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

摘要

A new method, based on the measurement of a direct and specific chromatographic signal obtained by hydrophobic interaction chromatography of some glycolytic enzymes from rabbit skeletal muscle, is presented. The enzymes examined were: aldolase, glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, 3-phosphoglyceric phosphokinase, enolase, phosphoglucomutase, phosphoglucose isomerase, L-lactate dehydrogenase, pyruvate kinase. The possibility of simultaneous determination of six enzymes in a synthetic mixture has been tested. In this approach to the quantitation of such enzymes the use of the substrate is avoided: 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 method has been applied to three commercial crudes: by a simple procedure, the identification of more enzymes has been possible and the simultaneous determination of some of them has been performed. For such enzymes recoveries were quantitative and their determination is of good precision with a mean coefficient of variation 2.43-3.0 %. [References: 15]