Assay of Amino Acid Racemases

Abstract

D-Amino acids play important physiological roles in the mammalian body. Recent investigations revealed that, in mammals, D-amino acids are synthesized from their corresponding L-enantiomers via amino acid racemase. This article describes a method used to measure amino acid racemase activity by high-performance liquid chromatography (HPLC). The assay involves fluorogenic chiral derivatization of amino acids with a newly developed reagent, and enantioseparation of D- and L-amino acid derivatives by HPLC. The method is accurate and reliable, and can be automated using a programmable autosampling injector.

A sensitive assay for measuring aspartate-specific amino acid racemase activity

“…The data obtained were fitted to the Michaelis-Menten equation, and the V max and K m values were estimated using the non-linear least-squares fitting algorithm in pro Fit 6.1 software (Quantum Soft, Zürich, Switzerland; http://www.quansoft.com). The molecular activity (k cat ) values were calculated from the V max values and the estimated molecular mass of the recombinant S. thermophilus Asp racemase (27,945 Da).…”

“…High-performance liquid chromatography (HPLC) is the most widely used technique for the determination of Asp racemase activity by measuring d-or l-Asp formed from the corresponding enantiomer in the reaction mixture [27]. This technique, however, is time-consuming, requires costly instruments, and is not highly efficient.…”