Spatial-Conformational Modifications of Proteins in Membrane-Cytoskeleton Complex of Cryopreserved Erythrocytes

A selective saving action of cryoprotective agents (CPAs) may be stipulated by peculiarities of cellular element modifications during freeze-thawing. Spatial-conformational changes in proteins of the membrane-cytoskeleton complex of human erythrocytes under cryopreservation in the presence of various CPAs were studied using a protein-crosslinking reagent diamide. Measurements of the level of high-molecular weight polypeptide complexes (HMPC) induced by diamide and analysis of changes in the erythrocyte ghost protein profiles were performed with sodium dodecyl sulphate PAGE. Exposure of erythrocytes to a solution with high ionic strength or freezing without CPA increased the amount of HMPC due to an augmented availability of the protein sulfhydryl groups for the reagent. Spectrin and band 3 mainly contributed to the observed increase in the amount of HMPC under those conditions. The formation of HMPC in erythrocytes frozen under the protection of glycerol, dimethylsulfoxide (DMSO), sucrose, dextran, and PEG largely reflected their protective efficiency. At the stage of cell thawing, a higher level of hemolytic damage in the presence of sucrose and dextran, as compared to DMSO, PEG and glycerol, corresponded to a significant increase in the HMPC amount. Transfer of cells to physiological conditions after the removal of CPAs showed that only in erythrocytes frozen under the glycerol protection the distribution of protein fractions and the HMPC amount corresponded to the respective parameters of control cells. Modifications of proteins in the membrane- cytoskeletal complex under extreme environmental factors may also be related to oxidative stress development in case of hemoglobin structure distortion, which was confirmed by a change in the amount of peroxiredoxin in ghost protein profiles. Thus, effective protection of cells upon cryopreservation can be associated with a restriction of spatial-conformational modifications of proteins in membrane-cytoskeleton complex and the reversibility of such changes under physiological conditions after the removal of CPAs.