一种利用二氧化碳与水之间的氧-18交换来测量碳酸酐酶活性的微量方法。

A micromethod for measuring carbonic anhydrase activity using 18O exchange between CO2 and H2O.

作者信息

Bitterman N, Lin L, Forster R E

机构信息

Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6085.

出版信息

J Appl Physiol (1985). 1988 Oct;65(4):1902-6. doi: 10.1152/jappl.1988.65.4.1902.

DOI:10.1152/jappl.1988.65.4.1902

PMID:2846500

Abstract

We have developed a method of measuring the activity and characteristics of carbonic anhydrase (CA) using the disappearance of 18O from CO2 in 1 ml of gas contained in a glass chamber as it exchanges with H2O in 0.01 ml 0.25 M NaHCO3 solution in a thin (25 micron) porous membrane. Serial gas samples (approximately 0.02 ml) are analyzed in a mass spectrometer to obtain the rate of disappearance of the label. The enzyme activity can be measured inside intact cell or particle membranes. As little as 10(-15) mol of high-activity type CA can be detected at 25 degrees C, and the activity of 200 times this amount can be measured. The uncatalyzed hydration reaction velocity constant was 0.056 +/- 0.004 s-1, in agreement with published data.

摘要

我们开发了一种测量碳酸酐酶（CA）活性和特性的方法，该方法利用玻璃腔中1 ml气体中的CO₂与0.01 ml 0.25 M NaHCO₃溶液中的H₂O在薄（25微米）多孔膜中交换时¹⁸O从CO₂中的消失情况。在质谱仪中分析系列气体样本（约0.02 ml）以获得标记消失的速率。酶活性可以在完整的细胞膜或颗粒膜内进行测量。在25℃时，低至10⁻¹⁵摩尔的高活性型CA都可以被检测到，并且可以测量比此量多200倍的活性。未催化的水合反应速度常数为0.056±0.004 s⁻¹，与已发表的数据一致。