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PURIFICATION AND CHARACTERIZATION OF RHODANESE FROM THE LIVER OF MANGO TILAPIA (Sarotherodon galilaeus) FOUND IN ERINLE RESERVOIR, SOUTH WEST, NIGERIA

ABSTRACT

Rhodanese, a ubiquitous enzyme, catalyzes the detoxification of cyanide by sulphuration reaction. Cyanide is a highly toxic substance and an ineffective detoxification of this substance may lead to inhibition of respiration, production of ATP and its dependent processes, liver damage and necrosis in aquatic organisms. Considering the habitat of this fish which is a reservoir, it is exposed to toxic substances and waste products such as cyanide, ammonia etc. If the fishes can survive despite being exposed to cyanide, then we can assume that the fish has cyanide-detoxifying mechanism and enzyme. In this research, rhodanese was purified and characterized from the liver of Sarotherodon galilaeus using ammonium sulphate precipitation and Reactive blue-2-agarose affinity chromatography. The specific activity of the enzyme was found to be 121RU per milligram of protein. The optimum pH of the enzyme was found at pH 8.5 while the optimum temperature was 40°C. The Km values of the substrates; KCN and Na2S2O3 were 40mM and 11.76mM respectively. Studies on the effect of cations on the enzyme showed that the activity of the enzyme was not affected by Mn2+and Ni2+., however, 0.01mM concentration of Mg2+, Ca2+, and Zn2+ inhibited the enzyme considerably. The results of this research showed that there is activity of the enzyme; rhodanese in the liver of the fish and it implies that the organism has a cyanide-detoxifying mechanism. Thus, a prolonged exposure to cyanide will overwhelm the detoxifying mechanism eventually leading to liver damage and inhibition of respiration. Also, at very high temperature (above 70°C) and pH, the enzyme is inactivated.

TABLE OF CONTENTS

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW

CHAPTER ONE                                                                                                                 

1.0   INTRODUCTION AND LITERATURE REVIEW                                           

1.1         Occurrence and Distribution

1.2         Assay Method

1.3       Methods of Purification

1.4       Structural properties of rhodanese

1.4.1        Molecular Weight

1.4.2       Amino Acid Composition

1.4.3       Amino Acid Sequence

1.4.4     Tertiary Structure of Rhodanese

1.4.5       Active site of Rhodanese

1.4.6     Mechanism of Action of rhodanese

1.5       Physical and Catalytic properties of rhodanese

1.5.1   Isoelectric point

1.5.2     Effect of pH

1.5.3     Effect of Temperature

1.5.4       Substrate specificity

1.5.5     Turnover number

1.5.6     Inhibition

1.5.7     Effect of Sulphyldryl reagent

1.6      Cloning of rhodanese gene

1.7     Biological importance of rhodanese

1.8     Background information

1.9     Specific Aims of research

1.10   Justification of research

CHAPTER TWO

2.0        MATERIALS AND METHODS                                                           

2.1         Materials

2.2          Methods

2.2.1       Preparation of Buffer and Reagents

2.2.1.1   Preparation of Borate buffer

2.2.1.2   Preparation of 15% formaldehyde from 40% formaldehyde

2.2.1.3   Preparation of sorbo reagent

2.2.1.4   Preparation of Bradford reagent

2.2.1.5   Preparation of 0.25M KCN

2.2.2       Enzyme source

2.2.3    Isolation and Purification of the enzyme

2.2.3.1   Homogenization

2.2.3.2   Ammonium sulphate precipitation

2.2.3.3   Reactive Blue-2-Agarose Affinity chromatography

2.2.4     Enzyme assay

2.2.5      Protein determination

2.2.6       Determination of kinetic parameters

2.2.7       Effect of pH on enzyme activity

2.2.8       Effect of Temperature on enzyme activity

2.2.9       Effect of Cations on enzyme

CHAPTER THREE

RESULTS                                                                                                                

3.1       Purification of Rhodanese

3.2       Kinectic Parameters

3.3       Substrate Specificity

3.3.1    Effects of pH and temperature on enzyme activity

3.3.2    Effects of Cations

CHAPTER FOUR

4.0          Discussion and conclusion

4.1         Discussion

4.2         Conclusion

4.3          Recommendation