Medium/high throughput D-amino acid oxidase colorimetric method for determination of D-amino acids. Application for amino acid racemases

Abstract

Free amino acids detection and quantification in samples still calls for elaborated, powerful and usually high cost and sometimes time-consuming technologies such as gas chromatography, HPLC or highly selective and sensitive column-switching techniques. Here, we undertook to develop a simple, one-step, microtiter plate method to detect and/or quantify most D-amino acids in solutions based on the enzyme D-amino acid oxidase and the generation of stoechiometric amounts of hydrogen peroxide. Inclusion of horseradish peroxidase and OPD allows the determination of D- amino acid concentration through the absorbance of the reaction products as compared to serial dilutions of equimolar amounts of L/D amino acid standards. The methodology is easily adapted for a medium/high throughput analysis of amino acid racemase activities. © 2010 Berneman A, et al.