ANION-EXCHANGE ANALYSIS OF RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE REACTIONS - CO2/O2 SPECIFICITY DETERMINATION AND IDENTIFICATION OF SIDE PRODUCTS

被引：34
作者：
HARPEL, MR
LEE, EH
HARTMAN, FC
机构：
[1] Protein Engineering Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge
来源：
ANALYTICAL BIOCHEMISTRY | 1993年 / 209卷 / 02期
关键词：
D O I：
10.1006/abio.1993.1136
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
An improved anion-exchange chromatographic method for determining the carboxylation/oxygenation specificity (τ) of ribulose 1,5-bisphosphate carboxylase/oxygenase is presented. This assay, which entails radiometric detection of [1-3H]ribulose-bisphosphate turnover products separated on MonoQ anion-exchange resin, is more convenient, less error-prone, and more generally applicable than previous methods of τ determination. It is suitable for both wild-type and site-directed mutant enzymes of widely varying activity and specificity levels and allows simultaneous visualization of various side products of ribulose-bisphosphate processing. A facile method for scrubbing dissolved O2 from carboxylase reaction solutions, which does not require extensive purging and exchange of dissolved gases, is also described. © 1994 Academic Press, Inc. All rights reserved.
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收藏
页码：367 / 374
页数：8
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