AN IMPROVED METHOD FOR MEASURING THE CO2 O2 SPECIFICITY OF RIBULOSEBISPHOSPHATE CARBOXYLASE-OXYGENASE/

A simple, but very reproducible, method for measuring the relative spe cificity of ribulosebisphosphate carboxylase-oxygenase for CO2, as opp osed to O-2, is described. The method uses [1-C-14]ribulose bisphospha te as substrate and combines the advantages of supplying both gaseous substrates from the gas phase with HPLC separation of the labelled pro ducts. 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 wi thout the need for normalisation. The average standard deviation was 1 .3% of the measured CO2/O-2 specificity. Use of very low ribulose bisp hosphate concentrations ensures that the gaseous substrates cannot be depleted appreciably during the reaction and enhances the attractivene ss of the procedure for measurements with crippled mutant enzymes. The procedure's ability to resolve small differences in relative specific ity is demonstrated by its easy detection of the 5% increase in specif icity that accompanies substitution of four residues at positions 338- 341 of the cyanobacterial large subunit with the analogous higher-plan t residues. This resolving power is essential for detecting small diff erences in the specificities of higher-plant ribulosebisphosphate carb oxylases which may be the signature of continuing evolutionary refinem ent.