Optimization of methods for aspartate aminotransferase and alanine aminotransferase.
Conditions for accurate measurement of catalytic activity of aspartate aminotransferase and alanine aminotransferase in human serum have been reinvestigated. The basic variables (kind of buffer, buffer concentration, pH, ion effects, and the influence of pyridoxal-5-phosphate) can now be considered optimized. On this basis, the kinetic parameters of both aminotransferases were determined, i.e., Michaelis and inhibitor constants for substrates and reaction products. With a mathematical approach for two-substrate enzyme reactions the substrate concentrations were calculated from the viewpoints "most economical," "most convenient," and "lowest variability." Also the conditions for the indicator reactions have been newly defined with respect to a kinetic model. All calculated data were rechecked experimentally and it can be shown that both approaches fully agree. Furthermore, we show that the mathematical approach allows more precise recommendations for optimized methods. For technical reasons, the catalytic activity of aspartate aminotransferase in human serum can only be measured as a 0.96 fraction of its theoretical maximum velocity, the catalytic activity of alanine aminotransferase as a 0.91 fraction. The assay conditions for a Reference Method are finally described and recommendations are made for optimized routine methods for determination of the catalytic activity of these transferases in human serum.
D000410:Alanine Transaminase / Q000037:antagonists & inhibitors / Q000097:blood*; D000838:Anions; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000097:blood*; D005971:Glutamates; D006331:Heart Diseases / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids; D007700:Kinetics; D008107:Liver Diseases / Q000201:enzymology; D008433:Mathematics; D008722:Methods; D009994:Osmolar Concentration; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D011773:Pyruvates
D000838:Anions; D005971:Glutamates; D007656:Ketoglutaric Acids; D011773:Pyruvates; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase