Simultaneous determinations of glycolytic enzymes in crude extract concentrate by hydrophobic interaction chromatography

Previously described methods for the simultaneous determination of some glycolytic enzymes by hydrophobic interaction chromatography (HIC) have been further developed in order to identify and quantitate similar enzymes in extract concentrates. Previously reported problems arising from difficult elution, broad peaks production and coelution have been overcome by collecting separate fractions of HIC eluate of the original sample. These fractions, which contain selected mixtures of troubling enzymes, are reinjected using an appropriate choice of operating conditions involving hydrophobic phase, sample treatment and elution conditions. Using slight changes to the previous procedures, mixtures of myokinase/triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase/3-phosphoglyceric phosphokinase, pyruvate kinase/L-lactate dehydrogenase/creatine phosphokinase and phosphoglucose isomerase/phosphoglucose mutase can be resolved. This allows easy identification and simultaneous determination of related enzymes. Results obtained in the application to commercial crudes produced near quantitative recoveries of examined enzymes. Determination of these compounds was found to be precise with a mean coefficient of variation ranging from 2.6% to 4.2%.