Reactions of spin-label cross-linking reagents with red blood cell proteins.

The reactions of two spin-label cross-linking reagents with components of red blood cells have been studied. A bifunctional bis(N-hydroxysuccinimide ester) reagent passes readily through the membrane of intact red cells and reacts almost exclusively with hemoglobin. The rotational motion of hemoglobin inside of red cells may be studied by electron paramagnetic resonance (EPR) by using this spin-labeling method. A second cross-linking molecule, a negatively charged, disulfide-exchange reagent, reacts both with membrane components and with hemoglobin when intact red cells are labeled. Upon reaction with membranes stripped of components other than band 3, this reagent readily produces dimers and higher oligomers of this protein. The EPR spectrum of band 3 labeled in this way shows that most of the spin-label molecules are highly immobilized on the protein. When the same reagent reacts with intact red cells, the cross-linked membrane products include components identified as band 3 dimers and a high molecular weight product consisting primarily of bands 1 and 2 but also containing lesser amounts of most of the other red cell membrane components and a slight amount of band 3. The reagent was made radioactive to demonstrate that the spin-label disulfide exchange reagent is indeed involved in formation of cross-linked products. Comparison of Coomassie Blue staining and radioactivity in the region where band 3 dimers are expected on a one-dimensional sodium dodecyl sulfate-polyacrylamide gel shows that there is approximately 0.5 cross-linking reagent per protein T e reactions of chemical reagents with components of the red blood cell have been used in studies of the geometry of erythrocyte membrane proteins, of the mobility of the band 3 protein in membranes, and of potential antisickling agents. Questions pertaining to the geometry of erythrocyte membranes have been addressed by using cross-linking reagents, ‘From the Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218. Received September 7, 1982. This work was supported by a grant from the National Institutes of Health (GM 28070). B.J.G. was a recipient of a Research Career Development Award (CA 000168) during part of the time the work was done. in the region of band 3 dimers. Further proof that the spinlabel reagent actually is involved in cross-linking some of the band 3 dimers is complicated by the possibilities of sulfhydryl oxidation and disulfide rearrangement. Chymotrypsin cleavage at the external, red cell face results in no significant crosslinked fragments on two-dimensional gels. However, chymotrypsin cleavage of red cell ghosts does reveal two peptides off the diagonal of a two-dimensional gel. The approximate molecular weights of these peptides are 13000 and 8000. There are two possible explanations of the results of the chymotrypsin treatments. The more likely is that the 13 000and 8000-dalton bands arise from an intramolecular cross-link or disulfide bond. The other explanation is that they do arise from an intermolecular cross-link, but this link is unstable during the chymotrypsin treatment of intact erythrocytes even though it is stable when cross-linked ghosts are treated with the protease. The 13 000and 8000-dalton fragments may arise from disulfide formation rather than from cross-linking by the spin-label reagent because they are also observed when intact red cells are treated with the monofunctional reagents 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) and [ (2-aminoethyl)dithio] -2-nitrobenzoic acid. The results are consistent with a new disulfide bond being formed between an -21 000-dalton, transmembrane portion of band 3 and a small fragment of the 35 000-dalton segment released by internal chymotrypsin cleavage of ghosts. in particular ones that can be cleaved for positive identification of the components of the cross-linked products (Wang & Richards, 1974), by application of reagents to which the membrane is impermeable (Staros et al., 1975, 1981) and by chemical modification of specific proteolysis products (Steck, 1978; Rao & Reithmeier, 1979; Ramjeesingh et al., 1980). It has been possible to use optical techniques to study the lateral mobility of band 3 in intact erythrocytes (Fowler & Branton, 1977; Koppel & Sheetz, 1981) and the rotational motion of this integral membrane protein in ghosts (Nigg & Cherry, 1979) because several fluorescein and eosin derivatives are highly specific for band 3 among the protein components 0006-2960/83/0422-0892$01.50/0