一种通过高效液相色谱法测定多巴胺-β-羟化酶活性的灵敏荧光测定法。

A sensitive fluorometric assay for dopamine-beta-hydroxylase activity by high-performance liquid chromatography.

作者信息

Matsui H, Kato N, Yamamoto C, Fujita K, Sakai H, Nagatsu T

出版信息

Biochem Med. 1984 Apr;31(2):140-6. doi: 10.1016/0006-2944(84)90019-x.

Abstract

A sensitive and specific fluorometric assay for dopamine-beta-hydroxylase (DBH) activity is described. The main natural substrate, dopamine (DA), was used and incubated under optimal conditions. Norepinephrine (NE) formed enzymatically from DA was isolated by an aluminum oxide column and was analyzed by high-performance liquid chromatography (HPLC) with trihydroxyindole fluorescence. Epinephrine (EN) was added to the incubation mixture as an internal standard after incubation, and this assay was therefore highly reproducible. HPLC conditions were settled to elute the product, NE, prior to the substrate, DA, and the internal standard, EN, between NE and DA. Only catecholamines produced significant peaks, and therefore, this assay is highly specific. We applied this method to measure the DBH activity in human serum and cerebrospinal fluid.

摘要

描述了一种用于检测多巴胺-β-羟化酶（DBH）活性的灵敏且特异的荧光测定法。使用主要天然底物多巴胺（DA），并在最佳条件下进行孵育。通过氧化铝柱分离由DA酶促形成的去甲肾上腺素（NE），并采用三羟基吲哚荧光高效液相色谱法（HPLC）进行分析。孵育后将肾上腺素（EN）作为内标添加到孵育混合物中，因此该测定具有高度可重复性。确定HPLC条件以在底物DA和内标EN之前洗脱产物NE，且NE在DA之前洗脱。只有儿茶酚胺产生显著峰，因此该测定具有高度特异性。我们应用此方法测量人血清和脑脊液中的DBH活性。