Trypsinolysis promotes disulfide formation between 21- and 50-kilodalton segments of myosin subfragment 1 during reaction with 5,5'-dithiobis(2-nitrobenzoic acid).
The reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with S1 and tryptic S1 has been examined to identify the sites of mixed and intramolecular disulfides formed in the initial and final stages of the reaction in these two forms of S1. With undigested S1, the two mixed disulfide bonds initially formed were found to be in the 21-kDa segment. The intramolecular disulfide bond, formed in the subsequent slow phase of the reaction, was also found to be mainly confined to the 21-kDa segment although a small fraction arose from disulfide formation between the 21-kDa and 50-kDa segments. Only 35% of the light chain was modified in undigested S1 after 24 h. For tryptic S1, the initial reaction also led to the formation of mixed disulfides in the 21-kDa segment. However, in the second slower phase, the formation of the intramolecular disulfide occurred primarily between thiols in the 21-kDa and 50-kDa fragments, and in this case, the light chain was labeled to about 60% after 24 h. The enhanced formation of disulfide links between 21-kDa and 50-kDa domains in tryptic S1 points to an increase in flexibility between the thiol-containing regions of these segments.
Rajasekharan
K N
KN
Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106.
Burke
M
M
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000032:analysis; D004228:Dithionitrobenzoic Acid / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism*; D009579:Nitrobenzoates / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism*
D004220:Disulfides; D015879:Myosin Subfragments; D009579:Nitrobenzoates; D010446:Peptide Fragments; D004228:Dithionitrobenzoic Acid; D014357:Trypsin