Jacobsohn G M, Iskandar R, Jacobsohn M K
Department of Biological Chemistry, Hahnemann University, Philadelphia, PA 19102.
Biochim Biophys Acta. 1993 Oct 6;1202(2):317-24. doi: 10.1016/0167-4838(93)90022-j.
A suspension of tyrosinase-coated glass beads in butanol effectively oxidizes catechol substrate. The enzyme is not soluble in the organic solvent and activity can be stopped by removal of the solid state enzyme after low-speed centrifugation or decantation. The product was assayed by HPLC and by its reactivity towards Besthorn's reagent, which gave a reaction typical for o-quinones. Addition of water to the extent of 0.5 to 4% raised the rate of substrate utilization but the accumulation of quinone first increased and then began to decrease. It is suggested that the product in dry butanol is prevented from reacting further by lack of water, which is necessary to promote secondary reactions causing free radical formation and leading ultimately to polymerization to melanin. Successive washes of the solid state enzyme with butanol increased enzyme activity, indicating presence of a butanol extractable inhibitor in the tyrosinase preparation. The enzyme on glass beads in butanol suspension was stabilized by the presence of substrate. 2-Hydroxyestradiol acted as an inhibitor of the tyrosinase-catalyzed oxidation of catechol. The data obtained can be interpreted to mean that the oxidation of the estrogen in the presence of tyrosinase, as previously reported, may be dependent upon the enzyme-catalyzed oxidation of catechol. The oxidation product of catechol, the o-quinone, is likely to function as oxidant towards 2-hydroxyestradiol.
酪氨酸酶包被的玻璃珠在丁醇中的悬浮液能有效氧化儿茶酚底物。该酶不溶于有机溶剂，低速离心或倾析除去固态酶后，其活性可被终止。产物通过高效液相色谱法（HPLC）以及其对贝斯索恩试剂的反应性进行测定，该反应给出了邻醌典型的反应。加入0.5%至4%的水可提高底物利用速率，但醌的积累先增加，然后开始减少。这表明在干燥的丁醇中，产物因缺水而无法进一步反应，水对于促进导致自由基形成并最终导致聚合形成黑色素的二次反应是必需的。用丁醇对固态酶进行连续洗涤可提高酶活性，表明酪氨酸酶制剂中存在可被丁醇提取的抑制剂。丁醇悬浮液中玻璃珠上的酶因底物的存在而稳定。2-羟基雌二醇作为酪氨酸酶催化儿茶酚氧化的抑制剂。所获得的数据可以解释为，如先前报道的那样，在酪氨酸酶存在下雌激素的氧化可能依赖于酶催化的儿茶酚氧化。儿茶酚的氧化产物邻醌可能作为2-羟基雌二醇的氧化剂。
