An Approach to Nearest Neighbor Analysis of Membrane Proteins

Abstract In experiments on isolated human erythrocyte membranes, cross-links between protein components have been produced both by the catalyzed oxidation of intrinsic sulfhydryl groups with the o-phenanthroline cupric ion complex and by reaction with dimethyl dithiobispropionimidate, a bisalkylimidate containing a disulfide linkage between the two reactive groups. The mixture of molecular species has been analyzed by a two-dimensional gel electrophoresis procedure in which the cross-links are split by reductive cleavage during an initial phase of the separation in the second dimension. Cross-linked complexes are recognized by the positions of the off-diagonal elements in the final pattern. Complexes apparently derived from stable oligomers of peptides of the same molecular weight have been found corresponding to many of the bands in the standard sodium dodecyl sulfate gel pattern. Heterocomplexes formed from peptides of different molecular weight have been tentatively identified in a few instances but are less common than oligomers from single bands. In addition cross-linking yields very high molecular weight complexes of spectrin and many of the lower molecular weight protein components of the membrane. The observed cross-linking patterns are consistent with the picture of transmembrane and cytoplasmic surface components anchored to the submembrane network of spectrin fibers. The cross-linking patterns of broken ghosts and intact cells treated with these membrane-permeable reagents are almost identical except for the addition of hemoglobin complexes in the latter.