Carbon dioxide hydration activity of carbonic anhydrase in mixtures of water and deuterium oxide.
Carbon dioxide hydration activity of carbonic anhydrase in mixtures of water and deuterium oxide.
复制标题
水和氧化氘的混合物中碳酸酐酶的二氧化碳水合活性。
DOI：
10.1021/bi00563a008
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发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
Silverman,DN
中科院分区：
文献类型：
--
作者：
Venkatasubban,KS;Silverman,DN
材料和方法酶和化学品。通过 Khalifah 等人 (1977) 描述的亲和层析程序从红细胞中分离牛碳酸酐酶，并用蒸馏水彻底透析。酶浓度根据 280 nm 处 5.6 X 104 M" 1 cm" 1 的摩尔消光系数估算（Coleman，1967）。所有缓冲液和指示剂均购自Aldrich Chemical Co.：咪唑从苯中重结晶两次； 1、减压蒸馏2-二甲基咪唑；对硝基苯酚和间甲酚紫按原样使用。通过将CO 2 气体鼓入水浴中保持在25℃的容器中的去离子蒸馏H 2 O或D 2 O中来制备CO 2 的饱和溶液。通过使溶液通过重力流入气密注射器来取出等分试样。 Pocker & Bjorkquist (1977b)报道了在25°C下在H 2 O (3.38 X 10” 2 M)和D 2 O (3.81 X 10” 2 M)中饱和时CO 2 的浓度。通过用脱气的H 2 O或D 2 O稀释饱和CO 2 溶液来制备不同CO 2 浓度的溶液。该程序是通过使用两个具有气密连接的注射器来进行的。停流方法。 CO 2 水合的初始速度测量在Durrum-Gibson停流分光光度计上进行。通过使用方程v d (CO 2 ) I dZ Jinitial 计算水合的初始速度，其中dA/dt通过对接口的Hewlett-Packard 9835B计算器中的吸光度值和累积时间的最小二乘分析确定。对硝基苯酚的吸光度在 400 nm 处跟踪，间甲酚紫在 578 nm 处跟踪。注意仅在t=0附近的区域测量dA/dt。获取整个反应的数据，但仅使用从t-5ms直到完成10%的数据来测量初始速度。通过 Khalifah (1971) 的方法，针对咪唑与对硝基苯酚和 1, 2-二甲基咪唑与间甲酚紫的缓冲指示剂对，估计与吸收变化与 (H+) 变化相关的缓冲因子 Q0。
Materials and MethodsEnzyme and Chemicals. Bovine carbonic anhydrase was isolated from erythrocytes by the affinity chromatography procedure described by Khalifah et al.(1977) and dialyzed exhaustively against distilled water. Enzyme concentrations were estimated from the molar extinction coefficient at 280 nm of 5.6 X 104 M" 1 cm" 1 (Coleman, 1967). All buffers and indicators were obtained fromAldrich Chemical Co.: imid-azole was twice recrystallized from benzene; 1, 2-dimethylimidazole was distilled underreduced pressure; p-nitrophenol and w-cresol purple were used as obtained. Saturated solutions of C02 were prepared by bubbling C02 gas into deionized, distilled H20 or D20 in a vessel maintained at 25 C in a water bath. Aliquots were withdrawn by al-lowing the solution to flow by gravity into a gas-tight syringe. Pocker & Bjorkquist (1977b) have reported the concentration of C02 when saturated in H20 (3.38 X 10" 2 M) and inD20 (3.81 X 10" 2 M) at 25 C. Solutions of different C02 con-centration were made by dilution of saturated C02 solutions with degassed H20 or D20. This procedure was carried out by using two syringes with a gas-tight connection. Stopped-flow Methods. Initial velocity measurements of the hydration of C02 were carried out on a Durrum-Gibson stopped-flow spectrophotometer. The initial velocity of hy-dration was calculated by using the equation v d (C02) I dZ J initial with dA/dt determined by least-squares analysis of values of absorbance and time accumulated in an interfaced Hewlett-Packard 9835B calculator. The absorbance of p-nitrophenol was followed at 400 nm and m-cresol purple was followed at 578 nm. Care was taken to measure dA/dt only in the region near t= 0. Data were taken for the entire reaction but only data from t— 5 ms up to 10% of completion were used to measure initial velocity. The buffer factor Q0 relating changes in absorption to changes in (H+) was estimated by the method of Khalifah (1971) for the buffer-indicator pairs of imidazole with p-nitrophenol and 1, 2-dimethylimidazole with m-cresol purple.