Effects of heat and freeze on isolated erythrocyte submembrane skeletons.
In this study we heated insoluble residues, obtained after Triton-X-100 (0.1 v/v%) extraction of erythrocyte ghost membranes (EGMs). Specific heat capacity, electric capacitance and resistance, and optical transmittance (280 nm) sustained sharp changes at 49°C (TA) and 66°C (TC), the known denaturation temperatures of spectrin and band 3, respectively. The change at TA was selectively inhibited by diamide (1 mM) and taurine mustard (1 mM) while its inducing temperature was selectively decreased by formamide in full concert with the assumed involvement of spectrin denaturation. In the residues of EGMs, pretreated with 4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid (DIDS), the change at TC was shifted from 66 to 78°C which indicated the involvement of band 3 denaturation. The freeze and rapid thaw of EGM residues resulted in a strong reduction of cooperativity of band 3 denaturation while the slow thaw completely eliminated the peak of this denaturation. These effects of freeze-thaw were prevented in residues obtained from DIDS-treated EGMs. The freeze-thaw of residues slightly affected spectrin denaturation at 49°C although an additional denaturation appeared at 55°C. The results indicate preserved molecular structure and dynamics of the membrane skeleton in Triton-X-100 extracts of EGMs. The freeze-thaw inflicted strong damage on band 3 and spectrin-actin skeleton of EGM extracts which is relevant to cryobiology, cryosurgery and cryopreservation of cells.
Department of Physics and Biophysics, Medical Faculty of Thracian University, Stara Zagora 6000, Bulgaria. ivanov_it@gbg.bg.
Ivan T
IT
Ivanov
Boyana K
BK
Paarvanova
Veselin
V
Ivanov
Kathrin
K
Smuda
Hans
H
Bäumler
Radostina
R
Georgieva
D015925:Cryopreservation / Q000379:methods*; D004910:Erythrocyte Membrane / Q000737:chemistry*; D005615:Freezing; D006358:Hot Temperature; D006801:Humans; D017830:Octoxynol / Q000737:chemistry*; D019188:Organ Preservation Solutions / Q000737:chemistry; D011489:Protein Denaturation; D013049:Spectrin / Q000737:chemistry*
D019188:Organ Preservation Solutions; D013049:Spectrin; D017830:Octoxynol