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Protein Denaturation: a Different Point of View

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Protein Denaturation: a Different Point of View Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 1972 Protein Denaturation: A Different Point of View Robert Orman Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Medicine and Health Sciences Commons Recommended Citation Orman, Robert, "Protein Denaturation: A Different Point of View" (1972). Dissertations. 1122. https://ecommons.luc.edu/luc_diss/1122 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1972 Robert Orman PROTEIN DENATURATION; A DIFFERENT POINT OF VIEW by Robert Orman A Dissertation Submitted to the Faculty of the Graduate School of Loyola University' in Partial Fulfillment of the Requirements for· tre Degree of Doctor of Philosophy February 1972 ACKNOWLEDGEMENTS The author wishes to express his great appreciation to the entire faculty of the Biochemistry Department at the Stritch School of Medicine for its consideration, guidance, and general presence in times of need. The author wishes to thank Dr. William Hollemann and Abbott Laboratories for the use of the ultracentrifuge as described herein. The author wishes to thank and commend Mrs. Peter Oeltgen for the excellent typing and "deciphering" services rendered. The author especially wishes to acknowledge the efforts of Professor Norton C. Melchior "beyond the call of duty." It is of particular comfort to know the "correct answer" lies just across the hall. Finally and most importantly, the author wishes to express deepest appreciation to Professor Steven Kere~ztes-Nagy, not only for his expertise pertaining to the multitude of particular problems encountered by every graduate student, but for his example. In an advisor-student relationship, the most significant and effective teaching is done through example. In this regard and specifically as a thinker (for thinking is the one indispensible aspect which differentiates science from other walks of life), Dr. Nagy represents the consumate. His influence, when correctly perceived, and properly understood, can only lead to self improvement. TABLE OF CONTENTS CHAPTER PAGE I INTRODUCTION • l • • • • • • • • • • • • • • • • • • • • • Experimental Aspects of Denaturation • • • • • • • • 1 Thermodynamics od Denaturation • • • • • • • • • • • 8 Kinetics of Denaturation • • • • • • • • • • • • • • 11 ' Dissociation and Denaturation • .~ . • • • • • • 12 Yeast Enolase, A Brief Summary • • • • • • • • • • • lJ II MATERIALS AND METHODS • • • • • • • • • • • • • • • • • 15 Purification of Yeast Enolase • • • • • • • • • • • 15 . Reversibility of Thermal Denaturation . ·• • • • • • 15 Bromide Effects on Reversibility ••• • • • • • • • 18 Thermodynamic and Kinetics of Denaturation •• • • • 18 Establishment of Monom~ric Activity • • • • • • • • 20 Chromatography • • • • • • • • • • • • • • • • • 20 Ultracentrifugation • • • • • • • • • • • • • • • 22 Kinetic Properties of Monomer and Dimer • • • • • • 25 III RESULTS • • • • • • • • • • • • • • • • • • • • • • • • 28 Purification of Yeast Enolase • • • • • • • • • • • 28 . Reversibility of Denaturation • • • • • • • • • • • 28 Effect of Bromide on Reversibility • • • • • • • • • JJ Thermodynamics • • • • • • • • • • • • • • • • • • • 42 Kinetics • • • • • • • • • • • • • • • • • • • • • • 44 The Active Monomer • • • • • • • • • • • • • • • • • 53 Chromatography • • • • • • • • • • • • • • • • • 53 Ultracentrifugation • • • • • • • • • • • • • • • 56 TABLE OF CONTENTS CONTINUED CHAPTER ~ Kinetic Properties of Monomer and Dimer • • • • • • 65 IV DISCUSSION • • • • • • • • • • • • • • • • • • • • • 72 Thermodynamics • • • • • • • • • • • • • • • • • • • 73 Ion-Protein Interactions • • • • • • • • • • • • • 77 Protein-Protein Interactions • • • • • • • • • • • 83 Kinetics of Denaturation • • • • • • • • • • • • • 88 Molecular Weight of Yeast Enolase • • • • • • • • • 90 Pathway of Denaturation • . .. •· . • • • • • • • • 91 v Red Blood Cell Enolase, A Special Problem • • • • • 94 APPENDIX • • • • • • • • • • • • • • • • • • • • • • 120 REFERENCES . ~ . 124 ·. LIST OF TABLES TABLE PAGE. Ia Thermodynamic Parameters for the Denaturation of Proteins. • • • • • • • • • • • • • • • • • • • • • 9 I Properties of Pure and. Puri~ied Yeast Enolase • • • JO II Reversibility of Thermal Denaturation • • • • • • • 32 III Effect of Bromide on Reversibility • • • • • • • • J4 I Va O. D. Changes Resulting from Thermal Denaturation • 45 IVb Thermodynamic and Kinetic Parameters for Thermal Denaturation. • • • • • • • • • • • • • • • • • • • 46 v Thermodynamic and Kinetic Parameters for Dena tura ti on in 0 .1 .!YI Br - • • • • • • • • • • • • • 47 VI Thermodynamic and Kineti£ Parameters for Denaturation in 0.2 .rt! Br • • • • • •••••••• 48 VII Summary of Kinetic and Thermodynamic Parameters for Thermal Denaturation • • • • • • • • • • • • • 52 VIII Calibration of Sephadex Column •• • • • • • • • • • 54 IX· Dependence of Elution Volume on Temperature for Yeast Enolase • • • • • • • • • • • • • • • • • 57 x State of Aggregation of Yeast Enolase at J7° as a Function of Concentration ••••••••••••• 66 XI Effects of Anions on km and Vmax• •••• • • • • • 71 XII l(m Determin~tion for Monomer in the Presence of Cl-~ Br-, and Ac • • • • • • • • • • • • • • • • • • • • 80 XIII Effect of Temperature on Aggregation of REC Enolase 108 XIV Variation in km for RBC Enolase Monomer as a Cl- Function. • • • • • • • • • • • • • • • • • • • • • 114 '~ .. ~, LIST OF FIGURES f.IGURE l The Optical Approach to Denaturation • • • • • • • 3 2 Correlation of Optical Changes and Activity Changes 4 3 The Archibald Approach to Sedimentation Velocity,., 24 4 Elution Pattern for Yeast Enolase Purification • • 29 5 The Implementation of Enzyme Activity in Denatura- tion Studies • • • • • • • • • • • • • • • • • • • 37 6 Arrhenius Plot for Yeast Enolase·Catalyzed Reaction • • • • • • • • • • • • • • • • • • • • • 7 Van•t Hoff Plot for Thermal Denaturation •• • • • 8 Arrhenius Plot for Thermal Denaturation Relating to kl. • • • • • • • • • • • • • • • • • • • • • • 50 9 Arrhenius Plot for Thermal Denautration Relating to k2. • • • • • • • • • • • • • • • • • • • • • • 51 10 Elution Patterns for Yeast Enolase at Various Temperatures • • • • • • • • • • • • • • • • • • • 55 11 Sedimentation Equilibrium Studies for Yeast Eno lase. • • • • • • • • • • • • • • • • • • • • • 59 12 Sedimentation Velocity Studies for Yeast Enolase • 60 13 Sedimentation Velocity Studies for Yeast Enolase Monitoring Substrate Appearance. • • • • • • • • • 62 14 Specific Activity Studies for Yeast Enolase Corrected for Time • • • • • • • • • • • • • • • • 63 15 Correlation of Sedimentation Velocities and Molecular Weights •••••••••••••• • • • 64 16 Lineweaver-Burke Plots for Enolase Monomer and Dimer. • • • • • • • • • • • • • • • • • • • • • • 67 17 Lineweaver-Burke Plots for Monomer and Dimer as a Function of Increasing Bromide • • • • • • • • • • 68 LIST OF FIGURES CONTINUED .EJGURE 18 Lineweaver-Burke Plots for Monomer as a Function of Increasing Bromide ~ • • • • • • • • • • • • • 20 Elution Pattern for RBC Enolase as a Function of Hb Concentration. • • • • • • • • • • • • • • • • 106 21 Hb 2 PGA Binding. • • • • • • • • • • • • • • • • 110 22 Cl- Inhibition of RBC Enolase Monomer • • • • • • 113 ABSTRACT The glycolytic enzyme enolase (isolated from yeast cells), which catalyzes the conversion of 2-phosphoglycerate (2-PGA) into phosphoenolpyruvate is known to exist as a dimer of molecular weight 80,000. In this thesis, the enzyme a,ssay,.which utilizes the ab­ sorption of product at 240 nm, was employed to study the thermal de­ na tura tion of enolase between 45° and 60°. Additional studies were performed to determine various physical and chemical properties of the enolase monomer and dimer. With regard to thermal denaturation, the use of the enzyme assay is somewhat unique in that it reflec.ts the presence of active enzym~ only, whereas other approaches (especially those of a spec­ trophotometric nature) monitor both native and denatured enzyme forms as well as the concomitant variations in solvent structure. Con- sequently, the thermodynamic parameters derived from such activity oriented studies would likely be of a well defined nature as opposed to the marked v~riation in 6H 0 and 6S0 observed as a function of temper~ture by other techniques. The difficulty in quantitatively assessing the denaturation process, regardless of the experimental procedure employed, lies in the demonstration of thermodynamic rever­ sibility. In this. study~ irreversible denaturation was found to affect the thermodynamic calculations to a considerable degree. 0 0 Nevertheless, the derived 6H and 6S values, based on certain qual­ ifying assumptions, were to remain essentially constant over the temperature range.studied. From the kinetic point of view, the ex­ perimental activation energy for denaturation based on activity loss was found to be 41 Kcal/mole. Upon addition of bromide, a denatur- ., ing anion, in concentrations of 0.1 M and 0.2 M, the experimental activation energy was seen to increase to 4J Kcal/mole and 52 Kcal/ mole respectively. In general, the rate constant governing dena- turation was also seen to increase with increasing bromide.
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