Radiofrequency dielectric spectroscopy study: Effects of pH, hydrogen bond donors and acceptors on the attachment of spectrin skeleton to the lipid membrane of erythrocytes

Band 3 protein and glycophorin C are the two major integral proteins of the lipid membrane of human red blood cells (RBCs). They are attached from below to a network of elastic filamentous spectrin, the third major RBC membrane protein. The binding properties of the attachments to spectrin affect the shape and deformability of RBCs. We addressed band 3 and glycophorin C attachments to spectrin by measuring the strength of two recently discovered radiofrequency dielectric relaxations, β_sp (1.4 MHz) and γ1_sp (9 MHz), that are observable as changes in the complex admittance of RBCs in medium. In medium at pH 5.2, and also in media with protic substances (formamide, methylformamide, or urea), the β_sp relaxation became inhibited that is attributable to detachment of glycophorin C from spectrin. In medium at pH 9.2, we observed inhibition of γ1_sp relaxation attributable to detachment of band 3 from spectrin, as also was seen in media with aprotic substances difluoropyridine, dimethylsolfoxide, dimethylformamide, acetone, sodium tetrakis(4-fluorophenyl)borate), chlorpromazine, thioridazine and trifluopiperazine. The viscogenic cosolvents (glycerol, ethylene glycol, or i-erythritol) inhibited both the β_sp and γ1_sp relaxations and significantly lowered their characteristic frequencies. Our observations indicate that the glycophorin C attachment to spectrin has nucleophilic centers whose saturation disconnects this attachment and inhibits the β_sp relaxation, whereas at band 3-spectrin attachment site, it is the saturation of electrophilic centers that weakens this attachment and inhibits the γ1_sp relaxation.