HYDROPHOBIC INTERACTION CHROMATOGRAPHY SEPARATION AND DETERMINATION OF GLYCOLYTIC-ENZYMES - METHOD VALIDATION

A new method, based on the measurement of a direct and specific chroma tographic signal obtained by hydrophobic interaction chromatography of some glycolytic enzymes from rabbit skeletal muscle, is presented. Th e enzymes examined were: aldolase, glyceraldehyde 3-phosphate dehydrog enase, triosephosphate isomerase, 3-phosphoglyceric phosphokinase, eno lase, phosphoglucomutase, phosphoglucose isomerase, L-lactate dehydrog enase, pyruvate kinase. The possibility of simultaneous determination of six enzymes in a synthetic mixture has been tested. In this approac h to the quantitation of such enzymes the use of the substrate is avoi ded: the method eludes the measurement of enzymatic activity, utilised only for check purposes, and allows direct determination in terms of molarity. For each enzyme, reproducibility, linearity range, recovery greater than or equal to 97 %) and detection limit are reported. The m ethod has been applied to three commercial crudes: by a simple procedu re, the identification of more enzymes has been possible and the simul taneous determination of some of them has been performed. For such enz ymes recoveries were quantitative and their determination is of good p recision with a mean coefficient of variation 2.43-3.0 %.