Purification and characterization of rhodanese from the liver of mango tilapia (sarotherodon galilaeus) found in erinle reservoir, south west, nigeria
Table Of Contents
- AND LITERATURE REVIEWCHAPTER ONE
- 1.0 INTRODUCTION AND LITERATURE REVIEW
- 1.1 Occurrence and Distribution1.2 Assay Method1.3 Methods of Purification1.4 Structural properties of rhodanese1.
- 4.1 Molecular Weight1.
- 4.2 Amino Acid Composition1.
- 4.3 Amino Acid Sequence1.
- 4.4 Tertiary Structure of Rhodanese1.
- 4.5 Active site of Rhodanese1.
- 4.6 Mechanism of Action of rhodanese1.5 Physical and Catalytic properties of rhodanese1.
- 5.1 Isoelectric point1.
- 5.2 Effect of pH1.
- 5.3 Effect of Temperature1.
- 5.4 Substrate specificity1.
- 5.5 Turnover number1.
- 5.6 Inhibition1.
- 5.7 Effect of Sulphyldryl reagent1.6 Cloning of rhodanese gene1.7 Biological importance of rhodanese1.8 Background information1.9 Specific Aims of research1.10 Justification of researchCHAPTER TWO2.0 MATERIALS AND METHODS
- 2.1 Materials2.2 Methods2.
- 2.1 Preparation of Buffer and Reagents2.2.
- 1.1 Preparation of Borate buffer2.2.
- 1.2 Preparation of 15% formaldehyde from 40% formaldehyde2.2.
- 1.3 Preparation of sorbo reagent2.2.
- 1.4 Preparation of Bradford reagent2.2.
- 1.5 Preparation of 0.25M KCN2.
- 2.2 Enzyme source2.
- 2.3 Isolation and Purification of the enzyme2.2.
- 3.1 Homogenization2.2.
- 3.2 Ammonium sulphate precipitation2.2.
- 3.3 Reactive Blue-2-Agarose Affinity chromatography2.
- 2.4 Enzyme assay2.
- 2.5 Protein determination2.
- 2.6 Determination of kinetic parameters2.
- 2.7 Effect of pH on enzyme activity2.
- 2.8 Effect of Temperature on enzyme activity2.
- 2.9 Effect of Cations on enzymeCHAPTER THREERESULTS
- 3.1 Purification of Rhodanese3.2 Kinectic Parameters3.3 Substrate Specificity3.
- 3.1 Effects of pH and temperature on enzyme activity3.
- 3.2 Effects of CationsCHAPTER FOUR4.0 Discussion and conclusion4.1 Discussion4.2 Conclusion4.3 RecommendationREFERENCES.
Thesis 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.
Thesis Overview
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