HPLC Estradiol, Estradiol 3-sulfate, Estrone sulfate KAT-I Inhibition

0.7 µg of KAT-I was incubated at 37°C for 10 minutes in a 50 µL reaction mixture containing PLP (50 µM), α-ketoglutarate (5 mM), L-KYN (5 mM) in PBS, pH 7.4, and the inhibitor studied (1-2000 µM). Following incubation, the reaction was terminated by adding formic acid (0.8 M) in a 1:1 ratio. 50 µL of the mixture was transferred into a vial with 950 µL of water for HPLC analysis.The production of KYNA was measured using HPLC with UV detection at a wavelength of 330 nM, using a C18 reverse-phase column, and 50% (v/v) water and 50% (v/v) methanol mobile phase. The data was collected in triplicate.

将0.7 μg的KAT-I置于50 μL的反应体系中，于37 ℃孵育10分钟。该反应体系包含磷酸吡哆醛（PLP，50 μM）、α-酮戊二酸（5 mM）、L-犬尿氨酸（L-KYN，5 mM），溶剂为pH 7.4的磷酸盐缓冲液（PBS），并加入浓度范围为1~2000 μM的待研究抑制剂。孵育完成后，按1:1体积比加入0.8 M甲酸终止反应。取50 μL反应混合液转移至盛有950 μL水的进样瓶中，用于高效液相色谱（HPLC）分析。采用C18反相色谱柱为固定相、体积比50%（v/v）水与50%（v/v）甲醇的混合溶液为流动相的高效液相色谱法，在330 nm波长处进行紫外检测，测定犬尿喹啉酸（KYNA）的生成量。实验设置三次重复以采集数据。