title: Tryptophan metabolism via transamination. In vitro aminotransferase assay using dinitrophenylhydrazine method.
abstract: Transamination of tryptophan belongs to minor pathways of amino acid metabolism. The present paper describes conditions for application of dinitrophenylhydrazine method, originally prepared for alanine aminortansferase and aspartate aminotransferase assay, to the measurement of tryptophan transamination catalysed by any of the enzymes mentioned above. The method was tested using purified pig heart AST. While the free enzyme showed a characteristic absorption profile with the maxima at 360 and 430 nm, the course of transamination of tryptophan was confirmed by the measurement of UV-VIS spectral changes of the coenzyme in the active site of the enzyme in the presence of the amino acid substrate only, when tryptophan caused a shift of the peak from 360 nm to 330 nm due to a change of the pyridoxal form to the pyridoxamine form (= the first step of ping-pong transaminating reaction). A general limitation of dinitrophenylhydrazine method is the interference of hydrazones formed from the coenzyme pyridoxal-5'-phosphate and from the oxo- substrate 2-oxoglutarate, showing the absorption maxima at 492 nm and 388 nm, respectively with the hydrazones formed by the oxo- products (pyruvate and/or oxaloacetate in the case of ALT/AST, the absorption maxima at 443 nm in our measurements). In the case of tryptophan transamination, indolepyruvate as the oxo- product of a catalysed reaction forms dinitrophenylhydrazone, which has, besides a maximum at 435 nm, a distinct peak at 542 nm, convenient for the product concentration measurement. This is favourable for resolution from other (interfering) hydrazones. Suitable conditions for tryptophan transamination in tissue and enzyme preparations were found. Reaching optimal conditions for tryptophan transamination measurements in vitro is generally limited by low solubility of the amino acid in water solutions: With AST preparation, the velocity of catalysed reaction at 5-50 x 10(-3) M tryptophan concentration was of 1st order to the amino acid substrate. Km for tryptophan was found > or = 2 x 10(-1) M. Therefore the enzyme activity measurement at two different tryptophan concentrations is recommended for unknown samples. Tryptophan transamination by purified pig AST was compared with that catalysed by preparations obtained from mammalian tissues.
authors.affiliation: Charles University Faculty of Pharmacy and the Research Centre LN00B125, Heyrovského 1203, Hradec Králové, Czech Republic. drsata@faf.cuni.cz
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Drsata
mesh_terms: D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001921:Brain / Q000201:enzymology; D006834:Hydrazines / Q000378:metabolism*; D066298:In Vitro Techniques; D007421:Intestine, Small / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D009206:Myocardium / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D034421:Sus scrofa; D000637:Transaminases / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*; D051279:Tryptophan Transaminase
chemical_list: D006834:Hydrazines; C446799:dinitrophenylhydrazine; D014364:Tryptophan; D000637:Transaminases; D001219:Aspartate Aminotransferases; D051279:Tryptophan Transaminase