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 (tau) 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 tau 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.
Harpel
M R
MR
Biology Division, Oak Ridge National Laboratory, Tennessee 37831-8077.
Lee
E H
EH
Hartman
F C
FC
D000837:Anion Exchange Resins; D002245:Carbon Dioxide / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods; D005988:Glyceric Acids / Q000302:isolation & purification; D006016:Glycolates / Q000302:isolation & purification; D010100:Oxygen / Q000737:chemistry*; D011773:Pyruvates / Q000737:chemistry; D019289:Pyruvic Acid; D012117:Resins, Synthetic; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D000837:Anion Exchange Resins; D005988:Glyceric Acids; D006016:Glycolates; D011773:Pyruvates; D012117:Resins, Synthetic; D002245:Carbon Dioxide; C005156:3-phosphoglycerate; C042855:Mono Q; D019289:Pyruvic Acid; D012273:Ribulose-Bisphosphate Carboxylase; C013193:phosphoglycolate; D010100:Oxygen