Fluorometric Studies on the Light Chains of Skeletal Muscle Myosin

Abstract

Light chains of skeletal muscle myosin were studied through the reactivity of their SH groups with a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM). The experiments were carried out by reacting the reagent with myosin for a short time and measuring the amounts of reacted dye by fluorometry after separating light chains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two classes of light chains, alkali light chains and DTNB light chain, were clearly distinguished by their manner of reactivity change, and differences in their environment and in their function were suggested. Although we found that the SH groups of the DTNB light chain were susceptible to very low concentrations of Mg ions (of the order of 10⁻⁵ M), we could not observe Ca²⁺-induced conformational change by our technique. We also estimated the stoichiometry of light chains in skeletal muscle myosin to be 1.37 mol alkali light chain 1, 1.95 mol of DTNB light chain and 0.77 mol of alkali light chain 2 per mole of myosin from the total amounts of our reagent that reacted with each light chain.