Anomalous Nonstoichiometry Detected by Dual Label Analysis of Ribulose 1,5-Bisphosphate Carboxylase

When ribulose-1,5-bisphosphate carboxylase is assayed under N(2) using [(3)H]ribulose 1,5-bisphosphate and (14)CO(2), [(3)H]3-phosphoglycerate and [(14)C]3-phosphoglycerate are produced in nonstoichiometric amounts in a ratio which approaches 7 at low concentrations of CO(2) (2 micromolar) assuming a 1:1 ratio at V(max) (280 micromolar). The log of the molar ratio varies as a linear function of log[CO(2)]. Nonstoichiometry could be explained by CO(2) contaminatio of the reactants or tritium contamination of the products. However, the magnitude of CO(2) contamination required (18 +/- 4 micromolar) is far in excess of controlled CO(2) (<0.1 micromolar), and the required tritium contaminant would have to vary from 30 to 85% of the purified 3-phosphoglycerate at the 58 and 2 micromolar CO(2) assay levels, respectively. This contrasts with detectable tritium contamination which is only 1 to 4% and correctable. Nonstoichiometry is evident using either 1 or 5 labeled [(3)H]ribulose 1,5-bisphosphate. When 3-phosphoglycerate is reisolated as glycerate the (3)H/(14)C ratio remains unchanged.