比色法快速测定酶转化反应中γ-氨基丁酸质量分数

Rapid Determination of γ-Aminobutyric Acid in Enzymatic Reaction by Spectrometric Method

建立了谷氨酸脱羧酶催化L-谷氨酸生成γ-氨基丁酸过程中产物质量分数快速测定的比色检测方法,其原理为Berthelot显色测定ω-氨基酸的反应。结果表明,该方法灵敏度较高,准确度好,快捷有效,可替代氨基酸分析仪分析法。取不同时段反应液300μL在冰浴中加200μL 0.2 mol.L-1硼酸缓冲液(pH10.0)终止反应,加入体积分数6%重蒸苯酚100μL和质量分数10%次氯酸钠400μL,沸水浴加热10 m in后冰浴冷却5 m in,630 nm测定吸光度值,以标准曲线法确定γ-氨基丁酸的质量分数。该方法适合大批量样品的快速分析,但需排除游离氨的影响。

A colorimetric method with high sensitivity, veracity and low cost for determining γ -aminobutyric acid （GABA） transformed by glutamate decarboxylase （GAD） from L - glutamate （ L - Glu） was established. It was developed based on Berthelot reaction of ω -amino acid. The enzymatic reaction was terminated by immersion in ice - water and addition of 0.2mol · L^-1 sodium borate buffer 200 μL, then 6% redistillated phenol solution 100 μL and 10% sodium hypochloride 400 μL were added to the liquid. Color development was carried out in boilingwater for 10 min then immediateh put in ice - water bath for 5 min. The optical density was read at 630 nm. The content of GABA produced was ealeulated using the standard eurve. This method is suitable for quick detection of large numbers of samples while disturbance of dissociated ammonia should be removed.