Graphical evaluation of alkylation of myosin's SH1 and SH2: The N- phenylmaleimide reaction

Previous assertions about the effect of alkylation of SIll and SH2 on the myosin high-salt calcium and EDTA ATPases have been summarized, and a simple procedure for obtaining the fractional labeling of SIll and SH2 after treatment of myosin with alkylating agents has been derived. A simple graphical procedure for illustrating the degree of preference of a particular alkylating agent for SIll over SH2 has also been developed. The procedures we developed were validated by applying them to two previously studied compounds, 4-(2-iodoacetamido)-TEMPO and 2,4-dinitrofluorobenzine, and then were used to determine a procedure for maximizing the extent of labeling of SIll alone by N-phenylmaleimide, a compound not previously studied in this manner. It was found that ~80% of the SIll sites could be alkylated without significant alkylation of SH2.