Abstract
Modification of tryptophanyl residues (Trps) of myosin subfragment 1 (S-l) was performed with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS). Under controlled conditions, pH 6 at 0 °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-l-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-l. The thiol groups of S-l 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-l. The effect of DHNBS modification on the intrinsic fluorescence of S-l 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-l 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.
Original language | English |
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Pages (from-to) | 2903-2909 |
Number of pages | 7 |
Journal | Biochemistry |
Volume | 26 |
Issue number | 10 |
DOIs | |
State | Published - 1987 |
Externally published | Yes |