Rodríguez-López J N, Bañón-Arnao M, Martinez-Ortiz F, Tudela J, Acosta M, Varón R, García-Cánovas F
Departamento de Química-Física, E.U. Politécnica de Albacete, Universidad de Castilla-La Mancha, Spain.
Biochim Biophys Acta. 1992 Nov 20;1160(2):221-8. doi: 10.1016/0167-4838(92)90011-2.
The oxidation of 3,4-dihydroxyphenylalanine (dopa) by O2 catalyzed by tyrosinase yields 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaquinone-H+). This evolves non-enzymatically through two branches (cyclization and/or hydroxylation), whose respective operations are determined by pH. The hydroxylation branch of o-dopaquinone-H+ only operates significantly at pH < or = 5.0 and involves the accumulation of 2,4,5-trihydroxyphenylalanine (topa), which has been detected by high-performance liquid chromatography (HPLC). This last compound is also a substrate of tyrosinase. The oxidation of topa by both tyrosinase and periodate yields 5-(2-carboxy-2-aminoethyl)-4-hydroxy-1,2-benzoquinone, with its amino group protonated (o-topaquinone-H+), which is red (RTQH) (lambda max 272-485 nm) at pH 7.0 and yellow (TTQH) (lambda max 265-390 nm) at pH 3.0. This is based on pKa 4.5 of the 2-OH group of the benzene ring of o-topaquinone-H+, as derived from spectrophotometric and HPLC assays. At physiological pH, RTQH undergoes deprotonation of the ammonium group of the side chain to yields RTQ, which cyclize into 2-carboxy-2,3-dihydroxyindolen-5,6-quinone (dopachrome), with a 1:1 stoichiometry and first-order kinetics. The evolution of RTQH has been analyzed by spectrophotometry, HPLC, cyclic voltammetry and constant potential electrolytic assays. From HPLC assays, the value of the first-order constant for the evolution of RTQH at pH 7.0 (kRTQHapp 4.83 x 10(-5) s-1), as well as of the rate constant for the cyclization step of RTQ (kRTQc 2.53 x 10(-3) s-1) were determined.
酪氨酸酶催化下，氧气将3,4 - 二羟基苯丙氨酸（多巴）氧化生成4 -（2 - 羧基 - 2 - 氨基乙基）-1,2 - 苯醌，其氨基质子化（邻多巴醌 - H⁺）。它通过两个分支（环化和/或羟基化）非酶促转化，各自的反应由pH决定。邻多巴醌 - H⁺的羟基化分支仅在pH≤5.0时显著发生，涉及2,4,5 - 三羟基苯丙氨酸（topa）的积累，已通过高效液相色谱（HPLC）检测到。最后这种化合物也是酪氨酸酶的底物。酪氨酸酶和高碘酸盐将topa氧化均生成5 -（2 - 羧基 - 2 - 氨基乙基）-4 - 羟基 - 1,2 - 苯醌，其氨基质子化（邻topa醌 - H⁺），在pH 7.0时为红色（RTQH）（最大吸收波长272 - 485 nm），在pH 3.0时为黄色（TTQH）（最大吸收波长265 - 390 nm）。这是基于邻topa醌 - H⁺苯环2 - OH基团的pKa为4.5，该值由分光光度法和HPLC分析得出。在生理pH下，RTQH侧链铵基团去质子化生成RTQ，其按1:1化学计量比和一级动力学环化生成2 - 羧基 - 2,3 - 二羟基吲哚 - 5,6 - 醌（多巴色素）。通过分光光度法、HPLC、循环伏安法和恒电位电解分析研究了RTQH的转化过程。通过HPLC分析，确定了pH 7.0时RTQH转化的一级常数（kRTQHapp 4.83×10⁻⁵ s⁻¹）以及RTQ环化步骤的速率常数（kRTQc 2.53×10⁻³ s⁻¹）。
