谷氨酸脱羧酶活力测定中GABA比色定量方法研究

Colorimetric Determination of GABA in GAD Activity Assay

建立了谷氨酸脱羧酶活力测定中γ-氨基丁酸比色测定方法,该方法灵敏度高、精确性和重现性好。测定程序为:取酶反应液0.4ml,加入Na2CO3(1mol/L)0.1ml、pH10.0硼酸盐缓冲液(0.2mol/L)0.5ml、6%苯酚1ml,混匀,于室温下(20℃)在5min内加入5.2%NaClO溶液1ml,混匀后放置4～8min,然后沸水浴l0min,立即冰浴20min,待溶液出现蓝绿色后,加入2.0ml60%乙醇溶液,混匀后于20℃水浴中放置20～40min,测定640nm处的吸收值。经采用比色法和高效液相色谱法对谷氨酸脱羧酶酶反应液进行测定,两种方法的测定结果相对误差为4.91%。

A colorimetric method of determining gama-aminobutyric acid （GABA） in glutamate decarboxylase （GAD） activity assay with high sensitivity, accuracy and reproducibility is proposed, and the standard procedure leading to the establishment of the method is described. The reaction mixture （0.4ml） of GAD and L-Glu is mixed with lmol/L Na2CO3 （0.1ml）, 0.2mol/L borate buffer （0.5ml） and 6% phenol （1.0ml）, then added lml of 5.2% sodium hypochlorite solution at 20℃ within 5min. After being kept at 20℃ for 4～8min, the mixture is heated by boiling water bath for 10min and promptly cooled in ice bath for 20rain, then added 2ml of 60% ethanol, and kept at 20℃C for 20～40min.The optical density of the mixture is measured by the absorbance at 640nm. The accuracy and sensitivity of the determination method are also discussed, indicating that the relative error is 4. 91% in comparison with HPLC method.