Intramolecular disulfide linked alphabeta and alphaalpha in oxidized tropomyosin: separation, identification, and process of formation.

The oxidation of rabbit skeletal tropomyosin (TM) by repeated cycles of freezing and melting in 0.3 mM Na bicarbonate was studied by electrophoresis and column chromatography. The oxidized TM showed two bands at ca. 70,000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Each band component was separated into disulfide-linked alphabeta and alphaalpha by carboxymethyl cellulose (CMC) column chromatography in urea. Oxidized TM before fractionation, as well as the alphabeta and alphaalpha components, was found to have a molecular weight of about 80,000 daltons, indicating the disulfide bonds to be primarily intramolecular. Oxidation of dilute TM in 1 M NaCl by exposure to air also produced disulfide-linked alphabeta. Partially oxidized TM was found to separate into beta, alphabeta, alpha, and alphaalpha on CMC chromatography, and these were eluted with a linear gradient of NaCl at molarities of ca. 0.09, 0.11, 0.12, and 0.14 M, respectively. The oxidation process was investigated by CMC chromatography, and a possible mechanism is presented. The alphabeta and alphaalpha components may exist as dominant component in TM in vitro rather than as a random mixture of two subunits. A splitting of the electrophoretic band of the alpha subunit into a doublet was observed.