Purification and characterization of a carbonic anhydrase II inhibitor from porcine plasma.
Purification and characterization of a carbonic anhydrase II inhibitor from porcine plasma.
复制标题
从猪血浆中纯化和表征碳酸酐酶 II 抑制剂。
DOI：
10.1021/bi00164a034
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Fierke,CA
中科院分区：
文献类型：
--
作者：
Roush,ED;Fierke,CA
材料和方法材料。从猪脖子的动脉和静脉中采集全血（Garrard Sausage Co., Durham， NC），放入一个4升的罐子中，其中含有42.8 mL 0.5 M EDTA， pH为8.0，并在冰上冷却。血浆和细胞部分分别在3000g、4℃下离心15分钟，并在20℃下保存，分别用于纯化异食异黄酮和猪碳酸酐酶。HCA II-Sepharose树脂采用Porath （Porath, 1974）的方法制备，但有以下例外：cnbr活化的Sepharose CL-4B购自Sigma，所有偶联和阻断步骤均在4℃下进行24 h，省略了树脂的甘氨酸洗涤。通过活化树脂孵育前后蛋白溶液在280 nm处的吸光度测定，偶联效率通常大于95%。使用5 g树脂和100 mg HCA II，通过HCA II催化的对硝基苯醋酸酯（PNPA）水解测定，该过程产生约15 mL树脂，6.7 mg HCA II偶联/mL树脂。
MATERIALS AND METHODSMaterials. Wholeblood was collected from the arteries and veins of the neck of a pig (Garrard Sausage Co., Durham, NC) into a 4-L jar containing 42.8 mL of 0.5 M EDTA, pH 8.0, and cooled on ice. Plasma and cellular fractions were separated bycentrifugation for 15 min at 3000g at 4 C and stored at-20 C for future use in the purification of pICA and porcine carbonic anhydrases, respectively. HCA II-Sepharose resin was prepared by the method of Porath (Porath, 1974), with the following exceptions: the CNBr-activated Sepharose CL-4B was purchased from Sigma, all coupling and blocking steps were done for 24 h at 4 C, and theglycine wash of the resin was omitted. Coupling efficiency, as determined by absorbance of the protein solution at 280 nm before and after incubation with activated resin, was routinely greater than 95%. Using 5 g of resin and 100 mg of HCA II, this procedure yields approximately 15 mL of resin with 6.7 mg of HCA II coupled/mL of resin as determined by assays of HCA II-catalyzed p-nitrophenyl acetate (PNPA) hydrolysis.