A membrane-impermeant, cleavable cross-linker. Dimers of human erythrocyte band 3 subunits cross-linked at the extracytoplasmic membrane face.

We have synthesized diisethionyl-3,3'-dithiobispropionimidate (DIDIT), a new membrane-impermeant, cleavable protein cross-linking reagent designed for probing protein organization at one face of a membrane. Rabbit muscle aldolase were reacted in solution with DIDIT and the products were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. When electrophoresed under nonreducing conditions, the gels contain bands corresponding to oligomers of aldolase, while pretreatment with dithiothreitol to cleave the cross-link prior to electrophoresis results in gels containing primarily the band corresponding to aldolase monomer. These experiments demonstrate that DIDIT is a cleavable protein cross-linker. Reaction of isolated human erythrocyte membranes with DIDIT leads to extensive cross-linking of spectrin, band 3, and band 6, and residual hemoglobin, consistent with results previously obtained with permeant cross-linkers. In contrast, when intact human erythrocytes are cross-linked with DIDIT, hemoglobin and the cytoplasmic face membrane proteins are not cross-linked, but band 3, which is accessible at the extracytoplasmic face of the membrane, is cross-linked to dimers.