THE EFFECT OF HYDROGEN PEROXIDE ON PEROXIDASE (EC 1.11.1.7) FROM GONGRONEMA LATIFOLIUM

ABSTRACT
Peroxidase (EC 1.11.1.7) extracted from Gongronema latifolium was purified, on a two-step purification process of ammonium sulphate precipitation followed by dialysis. The enzyme was purified 6.8 fold with a specific activity of 2.04 when o-dianisidine was used as substrate. When the enzyme was subjected to different concentrations of hydrogen peroxide and o-dianisidine, the peak activity was 17.75µ/ml at 5mM for hydrogen peroxide and for o-dianisidine the peak activity was 2.4µ/ml observed at 0.4mM. The optimum pH and temperature were at pH 7.0 and 30oC respectively. The Km and Vmax for hydrogen peroxide were 1.8mM and 20u/ml and o-dianisidine had Km of 0.12mM and Vmax of 3.3 µ/ml. The inactivation of peroxidase extracted from Gongronema latifolium by hydrogen peroxide was time dependent and it also showed a biphasic inactivation curve with the initial fast phase and a slower second phase. About 20% protection of the enzyme against inactivation was obtained when 1mM ascorbate was incubated in all the concentrations of hydrogen peroxide while o-dianisidine had above 15% in all the concentrations. Spectral studies, indicated the peak at soret band as 381 nm for the native enzyme, and when the enzyme was incubated with hydrogen peroxide, there was a shift in the soret band of the enzyme from 381nm to 389nm. Increases in the concentration of hydrogen peroxide lead to decreases in the absorbance peak at the soret band of the enzyme and also reduction of size of Soret band. There were elevations in the absorbance peak when1mM ascorbate and 0.4mM o-dianisidine were incubated with the enzyme at different concentrations of hydrogen peroxide

Table of Contents
Title page…………………..………………………………………………………………………i
Apporval page……………………………………………………………………………………..ii
Dedication…………………………………………………………………………………………iii
Acknowledgement………………………………………………………………………………..iv
Asbtract…………………………………………………………………………………v
Table of contents………………………………………………………………………………….vi
List of figures……………………………………………………………………………………..ix
List of tables………………………………………………………………………………………xi
CHAPTER ONE: INTRODUCTION
1.0 Introduction……………………………………………………………………………,,……..1
1.1Peroxidases…………………………………………………………………………………….2
1.1.2.Functional roles……………………………………………………………2
1.2 The structure of peroxidase………………………………………………………………2
1.2.1 The description of peroxidase (horseradish)…………………3
1.2.2 Three-dimensional representation of x-ray crystal structure of HRP………………….…….8
1.3 Mechanisms of oxidation of peroxidase…………………..………….9
1.3.1 Mechanisms of oxidation of indole-3-acetic acid with peroxidase………………………….9
1.3.2 Mechanism of oxidation of small phenolic substrates (ferulic acid) with peroxidase……..10
1.4 Classes of peroxidases………………………………….13
1.5 Plant peroxidase………………………………………14
1.5 .1 Plant peroxidases………………………………….14
1.5.2 Functions of plant peroxidase…………………………..14
1.5.3 Fuctions of peroxidase in pharmacology and fine chemistry………………………………15
1.5.4 The use of peroxidase for wastewater treatment………………………17
1.5.5 The use of peroxidase in textile industry……………………..18
1.5.6 The use of peroxidase in the dairy industry………………..18
1.6 Substrates ……………………………………………………………………………………19
1.7 Factors that affect peroxidase activity……………………….21
1.7.1 pH……………………………………………….21
1.7.2 Temperature………………………………………………..22
1.8 Inhibition and inhibitors of peroxidase……………………………22
1.8.1 Inhibitor of peroxidases/ peroxidase suppressor………………..23
1.9 Inactivation of the enzyme………………………………………..23
1.9.1 Inactivation of peroxidase……………………………..……..23
1.9.2 Inactivation of peroxidase by hydrogen peroxidase……………….23
1:10 Spectral studies……………….……………………….25
1:10:1 Spectral properties of protein…………………….25
1:10:2 Spectral properties of peroxidase ………………………..26
1.11 Gongronema latifilium (utazi)………………………………………………28
1.11.1Gongronema latifilium (utazi)………………….28
1.11.2 The physilogical properties of Gongronema latifolium plant…………………………….29
1.11.3 Chemical composition of Gongronema latifolium……….……31
1.11.4 Microbal studies on the Gongronema latifolium………….……31
1.11.5 Phytochemical compositions of Gongronema latifolium……………32
1.11.6 The uses of Gongronema latifilium………………………………………………………….. 32
1.12 Aim of study………………………………………………………………………………..33
1.13 Objective of the study……………………………………………………………………33
CHAPTER TWO : MATERIALS AND METHOD
2.0 Material and method
2.1 Material
2.1.1 Sample collection and location……………………34
2.1.2 Apparatus and instruments…………………….………..34
2.1.3Chemicals and reagents…….………………………34
2.2 Methods……………………………………………….… ..35
2.2.1 Preparation of buffer solutions……………………………..35
2.2.2 Measurement of protein contents.…………………………36
2.2.3 Extraction of enzyme (peroxidase)………………………………36
2.2.3.1 Preparation of enzyme extract………….………………….…….36

2.2.3.2 Preparation of substrate solution………………………………………….37
2.2.3.3 Peroxidase assay using o-dianisidine as substrate…….….37
2.2.3.4 Assay for peroxidase activity in G. latifolium using guaiacol…………..38
2.2.4 Purification of peroxidase from Gongronema latifolium………………….39
2.2.4.1 Ammonium sulphate precipitation…………………….………….……39
2.2.4.2 Dialysis…………………………………………………………………………………..39
2.2.5 Effect of H2O2 on peroxidase activity……………..…………….39
2.2.6 Effect of o-dianisidine on peroxidase activity.……………39
2.2.7 Characterization of the enzyme…………………………40
2.2.7.1 Determination of optimum pH………………………………….40
2.2.7.2 Temperature……….……………………….…………40
2.2.8 Inactivation of peroxidase by hydrogen peroxide………………………………………….40
2.2.9 The absorption spectrum of inactivation of peroxidase by different concentrations of hydrogen peroxide ………………………40
CHAPTER THREE : RESULTS
3.0 Results……………………………………………………………………………………..…41
3.1 Purification of peroxidase………………………………41
3.2: The effect of different concentration of o-dianisidine on peroxidase activity……….….…. 43
3.3 The effect of different concentrations of hydrogen peroxide on peroxidase activity…..……42
3.4: Study on optimum pH ………….………………………..……..44
3.5: Study on optimum temperature ……………………………….….45
3.6: Variation of peroxidase activity with different concentrations of hydrogen peroxide..…….46
3.7: Determination of Km and Vmax………………………………………….47
3.8: Variation of peroxidase activity with varing concentrations of o-dianisidine………………48
3.9: The Lineweaver-Burk plot of effect of different concentration of o-dianisidine on peroxidase activity………………………………..….49
3.10: The inactivation of peroxidase by hydrogen peroxide…………50
3.11: The comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by hydrogen peroxide (0.1 to 10mM) ……………………………………………..…51
3.11.1: Comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by hydrogen peroxide (0.1mM) …………………………………………………….51
3.11.2: Comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by hydrogen peroxide ( 1mM) …………………………………………………..…..52
3.11.3: Comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by hydrogen peroxide (5mM) ………………………………..………………………53
3.11.4: Comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by hydrogen peroxide ( 7.5mM) …………………………………….……………….54
3.11.5: Comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by hydrogen peroxide ( 10mM) ……………………………………………………..55
3.12.1: Spectral studies of native peroxidase…………………………….56
3.12.2: Spectral properties of inactivation of peroxidase by different concentrations of hydrogen peroxidase………………………..57
3.12.3: Spectral properties of the protective effect of ascorbate on inactivation of peroxidase by different concentrations of hydrogen peroxidase………………………………………….……..58
3.12.4: Spectral of the protective effect of o-dianisidine on inactivation of peroxidase by different concentrations of hydrogen peroxide…………………………………………………..59

CHAPTER FOUR: DISCUSSION

4.1 Discussion……………………………………………………………………………………60
4.2 Conclusions……………………………………………………………………………….…64
References……………………………………………………………………………………….65
Appendix1: Values for protein standard curve…………………….81
Appendix 2; Graph of protein standard curve……………………..82
Appendix3: Percentage residual activity of inactivation of peroxidase by different concentrations of hydrogen peroxide …………………………………83
Appendix4: Percentage residual activity of protective effect of ascorbate against inactivation of peroxidase by different concentrations of hydrogen peroxide………………….………………. 83
Appendix4: Percentage residual activity of protective effect of o-dianisidine against inactivation of peroxidase by different concentrations of hydrogen peroxide…………………………………84

LIST OF FIGURES
Figure 1: Catalytic cycle of peroxidase….……………………..…………2
Figure 2: Haem component of (HRPC) ……………………………..4
Figure 3: Calcium ions component of (HRPC) …………………….4
Figure 4: Carbohydrate component of (HRPC) …………….……….………5
Figure 5: Key amino acid residues in the haem-binding region of HRPC………………….……..7
Figure 6: Three –dimensional representation of the x-ray crystal structure of HRP …..….……….8
Figure 7: A mechanism proposed for the formation of 3-methylene-2-oxindole from horseradiperoxidase (HRP C) and indole-3-acetic acid…………………………….10
Figure 8: Proposed mechanism for substrate oxidation in plant peroxidases……………………12
Figure 9: The diverse function and role of class lll peroxidase……………………….…………15
Figure 10: Structure of reduced and oxidized guaiacol…………………………20
Figure 11: Structure of a reduced o-dianisidine and oxidized o-dianisidine…………………….21
Figure 12: Effect of different concnetration of o-dianisidine on peroxidase activity……………43
Figure 13: Effect of different concentrations of hydrogen peroxide on peroxidase activity….…42
Figure 14:Effect of pH on peroxidase activity…………………44
Figure 15:Temperature dependence of peroxidase activity………….….……….45
Figure 16: Michaelis-Menten plot of hydrogen peroxidase .…………..…46
Figure 17: Lineweaver-Burk plot of peroxidase activity on H2O2………………………………47
Figure 18: Michaelis-Menten plot of o-dianisidine…………………….………….48
Figure 19: Lineweaver-Burk plot of o-dianisidine….……………………49
Figure 20: Inactivation of peroxidase by hydrogen peroxide……..………50 Figure 21a: Comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by 0.1mM of hydrogen peroxide………………….51
Figure 21b: Comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by 1mM of hydrogen peroxide52
Figure 21c: Comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by 5mM of hydrogen peroxide……….……………..53
Figure 21d: Comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by 7.5 mM of hydrogen peroxide………………………………………..……………54
Figure 21e: Comparison of the protective effect of ascorbate and o-dianisidine on inactivation of peroxidase by 10mM of hydrogen peroxide..……………………………………………………55
Figure 22a: Spectral properties of native peroxidas……………………56
Figure 22b: Spectral studies of inactivation of peroxidase by hydrogen peroxide…..………….57
Figure 22c: Spectral studies of protective effect of ascorbate agianst inactivation by hydrogen peroxide…………………………………..58
Figure 22d: Spectral studies of the protective effect of o-dainisidine against inactivation of peroxidase by hydrogen peroxide………………………………………………………………..59

LIST OF TABLES

Table 1: Classification of peroxidases …….……………………13
Table 2: Various substrates that can react with peroxide and their respective products….…….19
Table 3: The Absorption maximum of the aromatic amino acid…………………………………25
Table 4: Absorption maxima for intestinal peroxidase, lacto-peroxidase, eosinophil peroxidase and myeloperoxidase with their derivatives……….28
Table 5: Phytochemical and anti-nutrient content of Gongronema latifolium…………………..32
Table 6: Purification table…………………………….41

CHAPTER ONE
INTRODUCTION
The super-family of haem peroxidases from plants, fungi and bacteria is a group of enzymes that utilize hydrogen peroxide to oxidize a second (reducing) substrate often aromatic oxygen donor. These enzymes share similar catalytic cycles where hydrogen peroxide reacts with the resting ferric enzyme to form the intermediate compound I (known as compound ES in cytochrome c peroxidase) which carries two oxidizing equivalents. Compound I is subsequently reduced by reactions with two reducing substrate molecules. The reaction of these reduction steps generate the intermediate, compound II, which is then further reduced back to the ferric enzyme (Hiner et al.,2000). Peroxidase forms part of the defense system of living organisms against radical-mediated peroxidation of unsaturated lipids. They are ubiquitous in nature and are involved in various physiological processes in plants. Studies have suggested that peroxidases play a role in lignification, suberization, cross-linking of cell wall structural protein, auxin catabolism and self –defense against pathogens and senescence (Hiraga et al., 2001). Currently, industrial application of peroxidase in chemistry, pharmacology and biotechnology is well developed. Peroxidase is used in waste treatment in order to remove aromatic phenols and amine from aqueous solution in the presence of hydrogen peroxide. In this treatment, phenolic compounds are polymerized in the presence of hydrogen peroxide through a radical oxidation-reduction mechanism (Nazari et al., 2005). As hydrogen peroxide concentration increases, an irreversible mechanism-based inactivation process becomes predominant (Rodriguez-Lopez et al., 1997) and it leads to the degradation of haem, the release of iron and the formation of two fluorescent products (Gutteridge, 1986). At a low concentration of hydrogen peroxide below 0.1mM, inactivation is predominately reversible, resulting to the formation and accumulation of catalytically inert intermediate compound III. This inactivation of peroxidase by hydrogen peroxide is dependent on the concentration of hydrogen peroxide. (Zheng et al., 2001) Inactivation reaction between hydrogen peroxide and the intermediate of the enzyme’s catalytic cycle that reduced the sensitivity and efficiency of peroxidase has been studied with different sources of peroxidase, but not that of Gongronema latifolium (utazi).

1.1 PEROXIDASES