An Improved Method for Measuring the CO₂/O₂ Specificity of Ribulosebisphosphate Carboxylase-Oxygenase

Abstract

A simple, but very reproducible, method for measuring the relative specificity of ribulosebisphosphate carboxylase-oxygenase for CO₂, as opposed to O₂, is described. The method uses [1-¹⁴C]ribulose bisphosphate as substrate and combines the advantages of supplying both gaseous substrates from the gas phase with HPLC separation of the labelled products. Volumetric or gravimetric accuracy is not required at any stage of the procedure and variations in ionic strength and pH have little effect on the measurements. This leads to excellent reproducibility without the need for normalisation. The average standard deviation was 1.3% of the measured CO₂/O₂ specificity. Use of very low ribulose bisphosphate concentrations ensures that the gaseous substrates cannot be depleted appreciably during the reaction and enhances the attractiveness of the procedure for measurements with crippled mutant enzymes. The procedure's ability to resolve small differences in relative specificity is demonstrated by its easy detection of the 5% increase in specificity that accompanies substitution of four residues at positions 338-341 of the cyanobacterial large subunit with the analogous higher-plant residues. This resolving power is essential for detecting small differences in the specificities of higher-plant ribulosebisphosphate carboxylases which may be the signature of continuing evolutionary refinement.