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Development of a satisfactory and general continuous assay for aminotransferases by coupling with (R)-2-hydroxyglutarate dehydrogenase
- Source :
-
Analytical Biochemistry . Dec2012, Vol. 431 Issue 2, p127-131. 5p. - Publication Year :
- 2012
-
Abstract
- Abstract: A continuous general spectrophotometric assay for measuring the activity of aminotransferases has been developed. It is based on the transamination of a keto compound (amino acceptor) and l-glutamate (amino donor), yielding the corresponding amino compound and 2-oxoglutarate. The rate of formation of 2-oxoglutarate is measured in a coupled reaction with overproduced recombinant nicotinamide adenine dinucleotide (NAD+)-dependent (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans, with the rate of absorbance decrease at 340nm indirectly reflecting the aminotransferase activity. This new method allows continuous monitoring of the course of transamination. Because glutamate and 2-oxoglutarate are obligatory participants in most biological transamination reactions, a coupled assay based on measuring the formation of 2-oxoglutarate has very wide applicability. The article demonstrates its utility with branched-chain amino acid aminotransferase and l-valine:pyruvate aminotransferase. [Copyright &y& Elsevier]
Details
- Language :
- English
- ISSN :
- 00032697
- Volume :
- 431
- Issue :
- 2
- Database :
- Academic Search Index
- Journal :
- Analytical Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 83167083
- Full Text :
- https://doi.org/10.1016/j.ab.2012.09.009