Effects on tryptophyl absorption of the ionization of the catalytic carboxyls in hen and turkey lysozymes.
The difference spectra of hen and turkey egg-white lysozymes [EC 3.2.1.17] produced by acidification were measured. The difference spectra of both lysozymes had peaks at 295 and 301 nm which are characteristic of tryptophyl residues. The pH dependence curves of the extinction differences (delta eplision) at 301 nm and 295 nm for hen lysozyme were identical with the corresponding curves for turkey lysozyme. The pH dependence of delta eplision at 301 nm was analyzed assuming that the extinction at 301 nm is due to Trp 108 only, which interacts with the catalytic carboxyls, Glu 35 and Asp 52.  The macroscopic pK values of Glu 35 and Asp 52 in both lysozymes thus determined were 6.0 and 3.3, respectively. These values were in excellent agreement with those determined by measuring the pH dependence of the circular dichroic band at 305 nm (Kuramitsu et al. (1974) J. Biochem, 76, 671-683; (1975) ibid. 77, 291-301). The pH dependence of delta eplision at 295 nm could not be completely explained in terms of the electrostatic effects of the catalytic groups on Trp 108.
Effects on tryptophyl absorption of the ionization of the catalytic carboxyls in hen and turkey lysozymes. The difference spectra of hen and turkey egg-white lysozymes [EC 3.2.1.17] produced by acidification were measured. The difference spectra of both lysozymes had peaks at 295 and 301 nm which are characteristic of tryptophyl residues. The pH dependence curves of the extinction differences (delta eplision) at 301 nm and 295 nm for hen lysozyme were identical with the corresponding curves for turkey lysozyme. The pH dependence of delta eplision at 301 nm was analyzed assuming that the extinction at 301 nm is due to Trp 108 only, which interacts with the catalytic carboxyls, Glu 35 and Asp 52.  The macroscopic pK values of Glu 35 and Asp 52 in both lysozymes thus determined were 6.0 and 3.3, respectively. These values were in excellent agreement with those determined by measuring the pH dependence of the circular dichroic band at 305 nm (Kuramitsu et al. (1974) J. Biochem, 76, 671-683; (1975) ibid. 77, 291-301). The pH dependence of delta eplision at 295 nm could not be completely explained in terms of the electrostatic effects of the catalytic groups on Trp 108.