ISOLATION AND PURIFICATION OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE FROM THE GUT OF RHINOCEROS LARVA (Oryctes rhinoceros)

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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

 

LIST OF FIGURES

 

Figure 1.1: Structure of 3-MST                                                            

8

Figure 1.2: Oryctes rhinoceros Larva                                                   

13

Figure 1.3: Life Cycle of Oryctes rhinoceros Larva                             

15

Figure 1.4: Palm Tree                                                                           

16

Figure 1.5: Decaying Palm Trunk                                                        

18

Figure 3.1: Graph Showing the Affinity of 3-MST Protein Activity 

41

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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