Changes in tryptophan microenvironment in horse heart myoglobin due to γ-irradiation

Abstract

Changes in microenvironment of the two tryptophan residues of horse heart myoglobin subjected to γ-irradiation at concentration of 1 mg/ml and doses of 25–100 krad when no aggregation took place were studied by ultraviolet fluorescence spectrometry. Increase in fluorescence of irradiated myoglobin accompanied by a long wavelength shift from 335 to 340 nm of fluorescence maximum indicated unfolding leading to greater exposure of tryptophan to solvent. Fluorescence quenching by NO 2 − and I − was also more for irradiated myoglobin than for myoglobin. But the degree of exposure of tryptophan in irradiated myoglobin was not complete as evidenced by a further shift of fluorescence maximum to 350 nm in 6 M guanidine hydrochloride (GuHCl). Also, in GuHCl relative fluorescence of both native and irradiated myoglobin increased considerably though the increase for irradiated protein was comparatively less. The Stern-Volmer constants for iodide quenching showed that the amount of fluorescence in native myoglobin accessible to the quencher (55%) increased upon irradiation to 74%. The two tryptophan residues are about equal in their degree of exposure to the solvent. But denaturation of native or irradiated myoglobin rendered the fluorescence of both tryptophan residues completely accessible to I −. The data suggest that the enhanced exposure of tryptophans caused by radiation-induced unfolding of myoglobin makes them susceptible to destruction.