ABSTRACT
Cyanide is known to be one of the
most toxic substances present in a wide variety of food materials that are
consumed by animals.
One of the cyanide detoxifying
enzymes is 3-mercaptopyruvate sulfurtransferase (3-MST). Indeed, recent studies
have clearly shown that 3MST is involved in the detoxification of cyanide.
Rhinoceros
(Oryctes rhinoceros) larva feeds
on dead, decayed and living plants, wood and palm. Plants are known to contain
cyanide as a defence mechanism for intruding/pesting organisms. Thus, for rhinoceros larva to be able to live on
plants, it must have possessed a cyanide-detoxifying enzyme.
3-MST, a cyanide-detoxifying enzyme
was purified from Rhinoceros (Oryctes rhinoceros) larva in this work.
The 3-MST enzyme was isolated from the gut
of Oryctes rhinoceros larvae and
purified using Ammonium Sulphate Precipitation, Bio-Gel-P-100 Gel Filtration
Chromatography and Reactive Blue-2-Agarose Affinity chromatography.
The specific activity of the enzyme
was 0.22U/mg.
The presence of this enzyme could be
exploited by including it in the diet of animals which would serve as a source
of protein and 3-MST. Perhaps, these rhinoceros
larva could be introduced on farmland with contaminated soil whereby they
will process the dead roots and plants into soil thereby providing more space
and manure for plants to grow healthy.
TABLE OF CONTENTS
Contents Pages
Table
of Content v
List
of Figures vi
List
of Tables vii
Abstract
viii
Chapter One
1.0. Introduction and Literature
Review 1
1.1. Introduction 1
1.2. 3-Mercaptopyruvate Sulfurtransferase 2
1.2.1. Distribution
of 3-MST 5
1.2.2. Occurrence
of 3-MST 5
1.2.3. Mechanisms
of Action 6
1.2.4. Molecular
Formula and Molecular Weight 7
1.2.5. Structure
of 3-MST 8
1.2.6. Amino
Acid Composition of 3-MST 8
1.2.7. Catalytic
Activity of 3-MST 9
1.2.8. Enzyme
Regulation of 3-Mercaptopyruvate sulfurtransferase 9
1.2.9. Stability
of 3-MST 9
1.3. Physicochemical Properties of 3-MST 9
1.3.1. Optimal
Temperature of 3-MST 9
1.3.2. Optimum
pH of 3-MST 10
1.3.3. Effect
of Metals/ions on 3-MST 10
1.3.4. Specific
Activity of 3-MST 10
1.3.5. Inhibitory
Studies of 3-MST 10
1.4. Cyanide 11
1.5. Oryctes
rhinoceros Larvae 13
1.5.1. Taxonomy of Oryctes rhinoceros
1.5.2. Nutritional
Qualities of Rhinoceros Larvae 13
1.5.3. Life
Cycle of the Rhinoceros larva 15
1.5.4. Damage
16
1.5.5. Natural
Enemies 16
1.5.6. Management
17
1.6. The Gut 18
1.7. Oryctes
rhinoceros 19
1.7.1. Description
of Development Stages 19
1.7.2. Distribution
of Oryctes rhinoceros 21
1.7.3. Hosts/Species
Affected 21
1.7.4. Economic
Importance 23
1.8. Purification of 3-MST 27
1.9. Justification of Studies 28
1.10. Objectives
of Research 28
Chapter Two
2.0. Materials And Methods 29
2.1. Materials 29
2.1.1. Reagents
29
2.1.2. Apparatus
Used 29
2.1.3. Study
Sample 30
2.2. Method 30
2.2.1. Preparation
of Buffer and Reagents 30
2.2.1.1. Preparation
of 0.25M Potassium Cyanide 30
2.2.1.2. Preparation
of 0.5M Potassium Cyanide 30
2.2.1.3. Preparation
of 38% Formaldehyde 30
2.2.1.4. Preparation
of 0.25M Ferric Nitrates (Sorbo Reagent)
31
2.2.1.5. Preparation
of Bradford Reagent 31
2.2.1.6. Preparation
of 0.38M Tris-HCl Buffer 31
2.2.1.7. Preparation
of 0.30M Mercaptoethanol 31
2.2.2. Preparation
of Crude Extract from the rhinoceros larva
gut 32
2.2.3. Protein
Concentration Determination 33
2.2.4. Assay
for 3-Mercaptopyruvate Sulfurtransferase
34
2.2.5. Enzyme
Purification 35
2.2.6. Substrate
Specificity 37
Chapter Three
3.0. Results
Chapter Four
4.0. Discussion,
Conclusion and Recommendation 52
4.1. Discussion 52
4.2. Conclusion 52
4.3. Recommendation 52
References 53
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LIST OF FIGURES
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Figure
1.1: Structure of 3-MST
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8
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Figure
1.2: Oryctes rhinoceros Larva
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13
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Figure
1.3: Life Cycle of Oryctes rhinoceros Larva
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15
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Figure
1.4: Palm Tree
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16
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Figure
1.5: Decaying Palm Trunk
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18
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Figure 3.1: Graph Showing
the Affinity of 3-MST Protein Activity
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41
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Figure
3.2: Graph Showing Gel-Filtration of 3-MST Protein Activity
LIST OF TABLES
Table 2.1: Protein Assay Using Bradford Method 33
Table
2.2: Assay for 3-Mercaptopyruvate
Sulfurtransferase
Table 3.1: Purification Table
CHAPTER ONE
1.0. INTRODUCTION AND LITERATURE REVIEW
1.1. INTRODUCTION
One of the major
metabolic enzymes that have gained so much interest of scientists is
3-Mercaptopyruvate sulfurtransferase (3-MST). This enzyme occurs widely in
nature (Bordo, 2002 and Jarabak, 1981).
It has been reported
in several organisms ranging from humans to rats, fishes and insects. It is a
mitochondrial enzyme which has been concerned in the detoxification of cyanide,
a potent toxin of the mitochondrial respiratory chain (Nelson et al., 2000). Among the several
metabolic enzymes that carry out xenobiotic detoxification, 3-mercaptopyruvate
sulfurtransferase is of utmost importance.
3-mercaptopyruvate
sulfurtransferase functions in the detoxifications of cyanide; mediation of
sulfur ion transfer to cyanide or to other thiol compounds. (Vanden et al., 1967). It is also required for
the biosynthesis of thiosulfate. In combination with cysteine aminotransferase,
it contributes to the catabolism of cysteine and it is important in generating
hydrogen sulphide in the brain, retina and vascular endothelial cells (Shibuya et al., 2009). It also acquired
different functions such as a redox regulation (maintenance of cellular redox
homeostasis) and defense against oxidative stress, in the atmosphere under oxidizing
conditions Nagahara et al (2005).
Hydrogen sulphide (H2S)
is an important synaptic modulator, signalling molecule, smooth muscle
contractor and neuroprotectant (Hosoki et
al., 1997). Its production by the 3-mercaptopyruvate sulfurtransferase and
cysteine aminotransferase pathways is regulated by calcium ions (Hosoki et al., 1997).
Organisms that are
exposed to cyanide poisoning usually have this enzyme in them. This could be in
food as in the cyanogenic glucosides being consumed. It has been studied from
variety of sources, which include bacteria, yeasts, plants, and animals (Marcus
Wischik, 1998).
Cyanide could be
released into the bark of trees as a defence mechanism. There are array of
defensive compounds that make their parts (leaves, flowers, stems, roots and
fruits) distasteful or poisonous to predators. In response, however, the
animals that feed on them have evolved over successive generations a range of
measures to overcome these compounds and can eat the plant safely. The tree
trunk offers a clear example of the variety of defences available to plants
(Marcus Wischik, 1998).
Oryctes rhinoceros larva is one of the
organisms that are also exposed to cyanide toxicity because of the environment
they are found.
1.2. 3-MERCAPTOPYRUVATE SULFURTRANSFERASE
3-Mercaptopyruvate
sulfurtransferase (EC. 2.8.1.2), is a member of the group, Sulfurtransferases
(EC 2.8.1.1 – 5), which are widely distributed enzymes of prokaryotes and
eukaryotes (Bordo and Bork, 2002).
3-Mercaptopyruvate
Sulfurtransferase is an enzyme that is part of the cysteine catabolic pathway.
The enzyme catalyzes the conversion 3mercaptopyruvate to pyruvate and H2S
(Shibuya et al., 2009). The
deficiency of this enzyme will result in elevated urine concentrations of
3-mercaptopyruvate as well as of 3-mercaptolactate, both in the form of
disulfides with cysteine (Crawhall et al.,
1969). It catalyzes the chemical reaction:
3-mercaptopyruvate + cyanide à pyruvate +
thiocyanate
3-mercaptopyruvate + thiol à pyruvate + hydrogen sulphide (Sorbo 1957).
It transfers
sulfur-containing groups and participates in cysteine metabolism (Shibuya et al., 2013). This enzyme catalyzes the
transfer of sulfane sulphur from a donor molecule, such as thiosulfate or 3-
mercaptopyruvate, to a nucleophile acceptor, such as cyanide or mercptoethanol.
3-mercaptopyruvate is the known sulphur-donor substrate for 3-mercaptopyruvate
sulfurtransferase (Porter &
Baskin, 1995).
3-mercaptopyruvate
sulfurtransferase is believed to function in the endogenous cyanide (CN)
detoxification system because it is capable of transferring sulphur from
3-mercaptopyruvate (3-MP) to cyanide (CN), forming the less toxic thiocyanate
(SCN) (Hylin and Wood, 1959). It is an important enzyme for the synthesis of
hydrogen sulphide (H2S) in the brain (Shibuya et al., 2009).
The systematic
name of this enzyme class is 3-mercaptopyruvate:
cyanide sulfurtransferase. It is also called beta-mercaptopyruvate sulfurtransferase (Vachek and Wood, 1972). It is one of three known H2S
producing enzymes in the body (Hylin and Wood, 1959). It is primarily localised
in the mitochondria (Cipollone et al., 2008).
The expression
levels of 3-MST in the brain during the fetal and postnatal periods are higher
than those in the adult brain (unpublished data) although the promoter region
shows characteristics of a typical housekeeping gene (Nagahara et al., 2004). The observation is
supported by the finding that 3-MST expression in the cerebellum is decreased
during the adult period (Shibuya et al.,
2013). On the other hand, its expression level in the lung decreases from the
perinatal period. These facts suggest that 3-MST could function in the fetal
and postnatal brain. It was reported that serotonin signaling via the 5-HT1A
receptor in the brain during the early developmental stage plays a critical
role in the establishment of innate anxiety during the early developmental
stage (Richardson-Jones et al.,
2011).
In rat, 3-MST
possesses 2 redox-sensing molecular switches (Nagahara and Katayama, 2005). A
catalytic-site cysteine and an intersubunit disulfide bond serve as a
thioredoxin-specific molecular switch (Nagahara et al., 2007). The intermolecular switch is not observed in
prokaryotes and plants, which emerged into the atmosphere under reducing
conditions (Nagahara, 2013). As a result, it acquired different functions such
as a redox regulation (maintenance of cellular redox homeostasis) and defense
against oxidative stress, in the atmosphere under oxidizing conditions
(Nagahara et al., 2005).
Moreover, 3-MST can
produce H2S (or HS−) as a biofactor (Shibuya et al., 2009), which cystathionine
β-synthase and cystathionine γ-lyase also can generate (Abe and Kimura, 1996).
Interestingly 3-MST can uniquely produce SOx in the redox cycle of
persulfide formed at the low-redox catalytic-site cysteine (Nagahara et al., 2012). As an alternate
hypothesis on the pathogenesis of the symptoms, H2S (or HS−)
and/or SOx could suppress anxiety-like behavior, and therefore,
defects in these molecules could increase anxiety-like behavior. However, no
microanalysis method has been established to quantify H2S (or HS−)
and SOx at the physiological level (Ampola et al., 1969).
MCDU was first
recognized and reported in 1968 as an inherited metabolic disorder caused by
congenital 3-MST insufficiency or deficiency. Most cases were associated with
mental retardation (Ampola et al,
1969) while the pathogenesis remains unknown.
Human MCDU was
reported to be associated with behavioral abnormalities, mental retardation
(Crawhall, 1985), hypokinetic behaviour, and grand mal seizures and anomalies
(flattened nasal bridge and excessively arched palate) (Ampola et al, 1969); however, the pathogenesis
has not been clarified since MCDU was recognized more than 40 years ago.
Macroscopic anomalies were associated in 1 case (Ampola et al, 1969); however, this could be an accidental combination.
3-MST deficiency also induced higher brain dysfunction in mice without
macroscopic and microscopic abnormalities in the brain. 3-MST seems to play a
critical role in the central nervous system, i.e., to establish normal anxiety
(Richardson et al., 2011)
1.2.1. DISTRIBUTION
3-MST is widely
distributed in prokaryotes and eukaryotes (Jarabak, 1981). It is localized in the cytoplasm and
mitochondria, but not all cells contain 3-MST (Nagahara et al., 1998).
1.2.2. OCCURRENCE
Human
mercaptopyruvate sulfurtransferase (MPST; EC. 2.8.1.2) belongs to the family of
sulfurtransferases (Vanden et al.,
1967). These enzymes catalyze the transfer of sulfur to a thiophilic acceptor
(Sorbo 1957), where MPST has a preference for 3-mercapto sulfurtransferase as
the sulfur-donor. MPST plays a central role in both cysteine degradation and
cyanide detoxification. In addition, deficiency in MPST activity has been proposed
to be responsible for a rare inheritable disease known as
mercaptolactate-cysteine disulfiduria (MCDU) (Hannestad et al, 2006).
1.2.3. MECHANISMS OF ACTION
3-Mercaptopyruvate
sulfurtransferase catalyzes the reaction from mercaptopyruvate (SHCH2C
(= O) COOH)) to pyruvate (CH3C (= O) COOH) in cysteine catabolism
(Vackek and Wood, 1972). The enzyme is widely distributed in prokaryotes and
eukaryotes (Jarabak, 1981).
This disulfide bond
serves as a thioredoxin-specific molecular switch. On the other hand, a
catalytic-site cysteine is easily oxidized to form a low-redox potential
sulfenate which results in loss of activity (Nahagara et al., 2005). Then, thioredoxin can uniquely restore the activity
(Nagahara, 2013).
Thus, a catalytic
site cysteine contributes to redox-dependent regulation of 3-MST activity
serving as a redox-sensing molecular switch (Nahagara, 2013). These findings
suggest that 3-MST serves as an antioxidant protein and partly maintain
cellular redox homeostasis. Further, it was proposed that 3-MST can produce
hydrogen sulphide (H2S) by using a persulfurated acceptor substrate
(Shibuya et al, 2009).
As an alternative
functional diversity of 3-MST, it has been recently demonstrated in-vitro that
3-MST can produce sulfur oxides (SOx) in the redox cycle of
persulfide (S-S-) formed at the catalytic site of the reaction intermediate
(Nagahara et al, 2012).
1.2.4. MOLECULAR FORMULA AND
MOLECULAR WEIGHT
The
molecular formula of 3-MST is C3H4O3S (Vachek
and Wood, 1972).
3-MST has a molecular weight of
120.127g/mol or 23800 Daltons (as summarized by PubChem compound).
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