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Purification and Characterization of Rhodanese From the Liver of Mango Tilapia (Sarotherodon Galilaeus) Found in Erinle Reservoir, South West, Nigeria

Purification and Characterization of Rhodanese From the Liver of Mango Tilapia (Sarotherodon Galilaeus) Found in Erinle Reservoir, South West, Nigeria

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DEDICATION

This research material, titled “Purification and Characterization of Rhodanese From the Liver of Mango Tilapia (Sarotherodon Galilaeus) Found in Erinle Reservoir, South West, Nigeria” is dedicated to God for His boundless grace and guidance. It is also a tribute to all computer enthusiasts whose contributions made my research journey smoother and enriched my documentation process, making the experience truly fulfilling.



ACKNOWLEDGEMENT

I am profoundly grateful to everyone who contributed to the successful completion of this project. I am especially grateful to my Supervisor (Name), the Head of Department (Name), and the Lecturers in the Department of Biochemistry for their invaluable guidance and support. I also acknowledge the contributions of authors and scholars whose works on Purification and Characterization of Rhodanese From the Liver of Mango Tilapia (Sarotherodon Galilaeus) Found in Erinle Reservoir, South West, Nigeria provided essential insights. Special thanks go to my study area (and any funding organizations, if applicable) for their financial assistance. I am equally thankful to stakeholders, including mentors, teachers, and colleagues, for their encouragement and support. Finally, I deeply appreciate my family and friends for their patience and unwavering support throughout this journey. Your contributions have been instrumental in making this research a reality.



TABLE OF CONTENTS

PRELIMINARY PAGES

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

REFERENCES

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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.


Purification and Characterization of Rhodanese From the Liver of Mango Tilapia (Sarotherodon Galilaeus) Found in Erinle Reservoir, South West, Nigeria

CHAPTER ONE

1.1 Introduction

… As a prelude to other parts of this study, this chapter will discuss the background upon which this study was initiated, the statement of problems that led to this study, the Aim and Objectives of the study. Others are Significance of the study, Scope of work, Research hypothesis and questions, Limitation of the study and Definition of technical terms.…

CHAPTER TWO

2.0 Literature Review

2.1 Introduction

This chapter focuses on the review of related literature. A literature review includes the current knowledge as well as theoretical and methodological contributions to a particular topic. It documents the state of the art with respect to the topic you are writing. It surveys the literature in the topic selected. In this research work the literature review includes the conceputal review, theoretical framework, the review of related literature …

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