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.

中文翻译：

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低温保护剂对红细胞膜-细胞骨架复合物蛋白质的影响

摘要—

该研究的目的是评估属于细胞内冷冻保护剂（CPA）类型的甘油和DMSO，以及与胞外CPA相关的聚乙二醇，右旋糖酐，蔗糖和甘露醇对膜蛋白的影响冷冻前人类红细胞的β-细胞骨架复合物（MCC）。通过使用不同的样品制备方法进行分析，通过SDS-PAGE评估蛋白质修饰。在增溶缓冲液中使用β-巯基乙醇显示，用CPA预孵育的红细胞的MCC多肽谱无变化，因此表明所检查物质具有良好的生物相容性。使用交联剂二酰胺评估蛋白质修饰没有发现结构异常，该结构异常会导致-SH基团的定位发生重大变化，并通过SDS-PAGE鉴定出的高分子量多肽复合物的产量增加不含β-巯基乙醇。但是，在二酰胺存在下与CPA孵育的红细胞中，带5蛋白电泳迁移率的变化证明肌动蛋白原丝的结构状态发生重组，这可能是由于肌动蛋白单体本身的改变或肌动蛋白的修饰引起的。在CPA存在的情况下结合蛋白。另外，发现与CPA和二酰胺一起孵育的红细胞的MCC图谱中位于条带5和6之间的蛋白质部分的量增加。尽管红细胞蛋白与不同CPA的反应相似，但是由于大分子结构的修饰，取决于MCC的细胞的性质可能会有所不同，这与蛋白的-SH-基团的位置变化无关。获得的结果表明，在CPA的作用下，红细胞MCC可能发生不确定的重排，需要进一步研究。