Direct spectrophotometric detection of ascorbate free radical formed by dopamine beta-monooxygenase and by ascorbate oxidase

Abstract

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.