Ethoxyformylation and photooxidation of histidines in transferrins

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Abstract
The chemical reactivity of histidines in chicken egg white ovotransferrin and human serum transferrin was studied utilizing 2 different reactions. Upon dye-sensitized photooxidation of ovotransferrin and ethoxyformylation of human serum transferrin and ovotransferrin, losses in histidine and Fe-binding activity were observed. All of the histidines in both apoproteins could be ethoxyformylated by the use of 170-400 molar excesses of reagent resulting in complete loss in activity. The histidines of human serum transferrin showed a greater reactivity toward the reagent than did those of ovotransferrin. The binding of each Fe protected 2 histidines from ethoxyformylation, and in both cases the proteins remained completely active. First-order losses in histidine and Fe-binding activity were observed when ovotransferrin was irridiated in the presence of methylene blue. Comparison of the first-order rates indicates the loss of 2 histidines per binding site accounts for the inactivation of the protein. However, Fe binding did not protect ovotransferrin from photoinactivation as expected. Evidence from both modification techniques indicates that histidines are essential for Fe-binding activity and that there are 2 essential histidines in each binding site. The advantages of using 2 modification reactions, ethoxyformylation and photooxidation, in the study of the functional role of histidines in proteins are demonstrated in this work.