University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Fall 1979 AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF VANADYL-LABELED CONALBUMIN AND IRON TRANSFERRINS JAMES DAVID CASEY JR. Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation CASEY, JAMES DAVID JR., "AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF VANADYL-LABELED CONALBUMIN AND IRON TRANSFERRINS" (1979). Doctoral Dissertations. 1231. https://scholars.unh.edu/dissertation/1231 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. 8009661 C a s e y, Ja m e s D a v id, Jr . AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF VANADYL- LABELED CONALBUMIN AND IRON TRANSFERRINS University o f New Hampshire PH.D. 1979 University Microfilms International300 N. Zeeb Road, Ann Arbor, MI 48106 18 Bedford Row, London WC1R 4EJ, England AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF VANADYL-LABELED CONALBUMIN AND IRON TRANSFERRINS BY JAMES DAVID CASEY JR. B.S., Boston College, 1968 A DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Chemistry September, 1979 This thesis has been examined and approved. Thesis director, N. Dennis Chasteen Professor of Chemistry Vu. ■ V c k , Helmut M. Haendler, Professor of Chemistry ^TTVl^Ywa . Morrison, Professor of Chemistry Q (7James A. Stewart, Associate Professor of Biochemistry Rudolf W. iwsitz, .assistant Professor of Chemistry Date/! To those for whom I care, have cared, and will care. iii TABLE OF CONTENTS LIST OF TABLES ..................................... V LIST OF F I G U R E S ......................................vi ABSTRACT ............................................ X I. INTRODUCTION TO TRANSFERRINS AND VANADYL I O N ................................ 1 Transferrin ..................................... 2 The Vanadyl P r o b e ......................... 9 II. METAL BINDING SITE CHARACTERISTICS .............. 16 Experimental Procedure ......................... 16 R e s u l t s ......................................... 25 Stoichiometry of Binding ..................... 25 Binding Studies .............................. 29 Frozen Solution Spectra ..................... 35 D i s c u s s i o n ....................................... 51 Summary ............................. 63 III. MIXED METAL AND ANION BINDING STUDIES ..... 66 Introduction ..................................... 66 Experimental ..................................... 66 R e s u l t s ......................................... 73 D i s c u s s i o n ......................................... 105 IV. VANADYL RABBIT SERUM TRANSFERRIN ................ Ill Introduction ..................................... Ill Experimental ..................................... Ill R e s u l t s ........................................... 113 D i s c u s s i o n ......................................... 115 V. A Q-BAND EPR STUDY OF IRON- SATURATED TRANSFERRINS ......................... 122 Introduction ..................................... 122 Experimental ..................................... 124 R e s u l t s ........................................... 126 D i s c u s s i o n ......................................... 132 REFERENCES ........................................... 133 iv i List of Tables 2.1 V0^+ Binding Capacity .................................31 2.2 Titratable Protons Released During V02+ Binding ................................ 33 2.3 EPR Parameters ....................................... 50 2.4 EPR and Electronic Transition Parameters for V02+ Complexes .....................60 2.5 Squared Molecular Orbital Coefficients for Vanadyl Conalbumin ......................... 61 2.6 Squared 4s Atomic Orbital Coefficients for Vanadyl Conalbumin ......................... 64 3.1 ^ C Scintillation C o u n t s ............................ 71 v List of Figures 2+ 1.1 Sketch of VO Ion in Square Pyramidal Geometry ................................ 11 1.2 X-band Spectra of VO^+ (aq) at Room Temperature and at 77 K ..................... 12 2+ 1.3 Q-band Spectrum of VO (aq) ....................... 14 1.4 Sketch of Non-coincident g-tensor and A-tensor A x e s ................................ 15 2.1 Sketch of Equipment Used in Purging Protein Solution with N2(g) 21 2.2 Ultraviolet Difference Spectrum of Vanadyl Conalbumin ............................ 26 2.3 Spectrophotometric Titration of Apoconalbumin with VC>2+ by Ultraviolet Difference Spectroscopy ........... 27 2.4 Room Temperature Spectrum of Divanadyl Conalbumin at X-band ......... .... 28 2.5 Spectrometric Titration of Apo­ conalbumin with V02+ by E P R ..................... 30 2.6 X-band Spectrum of Divanadyl Conalbumin at pH 7.1 and 7 7 K .................. 36 2.7 X-band Spectrum of Divanadyl Conalbumin at pH 9.0 and 7 7 K .................. 37 2.8 X-band Spectra of Divanadyl Conalbumin at pH 8 and pH 10, 77 K .............. 38 2.9 X-band Spectrum and Computer- simulated Spectrum of Divanadyl Conalbumin at pH 11.0 and 77 K .................. 39 2.10 Relative Peak Heights of A, B, and C Conformations of Divanadyl Conalbumin as a Function of p H .................. 41 2.11 Relative Peak Heights of A, B, and C Conformations of 0.6 V02T per CA as Function of p H .............................. 42 vi 2.12 Q-band Spectrum of Divanadyl Conalbumin at pH 7 . 1 ............................ 44 2.13 Q-band Spectrum of Divanadyl Conalbumin at pH 9 . 0 ............................ 45 2.14 Q-band Spectra of Divanadyl Conalbumin at pH 8.0 and 1 0 . 0 ................ 46 2.15 Q-band Spectrum and Computer- simulated Spectrum of Divanadyl Conalbumin at pH 1 1 . 0 ......................... 47 2.16 Computer-simulated Spectra at Different Angles of Non-coincidence ......... 49 3.1 Dependence of Intensity Patterns of (-7/2).. and (-5/2).. Lines on VC>2+ to Conalbumin Ratio at pH 9.0 ..... 74 3.2 Variation of A, B, and C Intensities with V02+ to Conalbumin Ratio in pH 8-11 Range ................................ 75 3.3 X-band Spectrum of 1 Fe^+, 1 VO^+ per Conalbumin at pH 8.0 and 77 K ........... 76 3.4 Q-band Spectrum of 1 Fe^+, 1 V0^+ per Conalbumin at pH 8. 0 ............................ 77 3.5 X-band Spectrum of 1 Fe^+, 1 VO^+ per Conalbumin at pH 10.0 and 77 K ........... 78 3.6 Q-band Spectrum of 1 Fe^+, 1 VO^+ per Conalbumin at pH 1 0 . 0 ..................... 79 3.7 X-band Spectra (g - 4.3) of Diferric Conalbumin and of 1 Fe3+, 1 V02+ C o n a l b u m i n ....................................... 80 3.8 X-band Spectrum of 0.8 Fe^+ , 1.2 VO^+ per Conalbumin at pH 11.0 and 77 K ........... 81 3.9 Room Temperature Spectrum of Divanadyl Conalbumin at X-band Without Bicarbonate at pH 8 . 5 ......................... 83 3.10 X-band Spectrum of Divanadyl Conalbumin Without Bicarbonate at pH 9 and 7 7 K ................................ 85 3.11 Q-band Spectrum of Divanadyl Conalbumin Without Bicarbonate at pH 9.4 and 77 K .............................. 86 vii 3.12 Dependence of Intensity Patterns of (-7/2) || and (— 5 / 2 ) |( Lines of Divanadyl Conalbumin on Bicarbonate to Conalbumin Ratio at pH 9 . 1 ..................................... 87 3.13 X-band Spectrum of Divanadyl Conalbumin in Oxalate at pH 10 and 77 K Without B i c a r b o n a t e ..................... 89 3.14 Room Temperature Spectrum of Divanadyl Conalbumin at X-band in Oxalate at pH 8 . 5 .............................. 90 3.15 Room Temperature Spectrum of Vanadyl Conalbumin at X-band at pH 9.5 in Presence of N T A .................................91 3.16 Room Temperature Spectrum of 0.8 Fe^+ , 1.2 VO^+ per Conalbumin at X-band in 0.1 M Tris-HCl, pH 8. 1 ...........................92 3.17 Iron(III) Binding to Conalbumin Monitored by Absorbance at 470 n m .................. 93 3.18 X-band Spectrum of 2 Fe^+ per Conalbumin in Oxygenated Solution Without and With Bicarbonate at pH 9.5 and 77 K .................................95 3.19 X-band Spectrum of Divanadyl Conalbumin in 0.4 F NaC104 at pH 8.0 and 77 K ............... 96 3.20 X-band Spectrum of Divanadyl Conalbumin in 0.85 F NaC104 at pH 8.0 and 77 K .............. 97 3.21 X-band Spectrum of Divanadyl Conalbumin in 1.9 F NaClO^ at pH 8.0 and 77 K ............... 98 3.22 Q-band Spectrum of Divanadyl Conalbumin in 0.4 F NaC104 at pH 8 . 0 .........................100 3.23 Q-band Spectrum of Divanadyl Conalbumin in 0.85 F NaCl04 at pH 8 . 0 ...................... 101 3.24 Q-band Spectrum of Divanadyl Conalbumin in 1.9 F NaCl04 at pH 8 . 0 .........................102 3.25 X-band Spectrum of Divanadyl Conalbumin in 1.0 F NaCl at pH 9.0 and 77 K ................ 103 3.26 X-band Spectrum of Divanadyl Conalbumin in 0.45 F NaHCOg at pH 9.3 and 77 K .............104 viii 3.27 X-band Spectrum of Divanadyl Conalbumin in D20 at pD 10.0 and 77 K ...................... 106 3.28 Q-band Spectrum of Divanadyl Conalbumin in D 20 at pD 9.5 and 77 K ...................... 107 3.29 Q-band Spectrum of Divanadyl Conalbumin in D~0 Without Bicarbonate at pH 9.4 and 77 K ......................................... 108 4.1 Room Temperature Spectrum of Divanadyl Rabbit Serum Transferrin at X-band ............ 114 4.2 Spectrometric Titration of Apo-rabbit Serum Transferrin with V02+ by E P R ............. 116