The effect of cryoprotective agents on proteins of the erythrocyte membrane-cytoskeleton complex

Abstract

The aim of the study was to evaluate of the effects of glycerol and DMSO, belonging to the endocellular type of cryoprotective agents (CPAs), as well as polyethylene glycol, dextran, sucrose, and mannitol, related to exocellular CPAs, on proteins of the membrane-cytoskeleton complex (MCC) of human erythrocytes at the stage preceding freezing. The assessment of protein modifications was performed by SDS-PAGE using different approaches when preparing samples for analysis. The use of β-mercaptoethanol in the solubilizing buffer showed no changes in the MCC polypeptide profile of erythrocytes preincubated with CPAs thus suggesting good biocompatibility of the studied substances. The use of the cross-linking reagent diamide for assessment of protein modifications did not reveal structural abnormalities that would result in significant changes in the localization of −SH groups and an increase in the production of high-molecular-weight polypeptide complexes identified by SDS-PAGE without β-mercaptoethanol. However, the recognized changes in the electrophoretic mobility of proteins in the area of band 5 in erythrocytes incubated with CPA in the presence of diamide suggest a reorganization of the structural state of actin protofilaments, which can be caused by alterations of actin monomers themselves or initiated by modifications of actin-binding proteins in the presence of CPAs. In addition, an increase in the amount of the protein fraction located between bands 5 and 6 in the MCC profiles of erythrocytes incubated with CPA and diamide was revealed. Despite the similarity of the reaction of erythrocyte proteins to different CPAs, the properties of cells depending on MCC, may differ due to modifications in the macromolecule structures, which are not associated with changes in the localization of the −SH-groups of proteins. The results obtained indicate that CPAs may have a significant impact on the erythrocyte MCC, and this requires further research.