Modification of myosin subfragment 1 tryptophans by dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide.
Modification of tryptophanyl residues (Trps) of myosin subfragments 1 (S-1) was performed with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS). Under controlled conditions, pH 6 at 0 degrees C and 10-min reaction with 10-100-fold molar excess, K+(EDTA) activity was reduced down to less than half, whereas Ca2+-ATPase activity increased and acto-S-1-ATPase was not affected. The number of modified Trps (up to 2.5) agreed well with the number of 2-hydroxy-5-nitrobenzyl moieties incorporated in S-1. The thiol groups of S-1 were not affected up to 50-fold molar excess of DHNBS, thus indicating that the modification was selective for Trps. The modification of as few as one Trp caused a blue shift of the emission spectrum, accompanied by a reduction in the fluorescence quantum yield. The accessibility of Trps to the fluorescence quencher acrylamide is drastically reduced upon modification, indicating that DHNBS-reactive Trps are more "exposed" than the DHNBS-refractive ones. DHNBS modification did not seem to affect the ATP-induced tryptophan fluorescence enhancement of S-1. The effect of DHNBS modification of the intrinsic fluorescence of S-1 indicates that the modified Trps are located in a polar environment and that they may be identical with the long-lifetime Trps of Torgerson [Torgerson, P. (1984) Biochemistry 23, 3002-3007]. The most reactive Trp is located in the N-terminal 27-kDa fragment of the S-1 heavy chain. It might also be inferred from the above data that the nonexposed and ATP-perturbed Trp(s) is (are) located in the 50-kDa fragment.
D000199:Actins / Q000378:metabolism; D000818:Animals; D007202:Indicators and Reagents; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*; D011817:Rabbits; D013452:Sulfonium Compounds / Q000494:pharmacology*; D014357:Trypsin; D014364:Tryptophan
D000199:Actins; D007202:Indicators and Reagents; D015879:Myosin Subfragments; D009861:Onium Compounds; D010446:Peptide Fragments; D013452:Sulfonium Compounds; C024586:dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium; D014364:Tryptophan; D014357:Trypsin; D009218:Myosins