高效液相色谱法测定鼠脑谷氨酸脱羧酶活性

Determination of Activity of Glutamic Acid Decarboxylase from Rat Brain by Reversed Phase High Performance Liquid Chromatography

应用高效液相色谱-电化学检测技术建立了鼠脑谷氨酸脱羧酶(GAD)活性的测定方法.方法快速,灵敏.总分析时间为9min.γ-氨基丁酸的最低柱上检测限为50fmol.天内及天间相对标准差分别小于4.3%和9.8%.测得CAD最大催化速率为0.723nmol/min/mg蛋白.讨论了最佳衍生化pH及色谱条件.

Gamma-aminobutyric acid (GABA) can be catalyzed from glutamic acid(Glu) by glutamic acid decarboxylase(GAD), which is in crude homogenate from whole rat brain obtained within 10 minutes after decollation. Quantitative method for GABA and Glu as well as delta-aminovaleric acid ( internal standard) based on the precolumn derivatisation of those amino acids with o-phthalaldehyde in the presence of sulfite ion was developed on our previous work through using high performance liquid chromatography with electrochemical detection. The method reported was rapid and repeated. The relative standard deviation for between-run and within-run are less than 4.3% and 9.8%, respectively. A single run is 9 minutes totally. Minimum detect limit for GABA was 50 fmol on column. Optimization of pH for derivatisation and of the chromatographic separation was discussed. A value of 0. 723 nmol/min/mg protein has been measured as maximum reactory velocity for GAD activity.