title: Anion exchanger and the resistance against thermal haemolysis.
abstract: 4,4'-Diiso-thiocyanato stilbene-2,2'-disulphonic acid (DIDS) is a membrane-impermeable, highly specific covalent inhibitor and powerful thermal stabiliser of the anion exchanger (AE1), the major integral protein of erythrocyte membrane (EM). Suspensions of control and DIDS-treated (15 µM, pH 8.2) human erythrocytes were heated from 20° to 70°C using various but constant heating rates (1-8°C/min). The cellular electrolyte leakage exhibited a sigmoidal response to temperature as detected by conductometry. The critical midpoint temperature of leakage, T(mo), extrapolated to low heating rate (0.5°C/min) was used as a measure for EM thermostability. T(mo) was greater for DIDS-treated erythrocytes, 63.2° ± 0.3°C, than for intact erythrocytes, 60.7° ± 0.2°C. The time, t(1/2), for 50% haemolysis of erythrocytes, exposed to 53°C was used as a measure for the resistance of erythrocytes against thermal haemolysis. The t(1/2) was also greater for DIDS-treated erythrocytes, 63 ± 3 min, than for intact erythrocytes, 38 ± 2 min. The fluorescent label N-(3-pyrenyl)maleimide and EPR spin label 3-maleimido-proxyl, covalently bound to sulphydryl groups of major EM proteins, were used to monitor the changes in molecular motions during transient heating. Both labels reported an intensification of the motional dynamics at the denaturation temperatures of spectrin (50°C) and AE1 (67°C), and, surprisingly, immobilisation of a major EM protein, presumably the AE1, at T(mo). The above results are interpreted in favour of the possible involvement of a predenaturational rearrangement of AE1 copies in the EM thermostability and the resistance against thermal haemolysis.
authors.affiliation: Department of Physics, Biophysics, Roentgenology and Radiology, Medical Faculty, Thracian University, 11 Armeyska str., Stara Zagora, Bulgaria. ivanov_it@gbg.bg
authors.forename: I T
authors.initials: IT
authors.lastname: Ivanov
authors.forename: A
authors.initials: A
authors.lastname: Zheleva
authors.forename: I
authors.initials: I
authors.lastname: Zlatanov
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D006461:Hemolysis; D006358:Hot Temperature; D006801:Humans; D013113:Spin Labels
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D013113:Spin Labels; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid