Nagatsu T, Oka K, Kato T
J Chromatogr. 1979 Jul 21;163(3):247-52. doi: 10.1016/s0378-4347(00)81411-5.
A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.
基于通过双柱程序快速分离酶促形成的多巴，设计了一种通过高效液相色谱（HPLC）和安培检测法对酪氨酸羟化酶（TH）活性进行高灵敏度测定的方法，这些柱子依次组装在一起（顶部为Amberlite CG - 50柱，底部为氧化铝柱）。多巴吸附在第二个氧化铝柱上，然后用0.5 M盐酸洗脱，并通过HPLC和安培检测法进行测定。使用D - 酪氨酸作为对照。孵育后将α - 甲基多巴作为内标添加到孵育混合物中。该测定法比放射测定法更灵敏，并且在酪氨酸和6 - 甲基四氢蝶呤饱和浓度存在的情况下，可以测量酶促形成的5 pmol多巴。2 mg人壳核中的TH活性可以很容易地测量，并且发现该方法特别适用于测定来自动物和人类大脑少量核中的TH活性。
