Location of SH1 and SH2 Along a Heavy Chain of Myosin Subfragment 1

Two reactive SH groups (SH1 and SH2) of myosin subfragment 1 (S-1) were selectively labeled with a fluorescent dye, N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM). When the DACM-S-1 was digested with trypsin, 95K heavy chain was cleaved into 50K, 25K, and 20K fragments, and the fluorescent labels incorporated into SH1 and SH2 were exclusively found in the 20K fragment, indicating that these SH groups were located in the fragment. When the trypsin-treated DACM-S-1 was subsequently fragmented with hydroxylamine in 6 M guanidine hydrochloride at pH 9.0, the fluorescent 20K fragment was cleaved into two fluorescent segments, the 13K segment containing SH1 and the 5K segment containing SH2. Since it is known that SH1 and SH2 are only 10 residues apart in the sequence and that SH1 is nearer the COOH terminus than SH2, the results show that these reactive SH groups are separated from the COOH terminus of S-1 heavy chain by a 13K-dalton stretch of polypeptide chain.