Ethoxyformylation and photooxidation of histidines in transferrins.
The chemical reactivity of histidines in ovotransferrin and human serum transferrin was studied utilizing two different reactions. Upon dye-sensitized photooxidation of ovotransferrin and ethoxyformylation of human serum transferrin and ovotransferrin, losses in histidine and iron-binding activity were observed. All of the histidines in both apoproteins could be ethoxyformylated by the use of 170 to 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 iron protected two histidines from ethoxyformylation, and in both cases the proteins remained completely active. First-order losses in histidine and iron-binding activity were observed when ovotransferrin was irradiated in the presence of methylene blue. Comparison of the first-order rates indicates the loss of two histidines per binding site accounts for the inactivation of the protein. However, iron binding did not protect ovotransferrin from photoinactivation as expected. Evidence from both modification technqiues indicates: (1) Histidines are essential for iron-binding activity. (2) There are two essential histidines in each binding site. The advantages of using two modification reactions, ethoxyformylation and photooxidation, in the study of the functional role of histidines in proteins are demonstrated in this work.
D000818:Animals; D001059:Apoproteins; D001665:Binding Sites; D002645:Chickens; D003207:Conalbumin / Q000528:radiation effects; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004527:Egg Proteins / Q000528:radiation effects; D006639:Histidine; D006801:Humans; D007501:Iron; D007700:Kinetics; D008027:Light; D010084:Oxidation-Reduction; D011485:Protein Binding; D014168:Transferrin / Q000528:radiation effects
D001059:Apoproteins; D004527:Egg Proteins; D014168:Transferrin; D003207:Conalbumin; D006639:Histidine; D007501:Iron; D004047:Diethyl Pyrocarbonate