Intramolecular Disulfide Linked αβ and αα in Oxidized Tropomyosin: Separation, Identification, and Process of Formation

Abstract

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 and aa by carboxymethyl cellulose (CMC) column chromatography in urea. Oxidized TM before fractionation, as well as the αβ and αα 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 αβ. Partially oxidized TM was found to separate into β, αβα, and αα 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 αβ and αα components may exist as dominant components in TM in vitro rather than as a random mixture of two subunits. A splitting of the electrophoretic band of the α subunit into a doublet was observed.