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

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.