Determination of quality of pyridoxal-5'-phosphate enzyme preparations by spectroscopic methods.
The present study evaluates purified aspartate transaminase (AST, EC 2.6.1.1) preparations from three commercial sources. The enzyme molecule contains pyridoxal-5'-phosphate coenzyme (PLP), which provides AST characteristic absorption spectra in the wavelength range of 300-500 nm. The coenzyme bound in the active site also shows circular dichroism (CD) spectra in the same range. Besides, AST like other proteins may be modified in vitro or in vivo by reactions with other molecules, e.g. reactive sugars, and may form fluorescent products (advanced glycation end products, AGE). Spectroscopic methods were used to assess the quality of AST preparations from three different sources, Serva, Roche, and Sigma. Absorption spectra showed that the peak 360 nm characteristic of the active PLP form of AST prevailed in the Serva and Sigma preparations, while 330 nm was the major peak in the Roche preparation. CD spectra demonstrated the major maximum at 360 nm in the Serva and Roche samples, thus suggesting the predominance of the active PLP form in both preparations. The Sigma sample showed a CD profile less characteristic of AST. Fluorescence measurements revealed formation of AGE in the case of the Roche preparation, while fluorescence of the other two preparations was low. In general, the Serva sample presented the most convenient properties of purified AST among the preparations tested. The results will be used for the selection of a commercial enzyme preparation applicable in our future spectroscopic studies of glycation of AST as a model protein and in our research of the influence of antioxidants on this process.
Drsata
Jaroslav
J
Department of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy, Heyrovského 1203, CZ-50005 Hradec Králové, Czech Republic. drsata@faf.cuni.cz
Bousová
Iva
I
Malon
Petr
P
D000818:Animals; D011732:Pyridoxal Phosphate / Q000032:analysis* / Q000138:chemical synthesis / Q000592:standards*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013552:Swine
D011732:Pyridoxal Phosphate