Skotland T, Ljones T
Biochim Biophys Acta. 1980 Jun 5;630(1):30-5. doi: 10.1016/0304-4165(80)90134-8.
A direct spectrophotometric method was used for detection of the ascorbate free radical formed during enzyme catalysis with dopamine beta-monooxygenase and with ascorbate oxidase. The optical absorption spectra in the range of 330-390 nm for the free radical formed by either of these enzymes were quite similar to the previously reported spectrum from pulse radiolysis experiments. The second order rate constant for dismutation of the radical generated by dopamine beta-monooxygenase at 23 degrees C was estimated from the levels of radical in the steady state, and the values of 2.4 .10(-6) M-1 . s-1 at pH 7.0 and 9.7 . 10(-6) M-1 . s-1 at pH 6.0 were in close agreement with reported values from experiments in which the radical had been generated with ascorbate oxidase or with pulse radiolysis. Moreover, the steady state radical levels at different levels of dopamine beta-monooxygenase or its substrate tyramine were also those predicted by a mechanism of nonenzymic dismutation of the radical. We conclude, in agreement with our earlier report with the cytochrome c scavenger method, that the radical is not an enzyme-bound intermediate, but a product of dopamine beta-monooxygenase catalysis.
采用直接分光光度法检测多巴胺β-单加氧酶和抗坏血酸氧化酶酶催化过程中形成的抗坏血酸自由基。这两种酶所形成的自由基在330 - 390nm范围内的光吸收光谱与先前脉冲辐解实验报道的光谱非常相似。根据稳态下自由基的水平估算了多巴胺β-单加氧酶在23℃时所产生自由基歧化反应的二级速率常数，在pH 7.0时为2.4×10⁻⁶ M⁻¹·s⁻¹，在pH 6.0时为9.7×10⁻⁶ M⁻¹·s⁻¹，这与用抗坏血酸氧化酶或脉冲辐解产生自由基的实验报道值非常一致。此外，不同水平的多巴胺β-单加氧酶或其底物酪胺时的稳态自由基水平也符合自由基非酶歧化反应机制的预测。我们得出结论，与我们早期用细胞色素c清除剂方法得到的报告一致，该自由基不是酶结合中间体，而是多巴胺β-单加氧酶催化的产物。
