RELATIONSHIP OF HYPERTHERMIA-INDUCED HEMOLYSIS OF HUMAN-ERYTHROCYTES TO THE THERMAL-DENATURATION OF MEMBRANE-PROTEINS
RELATIONSHIP OF HYPERTHERMIA-INDUCED HEMOLYSIS OF HUMAN-ERYTHROCYTES TO THE THERMAL-DENATURATION OF MEMBRANE-PROTEINS
复制标题
DOI：
10.1016/0005-2736(89)90399-4
复制
发表时间：
1989-04-14
期刊：
影响因子：
--
通讯作者：
MARKUS, J
中科院分区：
文献类型：
--
作者：
LEPOCK, JR;FREY, HE;MARKUS, J
人红细胞的溶血作为暴露于47.4 - 54.5 ℃的时间的函数。C的测量和相关的完整的红细胞的膜中的热转变，通过差示扫描量热法（DSC）确定。血红蛋白渗漏（溶血的量度）作为时间的函数的曲线具有不显示渗漏的肩部区域，指示累积亚致死损伤的能力（即，损伤不足以引起溶解），随后是接近伪一级动力学的泄漏区域。从47.4 - 54.5 ° C获得330-21分钟的反向泄漏率（Do）。C分别进行。相对较高的活化能为304 . ±. 22 kJ/mol的泄漏，消除了代谢过程的参与，但涉及的过渡作为限速步骤。膜蛋白的参与由非常低的速率（来自红细胞的速率的10-2）和低活化能（50 ± 10）表明。49 kJ/mol）的血红蛋白从不含膜蛋白的脂质体中渗漏。开发了预测临界目标（限速步骤）的转变温度（Tm）为60 ℃的模型。从完整的红细胞获得DSC扫描，并开发了一种程序来拟合和去除占主导地位的扫描的血红蛋白变性的转变。三个转变（转变A、B和C）保留，Tm值为50.0、56.8和63.8 °。C分别进行。这些对应于相同盐溶液中分离的红细胞膜的A、B和C转变，但发生在略有不同的温度下（Tm = 49.5、53-58和65.5 °）。C）。此外，三个转换的相对振幅不同的隔离膜和红细胞，表明膜的变化发生在隔离。因此，所有分析均在完整红细胞的DSC扫描上进行。B跃迁非常宽，可能由几个跃迁组成。在四阶导数曲线中被视为明显峰（转变B3）的拐点出现在60.8 °处。并且与关键靶标的预测Tm良好相关。乙醇（2.2%）使B3的Tm降低4.0-4.5 K，接近从其对溶血的影响预测的3.3 K的偏移。甘油（10%）对溶血和B3的Tm两者具有非常小的影响，但其稳定血影蛋白（Δ Tm = 1.5K）以对抗热变性。因此，我们提出了一种高温溶血的模型，其中血影蛋白的主要变性（Tm = 50 ° C）发生在血液中。C），虽然可能是裂解所必需的，但这是不够的，并且限速步骤是在60 ℃的转变。C，可能是由于额外的膜蛋白的变性。
Hemolysis of human erythrocytes as a function of time of exposure to 47.4-54.5.degree. 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.degree. 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.degree. 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.degree. 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.degree. 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.degree. 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. Thus, we propose a model for hyperthermic hemolysis in which the major denaturation of spectrin (Tm = 50.degree. C), while possibly necessary for lysis, is not sufficient, and that the rate-limiting step is a transition at 60.degree. C, possibly due to the denaturation of an additional membrane protein.