反应详情

DOPA decarboxylase activity was routinely measured by liquid chromatography and electrochemical detection of catecholamine products. DOPA decarboxylase (1-5 micrograms, 1-20 units) was incubated with L-DOPA (2000 micromolar, 150 microliters) and PLP (10 micromolar) in 0.1M phosphate at pH 6.8 and 37° C. After 1-10 minutes, the reaction was quenched with citric acid (2M, 25 microliters), diluted two-fold with distilled water and 10 microliter aliquots were analyzed by HPLC on a reverse phase Brownlee RP-18 column. Isocratic elution with pH 4.35 buffer [0.1M NaOAc (63% v/v), 0.1M citric acid (32% v/v) and methanol (15% v/v)] at a flow rate of 2.5 ml/min. afforded baseline resolution of dopamine (RT=2.8 min.) from L-DOPA (RT=1.9 min.).

1. customthe reaction was quenched with citric acid (2M, 25 microliters)
2. additiondiluted two-fold with distilled water and 10 microliter aliquots
3. washIsocratic elution with pH 4.35 buffer [0.1M NaOAc (63% v/v), 0.1M citric acid (32% v/v) and methanol (15% v/v)] at a flow rate of 2.5 ml/min.