Impact of Permeant Cryoprotectors on Under-Membrane Skeleton of Human Erythrocytes.

Formamide (FA), N-methylformamide (MFA), N,N-dimethylformamide (DMFA) and dimethyl sulfoxide (DMSO) are frequently used as cryoprotectors of cells. We applied thermal dielectroscopy to study the impact of these membrane-permeable cryoprotectors on the segmental mobility and attachment of spectrin-based skeleton to the lipid membrane of human erythrocytes. Upon heating a suspension of erythrocytes, spectrin denatures at 49.5 °C causing sigmoid frequency-dependent changes in the complex impedance, ΔZ*=ΔZre+j.ΔZim, of suspension. The -ΔZim vs ΔZre plot depicts two semicircles which correspond to the beta (0.05-1.0 MHz) and gamma (1.0-10 MHz) dielectric relaxations on spectrin skeleton [1]. At concentrations (1 to 4 M), relevant to cryoprotection, DMFA and DMSO subdued gamma relaxation, while FA and MFA strongly inhibited the beta relaxation. These effects were eliminated on washing and did not depend on osmotic pressure produced. Applied in combination, DMSO and FA neutralized their opposite effects on the dielectric relaxations. These results are discussed in light of possible impact of tested cryoprotectors on the attachment sites of spectrin skeleton to the lipid membrane. [ABSTRACT FROM AUTHOR]