Two new bifunctional protein modification reagents and their application to the study of parvalbumin.
2-[(Trifluoroacetoxy)mercuri]-4-fluorophenol (MFP) and 4-(acetoxymercuri)phenyl azide (MPA) have been prepared and characterized. The sulfhydryl-specific reagents MFP and MPA have been used to study the structure of parvalbumin. The 19F resonance of the derivative of parvalbumin with MFP was studied at 94.6 and 235.2 MHz. At 94.6 MHz, the 19F signal line width is 25 Hz and T1 = 0.28 s, while at 235.2 MHz, the line width is 35 Hz and T1 = 0.39 s. T1 was not affected by substitution of D2O for H2O as a solvent. Removal of calcium from the parvalbumin derivative resulted in the upfield shift of the 19F signal and a decrease in T1 at 94.6 MHz. The pKa for phenolic titration of the MFP derivative was 10.75 compared with 9.5 for the free reagent. These results are interpreted in terms of lodgment of the aryl group in a cleft in the surface of the parvalbumin. The derivative with MPA was photolyzed and subjected to amino acid analysis. Comparison of the analysis of parvalbumin, parvalbumin-MPA, and the photolyzed product indicated destruction of aspartic acid during the photolysis. Asp-22 is a reasonable candidate for the residue attacked, based on comparison with the published crystal structure of parvalbumin.
D000818:Animals; D002347:Carps; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D005978:Glutathione; D009682:Magnetic Resonance Spectroscopy; D009124:Muscle Proteins; D010320:Parvalbumins; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000138:chemical synthesis*; D010663:Phenylmercury Compounds / Q000138:chemical synthesis*; D010782:Photolysis
D003432:Cross-Linking Reagents; D009124:Muscle Proteins; D010320:Parvalbumins; D010663:Phenylmercury Compounds; C037970:2-((trifluoroacetoxy)mercuri)-4-fluorophenol; C037971:4-(acetoxymercuri)phenyl azide; D005978:Glutathione; D010662:Phenylmercuric Acetate