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The Effect of Cryoprotective Agents on Proteins of the Erythrocyte Membrane-Cytoskeleton Complex

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Abstract—

The aim of the study was to assess 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. Protein modifications were evaluated by SDS-PAGE using different approaches of sample preparation 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 examined substances. The use of the cross-linking reagent diamide for assessment of protein modifications did not reveal structural abnormalities that would cause 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 changes found in the electrophoretic mobility of band 5 proteins in erythrocytes incubated with CPAs in the diamide presence attest 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 CPAs and diamide was found. 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 −SH-groups of proteins. The results obtained indicate that unspecified rearrangements can occur in the erythrocyte MCC under CPA effects that requires further investigation.

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Funding

The work was performed within the framework of budgetary funding of the research work no. 2.2.6.90 (State Registration no. 0114U0011320) of the National Academy of Sciences of Ukraine.

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  1. Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, ul. Pereyaslavskaya 23, 61016, Kharkov, Ukraine

    N. G. Zemlianskykh

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  1. N. G. Zemlianskykh
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Correspondence to N. G. Zemlianskykh.

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COMPLIANCE WITH ETHICAL STANDARDS

All procedures performed on human blood corresponded to the Ethical Standards of the Institutional and National Research Ethics Committee, as well as the 1964 Declaration of Helsinki and its subsequent amendments.

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The author declares no conflicts of interest.

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Translated by A. Medvedev

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Zemlianskykh, N.G. The Effect of Cryoprotective Agents on Proteins of the Erythrocyte Membrane-Cytoskeleton Complex. Biochem. Moscow Suppl. Ser. B 15, 132–138 (2021). https://doi.org/10.1134/S1990750821020128

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