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Nuclear Magnetic Resonance Studies of Protein Conformation. I. Ribonuclease S. II. Hemoglobin


Huestis, Wray Hughes (1972) Nuclear Magnetic Resonance Studies of Protein Conformation. I. Ribonuclease S. II. Hemoglobin. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/JZKA-C327.


Fluorine nuclear magnetic resonance techniques have been used to study conformational processes in two proteins labeled specifically in strategic regions with covalently attached fluorinated molecules. In ribonuclease S, the ϵ-amino groups of lysines 1 and 7 were trifluoroacetylated without diminishing enzymatic activity. As inhibitors bound to the enzyme, changes in orientation of the peptide segment containing the trifluoroacetyl groups were detected in the nuclear magnetic resonance spectrum. pH Titration of one of the histidines in the active site produced a reversal of the conformational process.

Hemoglobin was trifluoroacetonylated at the reactive cysteine 93 of each β chain. The nuclear magnetic resonance spectrum of the fluorine moiety reflected changes in the equilibrium position of the β chain carboxy terminus upon binding of heme ligands and allosteric effectors. The chemical shift positions observed in deoxy- and methemoglobin were pH dependent, undergoing an abnormally steep apparent titration which was not observed in hemoglobin from which histidine β 146 had been removed enzymatically. The abnormal sharpness of these pH dependent processes is probably due to interactions between several ionizing groups.

The carbon monoxide binding process was studied by concurrent observation of the visible and nuclear magnetic resonance spectra of trifluoroacetonylated hemoglobin at fractional ligand saturations throughout the range 0-1.0. Comparison of the ligand binding process observed in these two ways yields evidence for a specific order of ligand binding. The sequence of events is sensitive to the pH and organic phosphate concentration of the medium, demonstrating the delicately balanced control system produced by interactions between the hemoglobin subunits and the effectors.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:(Biophysics and Chemistry)
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biology
Minor Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Raftery, Michael A.
Thesis Committee:
  • Unknown, Unknown
Defense Date:26 May 1972
Funding AgencyGrant Number
Record Number:CaltechTHESIS:04182016-091216066
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9675
Deposited On:18 Apr 2016 17:44
Last Modified:18 Jul 2023 23:04

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