Study on the Destabilization of Lysozyme and the Chaperone-Like Activity of Alpha Crystallin from Sokoto Red Goat Eye Lens

Filed in Biochemistry Project Topics by on August 31, 2020

Study on the Destabilization of Lysozyme and the Chaperone-Like Activity of Alpha Crystallin from Sokoto Red Goat Eye Lens.

ABSTRACT

Destabilization of Lysozyme and chaperone like action of alpha crystallin isolated from goat’s eye lens was investigated at various temperature ranges in phosphate buffer (pH 7.1) solution  and dithiothretol (DTT). T

his was monitored spectrophotometrically at 260nm. The heat and DTT-induced destabilization of lysozyme was prevented by alpha crystallin in a concentration dependent manner.

Alpha crystallin like other chaperones, fulfils its chaperone like action in preventing aggregation of denatured proteins by the formation of complexes.

TABLE OF CONTENTS

TITLE PAGE……… i

DEDICATION……… ii

CERTIFICATION……… iii

ACKNOWLEDGEMENTS……….. iv

TABLE OF CONTENTS….. v

LIST OF TABLES……. viii

LIST OF FIGURES…… ix

ABSTRACT…… x

CHAPTER ONE INTRODUCTION AND LITERATURE REVIEW

1.0   INTRODUCTION… 1

  • PROTEIN FOLDING………….. 3
  • PROTEIN DENATURATION…… 5
  • JUSTIFICATION….. 9
  • AIM AND OBJECTIVES……… 9
  • LITERATURE REVIEW…… 10
    • α-CRYSTALLINS: PROPERTIES, OCCURANCE AND FUNCTIONS………… 10
    • FUNCTIONS OF ALPHA CRYSTALLIN AND ITS RELATION SHSPS….. 14
    • GENE STRUCTURE, EXPRESSION AND REGULATION…….. 15
    • QUATERNARY STRUCTURE MODELS OF ……………. 18
    • IN VITRO MODIFICATIONS OF ALPHA CRYSTALLIN……….. 19
    • FUNCTION OF CRYSTALLIN…………….. 19
    • DITHIOTHRETOL………25
    • LENS………………. 26

CHAPTER TWO MATERIALS AND METHODS

  • MATERIALS…. 28
    • REAGENTS AND EQUIPMENT.. 28
    • APPARATUS USED FOR ………. 28
  • METHODS…….. 28
    • PREPARATION OF ALPHA CRYSTALLIN……… 28
    • DETERMINATION OF ALPHA CRYSTALLIN……. 28
    • ESTIMATION OF PROTEIN BY FOLIN-CIOCALTEU

[LOWRY] METHOD………….. 29

  • PURIFICATION OF CRYSTALLIN USING GEL ……………….. 30
  • TEMPERATURE DESTABILIZATION OF OXIDIZED LYSOZYME AND CHAPERONE EFFECT OF ALPHA CRYSTALLIN……….. 32

CHAPTER THREE RESULTS, DISCUSSION AND CONCLUSION

  • RESULTS……… 33
  • DISCUSSION….. 42
  • CONCLUSION……….. 47

REFERENCES… 48

APPENDICES  58

INTRODUCTION

Proteins are the workhorses of the living cell. Although proteins may differ in sequence, shape and function, but have in common, the same stereo configuration (i.e. they all have to fold into specific three-dimensional structures) which are mandatory for proper function (Bruce et al., 2002).

Protein structures however are not rigid, but have a dynamic life style, which may involve unfolding and refolding, complex association and dissociation (Anfisen, 1972).

Stress and also many physiological events require proteins to surrender their structure or to regain it at a later stage.

A very large number of distinct conformations exist for the polypeptide chain of which a protein spends most of its time in the native conformation, which spans only an extremely small fraction of the entire configuration space.

Thus, the amino acid sequence of proteins must satisfy two requirements: one, thermodynamics and the other kinetic. The thermodynamics requirement is that the sequence must have a unique folded conformation, which is stable under physiological conditions.

Most proteins can be denatured by heat, which has complex effect on the weak interactions in proteins (Vandenberg et al., 2000).

REFERENCES

Abgar, S., Vanhoudt, J., Aerts, T. and Clauwaert, J. (2001) Study of the Chaperoning Mechanism of Bovine Lens α-Crystallin, a member of the a Small Heat Shock superfamily; Biophysics Research Group, Department of Biochemistry, University of Antwerp, B-2610 Antwerp, Belgium.

Alexander, P. A., He, Y., Chen, Y, Orban, J., Bryan, P. N. (2007) The design and characterization of two proteins with 88% sequence identity but different structure and function. Proc. Natl. Acad. Sci. USA 104(29):11963-11968.

Anfinsen, C. B. (1972) “The formation and stabilization of protein structure”. Biochem. J. 128 (4): 737–740

Anfisen, C. B. (1973) Principles that govern the folding of protein chains. Science 181(4096):223-230

Argos, P. and Siezen, R. J. (1983) Structural homology of lens crystallins. A method to detect protein structural similarity from primary sequences. Eur. J. Biochem. 131:143- 148.

Arrigo, A. P. and Welch, W. J. (1991) Characterization and purification of the small 28000Dalton mammalian heat-shock protein, J. Biol. Chem. 262:15359-15369

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