A method is described for preparation of a species of 88 tropomyosin that is sulfhydryl-blocked at C36 and disulfide-cross-linked at C190. Five steps are involved: (1) Rabbit skeletal muscle tropomywin, comprising aa and a8 species, is oxidized with ferricyanide, &sulfide-cross-linking both species at C190. (2) The product is treated with iodoacetamide, blocking the only remaining free sulfhydryl, i.e., C36 of the 8-chains. (3) The C36-blocked, C190-cross-linked product is reduced with dithiothreitol (D"), unfolded in urea, and a and 8 chains separated by ionexchange chromatography. (4) The C36-blocked 8 chains are refolded by dialysis. (5) The refolded, C36-blocked 88 species are cross-linked at C190 by ferricyanide oxidation. The resulting C36-blocked, ClSO-cross-linked 88 product is separated from contaminating species-mostly completely blocked 8-chains and multichain cross-linked molecules-by size-exclusion chromatography in denaturing (guanidinium chloride) solvent. The five-step process and the final product were monitored by titration of free sulfhydryls and by NaD&O4/poIyacrylamide gel electrophoresis (PAGE). Thermal unfolding curves from CD are reported for the resulting pure, C36-blocked, C190-cross-linked 88 species and for its D"-reduction product, the noncross-linked C36-blocked species. The latter shows almost the same thermal unfolding transition as intact, noncross-linked 88 species. The former shows a pretransition similar to, but larger in extent than, the one well known to occur in the analogous case of Cl90-cross-linked aa tropomydn. These unfolding transitions are compared with one another and with that previously reported for doubly cross-linked (at C36 and C190) 88 species. These comparisons are made in the light of current physical models for coiled-coil unfolding equilibria. I t is concluded that although no extent model is demonstrably satisfactory, any successful model must include strain at the cross-link, loop entropy, and regional nonuniformities as essential parts of the pbysics.
'To whom reprint requests should be a d d r d .