Itada N, Forster R E
J Biol Chem. 1977 Jun 10;252(11):3881-90.
We have used a stirred, temperature-regulated, reaction vessel separated by a Teflon membrane from the ion source of a mass spectrometer to monitor continuously the time course of disappearance of C18O16O, mass 46, at chemical equilibrium as the 18O exchanges with 16O in water. This instrument is sensitive to less than 0.01 mm Hg of partial pressure of C18O16O with a response time of less than 3 s. The equation of Mills and Urey was used to calculate the hydration velocity constant for uncatalyzed or catalyzed homogenous solutions from the exponential disappearance of mass 46. Addition of red blood cells to the reaction mixture produces biphasic (double exponential) disappearance curve for mass 46. A theory of this process has been developed which describes the time course of [C18O16O] as a function of the catalytic factor for intracellular carbonic anhydrase (A) and the permeability of the cell membrane to HCO3- (P) in addition to the known values; water volume of the cells in the suspension, extracellular pH, the extracellular hydration reaction velocity constant, ku, and dehydration reaction velocity constant, ku. Using this theory, A and P were estimated from the disappearance curve for mass 46 at different values of hematocrit in the reaction mixture, both by a trial and error curve fitting procedure and by a more convenient graphical linearization method. The values of A and P obtained were very sensitive to small amounts of lysis (less than 1%), but the graphical method of analysis minimized this effect. For the blood cells of five normal subjects suspended in 24 mM bicarbonate in 145 mM NaCl at pH 7.4 and 37 degrees, using the graphical method we obtained an average value of 9,906 for A as compared to 19,900 for a comparable concentration of hemolysate. Correcting for a lower pH and chloride concentration inside the cell the latter figure would reduce to 17,500, still 80% higher than the intracellular value. The reason for this discrepancy is not clear. The average permeability of the red cell to bicarbonate ion was 3 X 10(-4) cm/s.
我们使用了一个搅拌式、温度可控的反应容器，该容器通过聚四氟乙烯膜与质谱仪的离子源隔开，以连续监测在化学平衡状态下，随着(^{18}O)与水中的(^{16}O)发生交换，质量数为46的(C^{18}O^{16}O)消失的时间进程。该仪器对(C^{18}O^{16}O)分压的敏感度小于(0.01)毫米汞柱，响应时间小于3秒。利用米尔斯和尤里的方程，根据质量数46的指数消失情况，计算未催化或催化均相溶液的水合速度常数。向反应混合物中加入红细胞会使质量数46出现双相（双指数）消失曲线。已开发出一种关于此过程的理论，该理论除了已知值（悬浮液中细胞的水体积、细胞外pH值、细胞外水合反应速度常数(k_u)和脱水反应速度常数(k_d)）外，还将([C^{18}O^{16}O])的时间进程描述为细胞内碳酸酐酶的催化因子（A）和细胞膜对(HCO_3^-)的通透性（P）的函数。利用该理论，通过试错曲线拟合程序以及更简便的图形线性化方法，根据反应混合物中不同血细胞比容值下质量数46的消失曲线来估算A和P。所获得的A和P值对少量细胞裂解（小于1%）非常敏感，但图形分析方法可将这种影响降至最低。对于五名正常受试者的血细胞，悬浮于pH值为7.4、温度为37摄氏度的145毫摩尔氯化钠中的24毫摩尔碳酸氢盐溶液中，使用图形方法我们得到A的平均值为9906，而对于可比浓度的溶血产物，该值为19900。校正细胞内较低的pH值和氯离子浓度后，后者的值将降至17500，但仍比细胞内值高80%。这种差异的原因尚不清楚。红细胞对碳酸氢根离子的平均通透性为(3×10^{-4})厘米/秒。
