Lepock J R, Frey H E, Bayne H, Markus J
Guelph-Waterloo Program for Graduate Work in Physics, University of Waterloo, Canada.
Biochim Biophys Acta. 1989 Apr 14;980(2):191-201. doi: 10.1016/0005-2736(89)90399-4.
Hemolysis of human erythrocytes as a function of time of exposure to 47.4-54.5 degrees C was measured and correlated to thermal transitions in the membranes of intact erythrocytes as determined by differential scanning calorimetry (DSC). Curves of hemoglobin leakage (a measure of hemolysis) as a function of time have a shoulder region exhibiting no leakage, indicative of the ability to accumulate sublethal damage (i.e., damage not sufficient to cause lysis), followed by a region of leakage approximating pseudo-first-order kinetics. Inverse leakage rates (Do) of 330-21 min were obtained from 47.4-54.5 degrees C, respectively. A relatively high activation energy of 304 +/- 22 kJ/mol was obtained for leakage, eliminating the involvement of metabolic processes but implicating a transition as the rate-limiting step. Membrane protein involvement was suggested by the very low rate (10(-2) of the rate from erythrocytes) and low activation energy (50 +/- 49 kJ/mol) of hemoglobin leakage from liposomes containing no membrane protein. A model was developed that predicts a transition temperature (Tm) for the critical target (rate-limiting step) of 60 degrees C when measured at a scan rate of 1 K/min. DSC scans were obtained from intact erythrocytes and a procedure developed to fit and remove the transition for hemoglobin denaturation which dominated the scan. Three transitions remained (transitions A, B, and C) with Tm values of 50.0, 56.8, and 63.8 degrees C, respectively. These correspond to, but occur at slightly different temperatures than, the A, B, and C transitions of isolated erythrocyte membranes in the same salt solution (Tm = 49.5, 53-58, and 65.5 degrees C, respectively). In addition, the relative enthalpies of the three transitions differ between isolated membranes and erythrocytes, suggestive of membrane alterations occurring during isolation. Thus, all analyses were conducted on DSC scans of intact erythrocytes. The B transition is very broad and probably consists of several transitions. An inflection, which is seen as a distinct peak (transition B3) in fourth-derivative curves, occurs at 60.8 degrees C and correlates well with the predicted Tm of the critical target. Ethanol (2.2%) lowers the Tm of B3 by 4.0-4.5 K, close to the shift of 3.3 K predicted from its effect on hemolysis. Glycerol (10%) has very little effect on both hemolysis and the Tm of B3, but it stabilizes spectrin (delta Tm = 1.5 K) against thermal denaturation.(ABSTRACT TRUNCATED AT 400 WORDS)
测量了人红细胞在暴露于47.4 - 54.5摄氏度时的溶血情况随时间的变化，并将其与通过差示扫描量热法（DSC）测定的完整红细胞膜中的热转变相关联。血红蛋白泄漏曲线（溶血的一种度量）随时间变化，有一个无泄漏的肩部区域，这表明红细胞有积累亚致死损伤（即不足以导致裂解的损伤）的能力，随后是一个近似假一级动力学的泄漏区域。在47.4 - 54.5摄氏度下分别获得了330 - 21分钟的反向泄漏率（Do）。泄漏的活化能相对较高，为304±22 kJ/mol，排除了代谢过程的参与，但暗示有一个转变是限速步骤。不含膜蛋白的脂质体中血红蛋白泄漏速率极低（是红细胞速率的10⁻²）且活化能低（50±49 kJ/mol），这表明膜蛋白参与其中。建立了一个模型，当以1 K/分钟的扫描速率测量时，该模型预测关键靶点（限速步骤）的转变温度（Tm）为60摄氏度。从完整红细胞获得了DSC扫描结果，并开发了一种程序来拟合和去除主导扫描的血红蛋白变性转变。剩下三个转变（转变A、B和C），Tm值分别为50.0、56.8和63.8摄氏度。这些转变与相同盐溶液中分离的红细胞膜的A、B和C转变相对应，但发生温度略有不同（分别为Tm = 49.5、53 - 58和65.5摄氏度）。此外，分离膜和红细胞中这三个转变的相对焓不同，这表明在分离过程中发生了膜改变。因此，所有分析均在完整红细胞的DSC扫描上进行。转变B非常宽，可能由几个转变组成。在四阶导数曲线中作为一个明显峰值（转变B3）出现的拐点在60.8摄氏度，与关键靶点预测的Tm值相关性良好。乙醇（2.2%）使B3的Tm降低4.0 - 4.5 K，接近根据其对溶血的影响预测的3.3 K的变化。甘油（10%）对溶血和B3的Tm影响很小，但它能稳定血影蛋白（δTm = 1.5 K）防止热变性。（摘要截短为400字）
