Reactions of spin-label crosslinking reagents with red blood cell proteins

Abstract

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 mem- brane 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 spec- trum 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 con- sisting 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 ra- dioactive 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-di- mensional sodium dodecyl sulfate-polyacrylamide gel shows that there is approximately 0.5 cross-linking reagent per protein Te 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 mem- branes have been addressed by using cross-linking reagents,