Fluorescence spectra of hen, turkey, and human lysozymes excited at 305 nm.

Abstract

Fluorescence spectra of hen egg-white, turkey egg-white, and human urine lysozymes [EC 3.2.1.17] were measured at various pH values by using 305 nm light for the excitation. The pH dependence curves of the fluorescence intensity at the maximum wavelength for these lysozymes were very similar in shape to each other, though the intensities were different. The results of the pH dependence of the fluorescence intensity of two hen lysozyme derivatives, in each of which Asp 52 and Glu 35 had been selectively esterified, and of turkey lysozyme, in which Asp 101 in hen lysozyme is replaced by Gly, showed that only the ionization of Glu 35 and Asp 52 affects the fluorescence of Trp 108 in hen and turkey lysozymes. The pK values of Asp 52 and Glu 35 of hen and turkey lysozymes determined by analyses of the pH dependence of the fluorescence excited at 305 nm in the pH range of 2 to 8 were identical with those determined from the pH dependence of the extinction difference at 301.5 nm (Itani et al. (1975) J. Biochem. 78, 705-711; Kuramitsu et al. (1977) J. Biochem. 82, 585-597) and the circular dichroism at 305 nm (Kuramitsu et al. (1974) J. Biochem. 76, 671-683; (1975) ibid. 77, 291-301). The pH dependence of the fluorescence excited at 305 nm of human lysozyme, in which Trp 62 in hen lysozyme is replaced by Tyr, was also interpreted in terms of the ionization of Asp 52 and Glu 35, with pK values identical with those determined from the pH dependence of the circular dichroism at 305 nm (Kuramitsu et al. (1974) J. Biochem. 76, 671-683) and the extinction difference at 300 nm (Kuramitsu et al., to be published.) The fluorescence spectra of hen, turkey, and human lysozymes excited at 281 nm were different from the respective spectra excited at 305 nm, and the pH dependence of the fluorescence intensity excited at 281 nm could not be completely explained in terms of the ionization of the catalytic groups. The fluorescences of hen, turkey, and human lysozymes were quenched above pH 8. Comparison of the fluorescence of intact hen lysozyme and that of a lysozyme derivative, in which all the amino groups had been acetylated, showed that an amino group(s) in addition to a tyrosyl residue quenched the tryptophyl fluorescence above pH 8.