A simple and sensitive detection of glutamic-pyruvic transaminase activity based on fluorescence quenching of bovine serum albumin

Abstract

It is well known that Cu(ii) can coordinate with l-alanine (Cu–Ala), which can be destroyed through the addition of glutamic-pyruvic transaminase (GPT) since GPT can effectively catalyze the conversion of l-alanine into keto-acetic acid.

Transaminase biocatalysis: optimization and application

“…Recently, a method for detecting transamination from alanine to pyruvate was developed based on fluorescence quenching of bovine serum albumin (BSA) in a reaction system containing the metal coordinate complex Cu(II)-Ala (Method AV). 96 Coordination of Cu(II) with L-alanine will be destroyed by conversion of L-alanine into pyruvate. Then the free Cu(II) ion will combine with BSA and cause the detectable fluorescence quenching.…”

Transaminases for Green Chemistry: Recent Progress and Future Prospects

“…A similar assay for the conversion of alanine to pyruvate was applied to characterize glutamic-pyruvic transaminase. In the assay dissociation of the Cu(II)-L-alanine complex is observed which results in free Cu(II) ions thereby combining with BSA, leading to a fluorescence quench which is monitored at 340 nm [77]. Another colorimetric detection is carried out using amino acid oxidases (AAO) and horseradish peroxidase (HRP) where an alanine is deaminated by AAO to produce hydrogen peroxide (H 2 O 2 ), which can be further detected using HRP [78][79][80].…”

Discovery, Engineering, and Synthetic Application of Transaminase Biocatalysts

“…One of the first high-throughput transaminases screening methods relied upon monitoring the formation of colored complexes between Cu­(II) ions and α-amino acids (595 nm); however, the method suffered from interference from other amino acids in the cell extract and enzyme inactivation due to the chromogenic agent. An updated version of this assay was later applied in the characterization of a glutamic-pyruvic transaminase, which catalyzes the conversion of alanine to pyruvate (Scheme e). Dissociation of the Cu­(II)- l -alanine complex results in free Cu­(II) ions which combine with BSA leading to fluorescence quenching (monitor at 340 nm).…”