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 paralbumin 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 pK_a 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.