis of glutathione
esters by rat liver esterases present in microsomal and cytosolic fractions will be
examined For this purpose, five esters of glutathione propyl, ethyl, benzyl, isobutyl
and n-butyl have been constructed. Refer to Figures 1.9-1.13 for the structures of the
esters.
Figure 1.9: Glutathione Monopropyl Ester
H
O
O
O
+
K^NC-C%-C%-C-NH-CH-C-NH-C%CO-O^CHjC^,
COO CH,SH
Figure 1.10: Glutathione Monoethyl Ester
H
O
O
O
+
H3NCCH2CHjCNHCHCNHCH^COCHD;CH3
I
COO
CH,SH
Figure 1.11: Glutathione Monobenzyl Ester
H
O
O
O
+
HjN-C-CHjO^C-NH-CH-C-NH-Ct%0-00%
COO' CH,SH
25


Figure 1.12: Glutathione Monoisobutyl Ester
H
O
O
O
+
H3N-CCI%CH^C-NH-CH-C-NH-CHjcoCHjCH-CH3
COO CP^SH CH3
Figure 1.13: Glutathione Monobutyl Ester
H
O
O
O
H3N-CCHjCHjCNH-CHC-NH-CHjC-O-CHjCHjCH^CHj
COO
CR,SH
26


2. Procedure
2.1 Reagents/Equipment
P-Nicotinamide Adenine Di-nucleotide phosphate, reduced form (NADPH); Clelands
reagent, dithiothreitol (DTT); glutathione reductase; and ethylenediaminetetraacetic
acid (EDTA) were obtained from Sigma. 5,5 dithiobis(2-nitrobenzoic acid) (DTNB);
Glycerol; p-nitro phen acetate (PnPAC); and 5-sulfosalicylic acid dihydrate (SSA) were
obtained from Aldrich. Glutathione, reduced free acid was obtained from Calbiochem.
Acetonitrile and barium chloride were manufactured by J. T. Baker. The Bio-Rad
Protein Assay solutions and standards were obtained from Bio-Rad Laboratories. The
tris(hydroxymethyl)aminomethane (Tris Acetate) was manufactured by Boehringer
Mannheim. The acetic acid used was produced by Mallinckrodt. Dulbeccos Phosphate
Buffered Saline (PBS) was obtained from Gibco BrL. DE53 diethylaminoethyl
cellulose anion exchange resin; and methanol were obtained from Fisher. All other
previously unmentioned regents were of the highest quality available.
Since glutathione is capable of reacting with metals, as was previously mentioned,
only deionized water was used for the preparations, and care was taken to minimize
metal exposure (Anderson et al., 1994; Levy et al., 1993). The rat livers were obtained
from Harlan Bioproducts. The dialyzer tubing used had a molecular weight cutoff of
6000-8000 and was produced by Thomas.
The centrifuge used was a Beckman L5-50E. The UV-Vis spectrophotometer was a
Varian Cary IE.
27


2.2 Buffer Solutions
The following buffer solutions were used throughout the procedure:
Buffer A: 0.1 M Tris Acetate, pH 7.4; 1 mM EDTA; and 0.1 mM DTT
Buffer B: 0.1 M Tris Acetate, pH 7.4; 20% (w/v) glycerol; 1 mM EDTA; and 0.1 mM DTT
Stock Tietze Buffer: 143 mM sodium phosphate, 6.3 mM EDTA at pH 7.4
Daily Buffer: 0.248 mg/mL of NADPH in Stock Tietze Buffer
Pyrophosphate Buffer: 0.1 M sodium pyrophosphate, pH 7.4; 20% (w/v) glycerol, 1 mM
EDTA; 0.1 mM DTT
Pig Liver Esterase (PLE) Buffer: 50 mM sodium pyrophosphate, pH 7.0 and 0.1M NaCl
2.3 Preparation of Microsomes and Cytosolic Fraction
Microsomes from rat liver were prepared according to the procedure developed by
Juris Ozols (Ozols, 1990). Liver is typically used due to the thorough study on the
micosomal preparations (Ozols, 1990). A brief summary of the procedure appears here.
At a temperature of 4C, the rat livers were homogenized with Buffer A in a 1:5 ratio:
that is to say, 1 g of rat liver to 5 mL of Buffer A. The liver was centrifuged and the
resulting pellets were discarded. The supernate was again centrifuged. The cytosolic
fraction (the supernate) was removed and dialyzed overnight in 4 L of Buffer B to
remove cellular glutathione. The dialyzing solution was replaced once.
The pellet remaining after the removal of the cytosolic fraction was resuspended in
Pyrophosphate Buffer, which served to stabilize the microsomes and was centrifuged.
The resulting pellet was removed and resuspended in Buffer B. This was the
microsomal fraction. While under neutral conditions, the enzymes do not appear to
break down even specially when frozen (Heymann, 1980).
28


2.3.1 Determination of the Protein Content
The protein content of the cytosol was determined, using the Bio-Rad protein assay.
The samples were analyzed spectrographically at 595 nm. A standard curve was
generated, using the standards prepared from bovine gamma globulin. A protein
content of 102 mg/mL was obtained.
2.3.2 Determination of Esterase Activity
The esterase activity was determined in the dialyzed cytosol prior to initiation of
any experiments to ensure that the esterases were still viable after the separation
procedure. 600 pL of the cytosol was diluted to 900 pL with 10% SSA. 100 pL of sample
was diluted to 1000 pL with a 50/50 mixture of Buffer A and Buffer B. 950 pL of PLE
Buffer, 200 pL of sample and 50 pL of 60 mM p-nitrophenyl acetate in acetonitrile were
mixed in a cuvette. The sample was analyzed spectrographically at 412 nm. The
dialyzed supernate had an activity of 0.73 Abs/min/mg protein.
2.4 Preparation of the Esters
The esters were prepared according to the procedure by Anderson et al (Anderson et
al, 1994). Sulfuric acid is typically used to prepare glutathione esters since hydrochloric
acid can produce toxic impurities in the esters (Meister, 1991). Sulfate was removed
from all esters prior to their use in the esterase assays. The esters were dissolved in
approximately 5 mL of methanol. The solution was slurried with approximately 5 g of
DE53. The solution was centrifuged and the supernate was tested for the presence of
sulfur, using a 2% solution of BaCR. This procedure was repeated until the sulfate test
was negative. The procedure was then repeated two additional times. The DE53 was
removed through filtering. Each sample was freeze-dried.
29


2.5 Preparation of Glutathione Ester Substrate Samples
Known concentrations of glutathione esters were prepared in duplicate. The
appropriate amount of glutathione ester was dissolved in 10 mL of 3.33% SSA to achieve
a concentration of 5 mM. The stock sample solution was diluted to prepare 12 samples
with final concentrations of 0.00 mM, 0.008 mM, 0.016 mM, 0.032 mM, 0.040 mM, 0.080
mM, 0.120 mM, 0.160 mM, 0.320 mM, 0.480 mM, 0.640 mM, and 0.800 mM. The
samples were analyzed using the Tietze Assay method.
2.6 Preparation of the Hydrolysis Samples
Five 1.5 mL microeppendorf tubes were set up for each of the time points. The
dialyzed cytosol or microsomal fraction was defrosted in a 37 C water bath and then
placed on ice until needed. A blank solution was prepared by mixing 750 pL of PLE
Buffer with 250 pL of the dialyzed cytosolic fraction or microsomal fraction. A
glutathione ester control was prepared by mixing 750 pL of glutathione ester in PLE
Buffer with 250 pL of Buffer B. The glutathione ester esterase sample was prepared by
mixing 750 pL of glutathione ester in PLE Buffer with 250 pL of cytosol. 100 pL of the 0
minutes time point sample were removed and mixed with 850 pL of 3.33% 5-SSA and 50
pL of 10% 5-SSA to quench the reaction. The samples were placed in a 37C water bath
for the duration of the experiment. At the appropriate time point, 100 pL of the samples
were removed and mixed with 850 pL of 3.33% 5-SSA and 50 pL of 10% 5-SSA to quench
the reaction. Typical time points were 0, 15, 30, 45 and 60 minutes. The samples were
centrifuged at 4C for 5 minutes. The samples were stored on ice until the Tietze assay
could be performed.
30


2.7 Preparation of the Cytosolic Concentration Studies
750 pL of a 10.67 mM solution of the benzyl monoester in PLE buffer and 250 pL of
the dialyzed cytosobc fraction were placed in a 1.5 mL microeppendorf tube. (In the
case of blank solutions, an equivalent volume of Buffer A was substituted.) The samples
were mixed. A 100 pL sample was taken and mixed with 850 pL of 3.33% 5-SSA and 50
pL of 10% 5-SSA. The samples were heated to 37C in a water bath and additional 100
pL aliquots were removed at 15, 30, 45, and 60 minute intervals. The samples were
frozen until analysis. The benzyl glutathione ester samples were run at concentrations
of 8 mM, 12 mM, and 16 mM.
2.8 Tietze Assay
The analysis of the glutathione ester samples was performed using a modified
version of the glutathione assay method developed by F. Tietze (Tietze, 1969). 25 pL of
each sample were added to a cuvette containing 700 pL of the Daily Buffer, 175 pL of
water, and 100 pL of 6 mM DTNB. A 1 in 4 solution of glutathione reductase was
prepared using the Stock Tietze Buffer. When not in use, the enzyme solution was
stored on ice. The samples were incubated in a 30 C water bath for at least 10 minutes.
Immediately, prior to analysis, 10 pL of glutathione reductase were added to the sample
cuvette and the solution was mixed. The sample was analyzed on the Cary UV for a
period of 3 minutes at 412 nm.
31


3. Results
3.1 Tietze Assay Theory
The concentration of glutathione in solution was determined using the Tietze assay
method. In this procedure, glutathione and 5,5 dithiobis (2-nitrobenzoic acid) or DTNB
react to form oxidized glutathione, glutathione disulfide, (GSSG) and 2-nitro-5-
thiobenzoic acid (TNB) via the following reaction. (Refer to Figure 3.1 for the structure
of DTNB.)
2GSH + DTNB -> GSSG + TNB
Figure 3.1: 5,5 dithiobis (2-nitrobenzoic acid)
o
II
HOC
O
II
COH
o2n-|^s s<^-no2
Glutathione disulfide is converted back to glutathione by the enzyme glutathione
reductase and NADPH. The 2-ntiro-5-thiobenzoic acid is yellow in coloring, the
intensity of which corresponds to concentration. This color change can be monitored
spectroscopically over time. Provided there is sufficient NADPH and glutathione
reductase present, the reaction will produce a linear response for absorbance versus
time. The rate of the reaction is proportional to the concentration of glutathione. A
32


series of glutathione standards of known concentration can be rim to produce a standard
curve which equates the slope of the line to the concentration of glutathione present in a
sample. Incidentally, independent work on the effectiveness of Tietze assay versus an
HPLC assay produced similar results (Eady et al., 1995). The Tietze method has the
advantage of being the quicker to perform. While this procedure seems relatively
straightforward, certain problems which have a drastic influence on the results must be
addressed prior to using the Tietze assay method.
The rate of hydrolysis can be affected by metal contamination, and by the pH of the
solution (Wellner & Anderson, 1984). If the pH of the solution rises above 7.6, the ester
will easily hydrolyze (Wellner & Anderson, 1984). During the experiment, the pH of the
solution was maintained below this level. The sample was acidified prior to the Tietze
assay to remove the protein in the sample which can also influence the results.
Samples of esters without enzymes were rim in order to monitor for hydrolysis of the
esters. Refer to section 3.3 for the hydrolysis data. Metal contamination was reduced
through the use of pure solvents and adequate rinsing of the glassware with deionized
water and inclusion of EDTA. Another problem would occur if the esters were
substrates of glutathione reductase. Naturally, this would proceed inaccurate results
using the Tietze assay method. This problem will be examined in section 3.2.
3.2 Glutathione Ester Substrate Studies
Concentration studies were performed on the glutathione esters in order to
ascertain whether the glutathione esters were substrates of the enzyme glutathione
reductase used in the Tietze assay. If this were the case, it would be impossible to
differentiate between the glutathione generated from the enzymatic hydrolysis of the
33


esters and the esters themselves reacting during the analysis step. The studies would
also determine whether the glutathione remaining from the synthesis step would
interfere with the experiments by providing too high of a background reading.
The results were evaluated by examining the percent hydrolysis of the esters at
varying concentration levels. Background corrections have been applied to the results.
Refer to Tables 3.1 3.5 for the results of the ethyl, n-propyl, n-butyl, isobutyl and
benzyl esters. The overall percent hydrolysis was also obtained graphically. The slopes
of the absorbance versus time plots obtained from the Tietze runs were plotted against
the concentration of the ester used to prepare the sample. The value was divided by the
slope from the standard curve to obtain the percent hydrolysis. Refer to Figures 3.2 -
3.6 for the slope versus concentration graphs for the ethyl, propyl, butyl, isobutyl, and
benzyl esters.
The individual percent hydrolysis results had a fair amount of fluctuation at the
lower concentrations of the esters where the signal to noise was a more important factor
in the results. The percent hydrolysis for individual values at concentration levels
greater than 0.08 mM compares favourably with the percent hydrolysis obtained via the
slope method. The individual percent hydrolysis values for the concentration levels
greater than 0.08 mM of ester did not appear to increase over time nor did the
concentration levels rise above 7%. Hence, none of the esters appear to be good
substrates for the enzyme glutathione reductase. The amount of glutathione present in
the esters is residual glutathione from the synthesis step. Separate HPLC analyses
were performed to determine the residual amount of glutathione (Levy et al.,
unpublished).
34


Table 3.1: Individual Ethyl Ester Hydrolysis Results
Ethyl Ester
Cone. (mM) % Hydrolysis
0.008 4.0
0.016 8.0
0.032 6.7
0.040 9.1
0.080 4.8
0.120 5.9
0.160 6.3
0.320 6.5
0.480 5.9
0.640 5.9
0.800 5.5
Figure 3.2: Ethyl Ester Concentration Study
Ethyl Ester
Hydrolysis estimate from slopes = 5.6%
35


Table 3.2: Individual n-Propyl Ester Hydrolysis Results
n-Propyl Ester
Cone. (mM) % Hydrolysis
0.008 0.7
0.016 3.1
0.032 2.9
0.040 3.2
0.080 2.2
0.120 2.7
0.160 2.0
0.320 2.3
0.480 2.2
0.640 2.2
0.800 2.2
Figure 3.3: n-Propyl Ester Concentration Study
n-propyl Ester
Hydrolysis estimate from slopes = 2.2%
36


Table 3.3: Individual n-Butyl Ester Hydrolysis Results
n-Butyl Ester
Cone. (mM) % Hydrolysis
0.008 3.9
0.016 5.1
0.032 1.7
0.040 2.5
0.080 1.9
0.120 2.3
0.160 2.0
0.320 2.5
0.480 2.8
0.640 2.5
0.800 2.0
Figure 3.4: n-Butyl Ester Concentration Study
n-Butyl Ester
Hydrolysis estimate from slopes = 2.3%
37


Table 3.4: Individual Isobutyl Ester Hydrolysis Results
Isobutyl Ester
Cone. (mM) % Hydrolysis
0.008 -0.5
0.016 -3.6
0.032 -0.5
0.040 2.0
0.080 1.1
0.120 1.3
0.160 1.1
0.320 1.4
0.480 1.4
0.640 1.4
0.800 1.4
Figure 3.5: Isobutyl Ester Concentration Study
Isobutyl Ester
0.0 0.2 0.4 0.6 0.8
Cone. Ester (mM)
Hydrolysis estimate from slopes = 1.4%
38


Table 3.5: Individual Benzyl Ester Hydrolysis Results
Benzyl Ester
Cone. (mM) % Hydrolysis
0.008 7.1
0.016 11.3
0.032 3.2
0.040 3.0
0.080 2.9
0.120 2.8
0.160 2.9
0.320 2.6
0.480 2.8
0.640 3.5
0.800 2.7
Figure 3.6: Benzyl Ester Concentration Study
Benzyl Ester
Hydrolysis estimate from slopes = 2.9%
39


At about the 0.08 mM ester concentration, the individual values began to show
internal consistency. The isobutyl ester had the lowest percent hydrolysis of all the
ester which accounts for the greater fluctuation of its data points. The highest percent
hydrolysis was obtained for the ethyl ester which averaged about 6% hydrolysis. This
was not too high of a background to complete the experiments.
3.3 Hydrolysis of Glutathione Esters
Each of the five glutathione esters was examined to see if enzymatic hydrolysis,
using microsomes or cytosolic fractions, would occur. Initial experiments were
performed on 12 mM isobutyl ester and 12 mM benzyl ester, using only cytosol.
Samples were taken at intervals of 0, 40, 80, and 120 minutes. The analysis was
performed in duplicate on separate days. A blank cytosol solution, a control ester
sample, and the ester in the presence of cytosol were run for each time point. The
average of the data is presented in Tables 3.6 3.9 for the data. Refer to Figures 3.7 -
3.8 for the graphical presentation of the data.
The cytosol blank solution had less than a one percent hydrolysis and did not appear
to have an increase in concentration of glutathione over time, but exhibited small
fluctuations in both directions. As with the concentration study, hydrolysis occurred in
the ester control samples. For the isobutyl ester concentrations, there was
approximately a one percent spontaneous hydrolysis that increased slightly over time.
The percent hydrolysis of the benzyl ester control increased from 2.9% to 15% over the
duration of the experiment. This is greater than the 3% hydrolysis observed during the
concentration studies. The isobutyl results were consistent with the concentration
studies. The conditions of the assay could be causing spontaneous hydrolysis over time.
40


In the presence of the cytosol, a significant increase in hydrolysis over the time of the
analysis was observed. The hydrolysis increased to a high of 27% for the isobutyl ester
and 53% for the benzyl ester. The benzyl ester clearly reacts at a much faster rate than
the isobutyl ester. By 120 minutes, the isobutyl ester was still reacting, though the
reaction rate had decreased whereas the benzyl ester percent hydrolysis remained fairly
constant after about 40 minutes.
The ability of a carbonyl esterase to hydrolyze an ester is based upon the functional
group rather than upon the atom to which it is attached (Heymann, 1980). Therefore,
the same esterase could react at varying rates with each of the different glutathione
esters. The time period chosen for analysis is clearly too long to have a linear increase
in the percent hydrolysis. Also, at an ester concentration of 12 mM spontaneous
hydrolysis appears to be problematic for the benzyl ester. The time period was reduced
to one hour and the concentration of the glutathione esters was concurrently reduced to
8 mM. The initial experiment was also performed with a 1:3 glutathione reductase
dilution which was subsequently changed to a 1:4 dilution.
Table 3.6: Concentration of Glutathione in Isobutyl Ester Samples
Time (mins) Cytosol Blank GSH (mM) Ester Control GSH (mM) Ester w/ Cytosol GSH (mM)
0 0.037 0.11 0.27
40 0.080 0.092 1.9
80 0.074 0.12 2.7
120 0.043 0.18 3.2
41


Table 3.7: Hydrolysis of Glutathione Isobutyl Ester
Time (mins) Ester Control % Hydrolysis Ester w/ Cytosol % Hydrolysis
0 0.95 2.1
40 0.76 16
80 1.0 22
120 1.5 27
Table 3.8: Concentration of Glutathione in Benzyl Ester Samples
Time (mins) Cytosol Blank GSH (mM) Ester Control GSH (mM) Ester w/ Cytosol GSH (mM)
0 0.037 0.40 0.65
40 0.080 0.45 6.3
80 0.074 0.45 6.3
120 0.043 0.51 6.4
Table 3.9: Hydrolysis of Glutathione Benzyl Ester
Time (mins) Ester Control % Hydrolysis Ester w/cytosol % Hydrolysis
0 2.9 5.4
40 9.3 53
80 13 53
120 15 53
42


Figure 3.7: Hydrolysis of Glutathione Isobutyl Ester
Isobutyl ester
60
50
& 40
iao
x 20
sP
* 10
0
0 20 40 60 80 100 120
o Ester w/ cytosol
Control
Time (mins)
Figure 3.8: Hydrolysis of Glutathione Benzyl Ester
Benzyl ester
60
.2 50
J? 40
*0
£ 30
X 20
10
0
o Ester w/ cytosol
Control
0 20 40 60 80 100 120
Time (mins)
43


For the second set of hydrolysis experiments, each of the esters was run. Both
cytosol and microsomes were separately used. A buffer blank, microsome or cytosol
blank, ester control, and ester either in the presence of microsomes or cytosol samples
were run for each time point. Refer to Tables 3.10 3.13 and Figures 3.9 3.17 for the
data.
44


Table 3.10: Concentration of Glutathione in Ester Samples with Microsomes
Blank
Time (mins) GSH (mM) Benzyl Ester GSH (mM) n-Butyl Ester GSH (mM) Isobutyl Ester
0 -0.026 -0.013 -0.013
15 -0.016 -0.014 -0.014
30 -0.024 -0.012 -0.012
45 -0.019 -0.014 -0.014
60 -0.013 -0.014 -0.014
Blank
Time (mins) GSH (mM) GSH (mM)
Ethyl Ester n-Propyl Ester
0 -0.056 -0.056
15 -0.062 -0.062
30 -0.044 -0.044
45 -0.057 -0.057
60 -0.041 -0.041
Microsome Blank
Time (mins) GSH (mM) GSH (mM) GSH (mM)
Benzyl Ester n-Butyl Ester Isobutyl Ester
0 -0.018 -0.0033 -0.0033
15 -0.015 -0.014 -0.014
30 -0.019 -0.003 -0.003
45 -0.013 -0.017 -0.017
60 -0.017 -0.013 -0.013
45


Table 3.10 (Cont.)
Microsome Blank
Time (mins) GSH (mM) Ethyl Ester GSH (mM) n-Propyl Ester
0 -0.044 -0.044
15 -0.048 -0.048
30 -0.036 -0.036
45 -0.044 -0.044
60 -0.041 -0.041
Ester Control
Time (mins) GSH (mM) Benzyl Ester GSH (mM) n-Butyl Ester GSH (mM) Isobutyl Ester
0 0.14 0.13 0.072
15 0.18 0.14 0.070
30 0.23 0.15 0.080
45 0.22 0.15 0.079
60 0.20 0.16 0.087
Ester Control
Time (mins) GSH (mM) GSH (mM)
Ethyl Ester n-Propyl Ester
0 0.32 0.080
15 0.33 0.061
30 0.31 0.10
45 0.34 0.038
60 0.31 0.090
46


Table 3.10 (Cont.)
Ester with Microsomes
Time (mins) % Hydrolysis Benzyl Ester % Hydrolysis n-Butyl Ester % Hydrolysis Isobutyl Ester
0 0.16 0.16 0.071
15 1.2 0.45 0.33
30 2.5 0.71 0.54
45 3.2 0.94 0.65
60 3.9 1.0 0.88
Ester with Microsomes
Time (mins) % Hydrolysis % Hydrolysis
Ethyl Ester n-Propyl Ester
0 0.60 0.11
15 0.36 0.22
30 0.38 0.44
45 0.43 0.48
60 0.54 0.54
47


Table 3.11: Hydrolysis of Glutathione Esters with Microsomes
Ester Control
Time (mins) % Hydrolysis Benzyl Ester % Hydrolysis n-Butyl Ester % Hydrolysis Isobutyl Ester
0 1.8 1.7 0.9
15 2.2 1.8 0.9
30 2.8 1.9 1.0
45 2.8 1.9 1.0
60 2.6 1.9 1.1
Ester Control
Time (mins) % Hydrolysis % Hydrolysis
Ethyl Ester n-Propyl Ester
0 4.0 1.0
15 4.1 0.8
30 3.9 1.3
45 4.3 0.5
60 3.9 1.1
Ester with Microsomes
Time (mins) % Hydrolysis % Hydrolysis % Hydrolysis
Benzyl Ester n-Butyl Ester Isobutyl Ester
0 2.0 2.0 0.9
15 15 5.6 4.2
30 31 8.8 6.8
45 40 12 8.1
60 48 13 11
48


Table 3.11 (Cont.)
Ester with Microsomes
Time (mins) % Hydrolysis Ethyl Ester % Hydrolysis n-Propyl Ester
0 7.5 1.3
15 4.5 2.8
30 4.8 5.6
45 5.4 5.9
60 6.7 6.7
49


Figure 3.9: Hydrolysis of Glutathione Benzyl Ester with Microsomes
Benzyl Ester
D Microsomes
50 ,
I 40
o 30
| 20
^ 10
0
0 10 20 30 40 50 60
Time (mins)
Figure 3.10: Hydrolysis of Glutathione n-butyl Ester with Microsomes
n-butyl Ester
Control n Microsomes
50


Figure 3.11: Hydrolysis of Glutathione Isobutyl Ester with Microsomes
Isobutyl Ester
Control D Microsomes
50
.3 40
CO
o 30
J-l
rd
£20
* 10 -
0 -
0
10

D

[I
20
30
Time (mins)
40
50
60
Figure 3.12: Hydrolysis of Glutathione Ethyl Ester with Microsomes
50
s40
§ 30
u
nd
£20
^ 10
0
Ethyl Ester
Control D Microsomes

10 20 30 40
Time (mins)
50
60
51


Figure 3.13: Hydrolysis of Glutathione Propyl Ester with Microsomes
Propyl Ester
Control D Microsomes
52


Table 3.12: Concentration of Glutathione in Ester Samples with Cytosol
Blank
Time (mins) GSH (mM)
0 -0.0084
15 -0.0066
30 -0.011
45 -0.0055
60 -0.0080
Cytosol Blank
Time (mins) GSH (mM)
0 0.14
15 0.17
30 0.24
45 0.22
60 0.18
Ester Control
Time (mins) Benzyl Ester GSH (mM) n-Butyl Ester GSH (mM) Ethyl Ester GSH (mM) Propyl Ester GSH (mM)
0 0.31 0.26 0.56 0.12
15 0.35 0.26 0.49 0.15
30 0.35 0.29 0.42 0.16
45 0.39 0.36 0.38 0.17
60 0.36 0.39 0.35 0.19
53


Table 3.12: (Cont.)
Ester with Cytosol
Time (mins) Benzyl Ester GSH (mM) n-Butyl Ester GSH (mM) Ethyl Ester GSH (mM) Propyl Ester GSH (mM)
0 0.61 0.53 0.70 0.45
15 3.2 1.3 0.90 0.77
30 4.6 3.0 0.95 1.2
45 5.2 3.4 1.1 1.8
60 6.2 3.6 0.65 2.0
54


Table 3.13: Hydrolysis of Glutathione Esters with Cytosol
Ester Control
Time (mins) % Hydrolysis benzyl ester % Hydrolysis n-butyl ester % Hydrolysis ethyl ester % Hydrolysis propyl ester
0 3.9 3.2 7.0 1.5
15 4.4 3.2 6.1 1.8
30 4.3 3.6 5.3 2.0
45 4.9 4.6 4.8 2.2
60 4.5 4.8 4.4 2.4
Ester with Cytosol
Time (mins) % Hydrolysis benzyl ester % Hydrolysis n-butyl ester % Hydrolysis ethyl ester % Hydrolysis propyl ester
0 7.6 6.7 8.7 5.6
15 40 17 11 9.6
30 58 37 12 15
45 65 43 13 22
60 77 45 8.2 26
55


Figure 3.14: Hydrolysis of Glutathione Benzyl Ester with Cytosol
Benzyl Ester
Control D Cytosol
80 - ----------------------------------------------- ,
70
55 60
£ 50
-S 40
£30
S 20
10
0 '
0 10 20 30 40 50 60
Time (mins)
Figure 3.15: Hydrolysis of Glutathione n-butyl Ester
80
70
1 60
£ 50
'S 40
ffi 30
^ 20
10
0
0 10 20 30 40 50 60
Time (mins)
n-butyl Ester
Control d Cytosol
56


Figure 3.16: Hydrolysis of Glutathione Ethyl Ester with Cytosol
80
70
1 60
£ 50
'S 40
£ 30
^ 20
10
0
0 10 20 30 40 50 60
Time (mins)
Ethyl Ester
Control a Cytosol
Figure 3.17: Hydrolysis of Glutathione Propyl Ester with Cytosol
80-----------------------
70
I 60
-t 50
-S 40
£30
£ 20
10
0 >
0 10 20 30 40 50 60
Time (mins)
Propyl Ester
Control D Cytosol
57


In the microsomal experiments, the concentration of glutathione for the blank
solutions was approximately 0.0 mM for each time point, and demonstrated no
appreciable increase over the course of the experiments. The same was true for the
microsomal blank solutions. The cytosol blank solutions also had no definite increase
over time, but the concentration of glutathione was higher at approximately 0.2 mM.
The cytosol appears to have a small amount of residual glutathione that was not
removed during the dialysis step. The ester control samples for both the microsome and
the cytosol experiments ranged between 1% and 5%. Overall the control samples were
slightly higher for the cytosol samples, which could be due to day-to-day variability.
Previous preparations of glutathione esters have reported unreacted glutathione
remaining in the ester, following the esterification in amounts up to 5% which could
account for most the ethyl ester reactions (Anderson, 1985).
With the exception of the ethyl ester, all the esters had much greater percent
hydrolysis than the ester control. In both trials, the ethyl ester had a lower percent
hydrolysis for the ester samples in the presence of the microsomes than the other esters.
Conversely, the ethyl ester had a higher percent hydrolysis in the absence of the
microsome than the other esters.
In the presence of microsomes, the percent hydrolysis increased over time,
indicating the presence of esterases capable of aiding in the hydrolysis of each of the
esters. The rate of increase varies for each ester, though the n-butyl and isobutyl ester
had similar percent hydrolysis. After 60 minutes, the percent hydrolysis for the benzyl
ester had by far the highest increase. The benzyl ester results at 60 minutes were 35%
higher than n-butyl ester, which had the next largest percent hydrolysis. The propyl
ester and the ethyl ester had equivalent percent hydrolysis values; however, the ethyl
58


ester had a background of approximately four percent, which would mean that it only
grew to three percent over the control, whereas the propyl ester only had a blank of
approximately one percent. This is about a six percent increase over the ester control.
The higher reaction rate of the benzyl ester could be explained in several ways. The
benzyl group could he the substrate for the optimal operation of the esterase. Esterases
are in place to remove xenobiotic groups from cells. This particular esterase could
specifically target larger groups. Of the five esters examined, benzyl is the largest side
group, followed by the butyl group, which had the second highest percent hydrolysis.
The isobutyl ester results are similar to the butyl ester while the propyl and ethyl
groups, the two smaller groups, also had the lowest percent hydrolysis.
It could also indicate that multiple esterases are involved and the esterase suited to
attack benzyl ester could be present in much higher concentrations than the other
esterases. Or conversely, the results could also indicate that the benzyl ester can be
attacked by multiple esterases, while the other esters are only attacked by one specific
esterase or at least fewer esterases than the benzyl ester.
The results for the cytosol addition were more favorable than the microsome. The
percent hydrolysis grew for each of the samples over the microsome. The ethyl ester
again showed the lowest increase rising to a high of only 13%. At 60 minutes, the propyl
ester had increased to 26%, almost a 20% increase. The butyl ester and the benzyl ester
showed an approximately 30% increase over the microsomal results. Clearly, there
appears to be a greater number of esterases overall or a higher concentration of
esterases capable of hydrolyzing the glutathione esters in the cytosol. The mode of
attack in this case appears to be size related, the benzyl ester had the highest percent
hydrolysis followed by n-butyl ester, propyl ester and finally the ethyl ester. Although
59


the percent hydrolysis increased, the benzyl ester still shows about 30% more hydrolysis
than the butyl ester. Again, there is no clear indication of whether this is the result of
multiple enzymes or a single enzyme, though given the larger hydrolysis result for the
benzyl ester, it appears likely that multiple esterases are responsible.
3.4 Concentration Studies for Glutathione Benzyl Ester in the Presence of
Cytosol.
In order to characterize the enzymatic reaction, concentration studies of the
glutathione benzyl ester of glutathione in the presence of cytosol were conducted to
determine the Michaelis-Menten constant. The Michaelis-Menten equation relates
velocity of the reaction to substrate concentration.
V = (Vmax[S])/(Km+ [S]) Equation 3.1
V = Velocity
Vmax = The maximum initial velocity
[S] = Initial substrate concentration of the substrate
Km = Michaelis-Menten constant
The Michaelis-Menten equation graphically appears as a hyperbola. It is an idealized
situation that allows enzymes to be compared. Results will differ, depending upon the
temperature at which the analysis is performed and the substrate used for the analysis.
The maximum velocity represents the point at which additional substrate will not
increase the velocity for a given amount of enzyme.
The concentration studies were performed in duplicate at benzyl ester (GBZE)
concentrations of 8 mM, 12 mM, and 16 mM. Refer to Tables 3.14 3.15 for the average
60


of results. Refer to Figure 3.18 for a graphical representation of the data. The 8mM
percent hydrolysis data is comparable to the previous percent hydrolysis values for
benzyl ester. (Refer to Table 3.9). The buffer control has almost 0 mM glutathione, as is
to be expected since it possesses neither ester nor cytosol. The cytosol control has
concentration of glutathione of about 0.02 mM. However, neither the buffer nor the
cytosol control appeared to increase over time. The ester control had an average percent
hydrolysis of around 5%, and had a minute increase in percent hydrolysis over time.
The percent hydrolysis of the ester control remained fairly constant over the different
concentrations of benzyl ester, as the initial glutathione ester concentration studies
predicted.
61


Table 3.14: Concentration of Benzyl Ester Concentration Study Samples
Buffer Control
Time (mins) GSH (mM)
0 0.00073
15 0.00057
30 0.00020
45 -0.000058
60 0.00054
Cytosol Blank
Time (mins) GSH (mM)
0 0.017
15 0.020
30 0.019
45 0.020
60 0.022
Benzyl Ester Control
Time (mins) 8mM GBZE GSH (mM) 12 mM GBZE GSH (mM) 16 mM GBZE GSH (mM)
0 0.035 0.054 0.074
15 0.037 0.053 0.077
30 0.040 0.055 0.082
45 0.034 0.062 0.091
60 0.036 0.077 0.088
62


Table 3.14: Cont.
Benzyl Ester w/ Cytosol
Time (mins) 8mM GBZE 12 mM GBZE 16 mM GBZE
GSH (mM) GSH (mM) GSH (mM)
0 0.06 0.08 0.09
15 0.33 0.37 0.54
30 0.42 0.62 0.71
45 0.47 0.66 0.80
60 0.56 0.87 0.95
63


Table 3.15: The Hydrolysis of Benzyl Ester Concentration Study Samples
Benzyl Ester Control
Time (mins) 8 mM GBZE % Hydrolysis 12 mM GBZE % Hydrolysis 16 mM (GBZE) % Hydrolysis
0 4.4 4.5 4.6
15 4.7 4.4 4.8
30 5.0 4.6 5.1
45 4.3 5.2 5.7
60 4.5 6.4 5.5
Benzyl Ester w/ Cytosol
Time (mins) 8 mM GBZE 12 mM GBZE 16 mM (GBZE)
% Hydrolysis % Hydrolysis % Hydrolysis
0 7.5 6.4 5.6
15 41 31 34
30 53 52 45
45 59 55 50
60 70 73 60
64


Figure 3.18: Hydrolysis of Glutathione Benzyl Ester Concentration Studies
Benzyl Ester
8mM Control
o 8mM w/ Cytosol
* 12 mM Control
n 12 mM w/ Cytosol
6 16 mM Control
16 mM w/ Cytosol
80.0
70.0
60.0
t 50.0
o
I-40-0
w
30.0
20.0
10.0
0.0
0 10 20 30 40 50 60
Time (mins)
65


The percent hydrolysis results for the 8 mM, 12 mM, and the 16 mM concentrations
were similar in magnitude; however, the 16 mM had the lowest results. To determine
velocity, the change in concentration over time was determined. This can either he
expressed in relation to the amount of glutathione produced or the amount of benzyl
ester consumed. In this case, the latter was used, with velocity calculated as a negative
of the change in the concentration of the benzyl ester over time. Refer to Table 3.16 for
the raw data and Figure 3.19 for the graphical representation. The velocities thus
calculated are reported in Table 3.17.
Table 3.16: Change in Concentration of the Esters Over Time
Time Point 8 mM GBZE Final Cone. Ester (mM) 12 mM GBZE Final Cone. Ester (mM) 16 mM GBZE Final Cone. Ester (mM)
0 7.922 11.94 16.00
15 5.293 9.056 11.59
30 4.385 6.542 9.863
45 3.804 6.255 9.125
60 2.978 4.265 7.551
Figure 3.19: Change in Glutathione Benzyl Ester Concentration over Time
Change in Concentration of GSH Benzyl Ester over Time
66


Table 3.17: Velocity for the Benzyl Ester Concentration Study
Initial Ester Cone. (mM) Velocity (mM/min)
8 0.07586
12 0.1210
16 0.1291
Although the percent hydrolysis for the 16 mM benzyl ester solution was lower than the
8 mM or the 12 mM, the rate of the reaction was higher. The concentration of the esters
is reported with the background ester hydrolysis and the background cytosol hydrolysis
removed.
The Vmax and Km were calculated using the Lineweaver-Burk transformation to
convert the hyperbolic graph to a linear graph. For the Lineweaver-Burk
transformation, 1/V vs. 1/[S] is plotted where the y-intercept is 1/Vmax and the x-
intercept is -1/Km. Refer to Table 3.18 for the data and refer to Figure 3.20 for the 1/V
vs. 1/[S] plot. The concentrations were maintained at a relatively low level in order to
remain in the linear portion of the Michaelis-Menten curve. As with any method to
simplify the data, problems can arise. For instance, a relatively small error in the
measure of the velocity can be translated to a much larger error when the data is
converted to the reciprocal (Cornish-Bowden, 1995).
Table 3.18: 1/[S] and 1/V data
1/S (mM) 1/V (min/mM)
0.1250 13.2
0.08333 8.26
0.06250 7.75
67


Figure 3.20: 1/V vs. 1/[S]
1/V vs. 1/[S]
Calculated from the plot, Vmax is 0.68 mM/min while the value for Km is 62 mM. The
back extrapolation of the line to determine Vmax and Km introduces error into the
calculation. Therefore, the data should be considered in terms of magnitude instead of
as absolute numbers. Another way of defining Km relates to the following reaction
where E represents the enzyme, S the substrate and P the product:
E + S ES^E + P
k-i
Km is equivalent to the ratio of the rate constants. Km = (k-i + k2)/ki. Therefore, high
values of Km would indicate higher rates of k-i and/or k2 versus the ki. In this case, the
value for Km is high, indicating a relatively weak interaction between the substrate and
the enzyme. Either the formation of products occurs quickly, or the enzyme is rapidly
disassembled back to the starting products. However, given that product is being
68


formed, the reverse reaction does not occur exclusively. These results indicate that the
enzymatic attack appears to follow the Michaelis-Menten kinetics.
3.5 Conclusion
Based upon the data for the hydrolysis studies, the hydrolysis of the benzyl, propyl,
butyl, isobutyl, and ethyl glutathione esters is enzymatically produced. The percent
hydrolysis of the esters in the presence of the microsomes or the cytosol was clearly
higher than the blank solution, the ester control, and the cytosol/microsome control.
The cytosol results showed greater percent hydrolysis, however, the either due to
greater concentrations of esterases or due to the presence of esterases which had a
greater specificity to the esters. However, without knowing the specific concentrations
it is impossible to determine which of the assumptions is correct.
The initial data for the benzyl ester glutathione study would appear to indicate that
the esterase follows the Michaelis-Menten kinetics. The Km value of 62 mM was high.
Typically, low Km values denote a lower concentration of substrate in the cell. High Km
values, however, indicate the enzyme is prepared for a high concentration of substrate
which would be expected for an esterase designed to remove xenobiotics. However, this
is only preliminary. Additional research would need to be performed to separate
enzymes to establish which specific enzymes are responsible for hydrolysis of the esters.
Ultimately, with a better understanding of the enzyme kinetics involved in the
hydrolysis of glutathione esters, appropriate esters could be designed which would
maximize the therapeutic ability while using the minimal amount of glutathione ester,
so as to reduce potential side effects of the therapy. Perhaps in the future glutathione
69


treatment will represent an additional approach to dealing with radiation, xenobiotic
removal or even in the study of appropriate cancer treatments.
70


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Anderson, M. E., Naganuma, A., and Meister, A. (1990) FASEB 4: 3251-3254.
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Cooper, A.J.L. (1998) Role of Astrocytes in Maintaining Cerebral Glutathione
Homeostasis and in protecting the Brain Against Xenobiotics and Oxidative Stress. In
Glutathione in the Nervous System, ed., Shaw, C.A. 91-115. Washington: Taylor &
Francis.
Cornish-Bowden, A. (1995) Fundamentals of Enzyme Kinetics, Revised Ed. London:
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