Wimalasena K, Wimalasena D S
Department of Chemistry, Wichita State University, Kansas 67208.
Anal Biochem. 1991 Sep 2;197(2):353-61. doi: 10.1016/0003-2697(91)90404-h.
Based on the novel chromophoric electron donors, N,N-dimethyl-1,4-phenylenediamine (DMPD) and 2-amino-2-deoxy-L-ascorbic acid (2-aminoascorbic acid), two sensitive, convenient, and continuous spectrophotometric assays for dopamine beta-monooxygenase (EC 1.14.17.1) are described. Both, DMPD and 2-aminoascorbic acid are kinetically and stoichiometrically well-behaved electron donors for dopamine beta-monooxygenase with kinetic parameters comparable to the most efficient physiological electron donor, ascorbic acid. During dopamine beta-monooxygenase turnover, DMPD is converted to its chromophoric cation radical which is stable under the standard assay conditions. The rate of the enzyme-dependent formation of DMPD cation radical under standard assay conditions could easily be followed at 515 nm with high accuracy and reproducibility. Similarly, dopamine beta-monooxygenase-mediated oxidation of 2-aminoascorbic acid results in the formation of the known, stable chromophoric product, 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment), which has a very strong absorption maximum at 385 nm. Both the above assays are superior to the existing assays in their convenience, reproducibility, and sensitivity for routine kinetic analysis of dopamine beta-monooxygenase and may be adopted as a simple color test for the enzyme. We propose that the above assays could also be adopted to design continuous and sensitive spectrophotometric assays for ascorbate oxidase, peptidyl alpha-amidating monooxygenase, and the chromaffin granule electron transport protein, cytochrome b561, due to their remarkable similarity to dopamine beta-monooxygenase in the chemistry of catalysis with regard to the electron donor.
基于新型发色电子供体N,N-二甲基-1,4-苯二胺（DMPD）和2-氨基-2-脱氧-L-抗坏血酸（2-氨基抗坏血酸），描述了两种用于多巴胺β-单加氧酶（EC 1.14.17.1）的灵敏、便捷且连续的分光光度法。DMPD和2-氨基抗坏血酸在动力学和化学计量学方面都是多巴胺β-单加氧酶性能良好的电子供体，其动力学参数与最有效的生理性电子供体抗坏血酸相当。在多巴胺β-单加氧酶周转过程中，DMPD转化为其发色阳离子自由基，该自由基在标准测定条件下稳定。在标准测定条件下，依赖于酶的DMPD阳离子自由基形成速率可在515 nm处轻松高精度且可重复地进行跟踪。同样，多巴胺β-单加氧酶介导的2-氨基抗坏血酸氧化导致形成已知的稳定发色产物2,2'-亚氨基二-2(2')-脱氧-L-抗坏血酸（红色色素），其在385 nm处有非常强的最大吸收峰。上述两种测定方法在便捷性、可重复性和对多巴胺β-单加氧酶常规动力学分析的灵敏度方面均优于现有测定方法，可作为该酶的简单颜色测试方法采用。我们建议，由于上述测定方法在电子供体催化化学方面与多巴胺β-单加氧酶具有显著相似性，因此也可用于设计抗坏血酸氧化酶、肽基α-酰胺化单加氧酶和嗜铬颗粒电子转运蛋白细胞色素b561的连续且灵敏的分光光度法。
