Department of Physics, Biophysics, Roentgenology and Radiology, Medical Faculty, Thracian University, 11 Armeyska str., Stara Zagora, Bulgaria.
Int J Hyperthermia. 2011;27(3):286-96. doi: 10.3109/02656736.2011.554064.
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.
4,4'-二异硫氰酸基二苯乙烯-2,2'-二磺酸(DIDS)是一种膜不可渗透的、高度特异性的共价抑制剂，也是阴离子交换器(AE1)的强力热稳定剂，AE1 是红细胞膜(EM)的主要整合蛋白。使用各种但恒定的加热速率(1-8°C/min)，将对照和 DIDS 处理(15 μM，pH 8.2)的人红细胞悬浮液从 20°C加热至 70°C。细胞电解质泄漏通过电导率检测呈温度的 S 型响应。泄漏的临界中点温度，T(mo)，外推至低加热速率(0.5°C/min)，用作 EM 热稳定性的度量。DIDS 处理的红细胞的 T(mo)更大，为 63.2°C ± 0.3°C，而完整的红细胞的 T(mo)为 60.7°C ± 0.2°C。将红细胞暴露于 53°C 时，50%溶血的时间 t(1/2)，用作红细胞对热溶血的抗性的度量。DIDS 处理的红细胞的 t(1/2)也更大，为 63 ± 3 分钟，而完整的红细胞的 t(1/2)为 38 ± 2 分钟。荧光标记 N-(3-芘基)马来酰亚胺和 EPR 自旋标记 3-马来酰亚胺基-羟丙基，共价结合到主要 EM 蛋白的巯基上，用于监测在瞬态加热过程中分子运动的变化。这两种标记都报告了在血影蛋白(50°C)和 AE1(67°C)变性温度处分子运动动力学的增强，并且令人惊讶的是，主要 EM 蛋白，推测是 AE1，在 T(mo)处固定。上述结果有利于 AE1 拷贝在 EM 热稳定性和对热溶血的抗性中的可能涉及预变性重排的假设。
