Reaction of a lipid-soluble, unsymmetrical, cleavable, cross-linking reagent with muscle aldolase and erythrocyte membrane proteins.

The compound di-N-(2-nitro-4-azidophenyl)cystamineS,S-dioxide (DNCO) has been synthesized and characterized. In a dark reaction this reagent will undergo efficient disulfide-sulfhydryl exchange with protein -SH groups, thus providing a covalently bound photoactivatable group. The cross-linked products obtained in applying this reagent to the soluble protein rabbit muscle aldolase have pointed up some of the analytical difficulties associated with mixtures of peptide products of identical molecular weight but different Stokes radii. When applied to erythrocyte membranes, a series of cross-linked complexes were produced which were similar to, but not identical with, those produced by watersoluble bisimidates, or &II-orthophenanthroline (CUP)catalyzed air oxidation of intrinsic -SH groups. Low yields of complexes of Bands 2.2 and 2.3 with about 40,000 daltons of extra material were noted. Spots most easily explained as complexes of Band 3 with Bands 4.1,4.2, 4.4, and 6 were noted. A similar spot for Band 5 could have been a Band 3 complex or a Band 5 trimer. As with the other reagents no clear evidence has yet been obtained for a complex of Band 5 with spectrin. A spot showing faster movement in the first dimension of Band 4.2 after treatment with DNCO (or CUP) suggests the formation of a disulfide link between distant parts of that chain through -SH groups that are very close together in the intact membrane. This behavior is similar to that seen even more clearly in the aldolase experiments.