KINETIC-ANALYSIS OF PECTATE LYASES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Ion-pair reverse-phase high-performance liquid chromatography has been used to resolve and quantify unsaturated oligosaccharides generated by pectate lyases. Increasing resolution is obtained with increasing size of oligomer, and there is a proportional relationship between the molecular weight of a given oligomer and the logarithm of the retention time. The rates of formation of individual unsaturated oligogalacturonic acids ranging in degree of polymerization from 2 to 11 and in concentrations from 0.02 to 1.6mM have been measured as products of endolytic eliminative D-galacturonan depolymerases. This system has provided a convenient and sensitive method to assay the kinetic properties of pectate lyases (EC 4.2.2.2) secreted by Erwinia chrysanthemi, and has been used to clearly distinguish different activities based upon their respective mechanisms and the formation of individual limit products.