Tyrosine Residues in Unirradiated and γ-irradiated Dihydroorotate Dehydrogenase: Fluorimetric and Second-derivative Absorption Spectrophotometric Studies

Abstract

The effect of γ-radiation on tyrosine residues of dihydroorotate dehydrogenase under different conditions was investigated by means of fluorescence spectroscopy and second-derivative spectrophotometry. No change in the fluorescence spectral distribution was observed when unirradiated and irradiated enzyme were denatured in guanidine hydrochloride. However, decreases in fluorescence intensity in irradiated samples indicates a radiation-induced modification of tyrosine residues. The fluorescence intensity at 340 nm decreased exponentially with radiation dose in aerated medium but non-exponentially under argon and nitrous oxide-saturated conditions. The percentage loss of tyrosine fluorescence under different conditions was determined. The number of tyrosine residues left intact following irradiation at a dose for 50% inactivation under different conditions was measured by second-derivative absorption spectrophotometry. The results obtained from both these methods show that the hydroxyl radical is less efficient in inducing radiation damage of tyrosine in aerated conditions compared with that under deoxygenated conditions. This lower efficiency of the hydroxyl radical in aerated medium has been attributed to the protective effect of oxygen and/or the superoxide radical anion.