Date of Award

Fall 1982

Project Type

Dissertation

Program or Major

Chemistry

Degree Name

Doctor of Philosophy

Abstract

Electron paramagnetic resonance difference spectroscopy of diferric human serum transferrin indicates that certain inorganic anions alter the electronic environment of the ion centers of the protein by binding to specific anion sites. The binding of these anions (thiocyanate, perchlorate, adenosinetriphosphate, pyrophosphate, and chloride) is shown to occur pairwise with high positive cooperativity, an observation unique to nonstereospecific anion-protein interactions. It is shown that human serum transferrin has binding sites for four nonsynergistic anions, two in each domain. The association constants are reported.

It is unlikely that the anions bind to a significant extent at the sites occupied by the synergistic anion since none of them facilitates iron binding in the absence of bicarbonate. It is more likely that these anions bind to positively charged amino acids or possibly to amide dipoles of the protein. The free energy of association of these anions follows the lyotropic series which is approximately the same sequence for the affinity of anions for positively charged sites on proteins.

31-P NMR spectroscopy reveals that the locus of interaction is near the iron centers and confirms that both sites are effected. In solutions containing an NTA:transferrin concentration of 4:1 no paramagnetic enhancement in the 31-P relaxation rate is observed. NTA also negates the response of the EPR spectrum to the above anions. Cooperativity in anion binding is also observed at pH 7.5 but at pH 9, no cooperativity is evident.

A competitive binding study between pyrophosphate and transferrin for iron is presented. The results reveal that the thermodynamics of iron binding to transferrin is dependent on the concentration of pyrophosphate, the pH of the solution and the bicarbonate concentration.

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