Suiko M, Sakakibara Y, Awan-Khan R, Sakaida H, Yoshikawa H, Ranasinghe J G, Liu M C
Department of Biochemistry, University of Texas Health Center at Tyler, TX 75710, USA.
J Biochem. 1998 Oct;124(4):707-11. doi: 10.1093/oxfordjournals.jbchem.a022170.
Upon two-dimensional thin-layer separation, the sulfated L-3, 4-dihydroxyphenylalanine (L-DopaS) generated enzymatically was found to co-migrate with only one of the two ninhydrin-stained spots corresponding to the two sulfated forms (3-O-sulfate and 4-O-sulfate) of synthetic L-DopaS. To clarify precisely the identity of the enzymatically generated L-DopaS, the two sulfated forms of synthetic L-DopaS were separated and purified using high performance liquid chromatography. Purified L-Dopa 3-O-sulfate and L-Dopa 4-O-sulfate were identified by 1H-nuclear magnetic resonance (NMR) spectrometry and used as standards in the analysis of the L-DopaS generated during metabolic labeling of HepG2 human hepatoma cells or enzymatic assay using recombinant human monoamine (M)-form phenol sulfotransferase. The results obtained demonstrated unequivocally the generation of L-Dopa 3-O-sulfate, indicating the specificity of the M-form phenol sulfotransferase being for the meta-hydroxyl group of L-Dopa.
在二维薄层分离时，发现酶促生成的硫酸化L-3,4-二羟基苯丙氨酸（L-DopaS）仅与合成L-DopaS的两种硫酸化形式（3-O-硫酸盐和4-O-硫酸盐）中与茚三酮染色斑点之一共迁移。为了精确阐明酶促生成的L-DopaS的身份，使用高效液相色谱法对合成L-DopaS的两种硫酸化形式进行分离和纯化。通过1H-核磁共振（NMR）光谱法鉴定纯化的L-Dopa 3-O-硫酸盐和L-Dopa 4-O-硫酸盐，并将其用作分析HepG2人肝癌细胞代谢标记过程中生成的L-DopaS或使用重组人单胺（M）型酚磺基转移酶进行酶促测定的标准品。所获得的结果明确证明了L-Dopa 3-O-硫酸盐的生成，表明M型酚磺基转移酶对L-Dopa间位羟基具有特异性。
