Structure of Lysozyme: XII. Effect of pH on the Stability of Lysozyme*

Abstract

In order to know the role of ionizable groups in the stability of the lysozyme [EG 3.2.1.17] molecule, the effect of pH on the optical rotatory and ultraviolet spectral properties was investigated. The optical rotatory properties of lysozyme did not change at pH's between 8 and 1. However, the acid-vs-neutral difference spectra of lysozyme have peaks characteristic of the tryptophan residues. However, in the presence of 5 M GuCl, in which lysozyme molecule is completely denatured, such an acid-vs-neutral difference spectrum was not observed. This suggests that a strong interaction exists between tryptophan residues and ionizable groups in the native lysozyme molecule. In the presence of 3.58 M GuCl, the values of Δs at 292 mμ and the Moffitt parameters, a_o and b_o3, changed greatly at pH's below 4.0, with an apparent pK value of 3.2. The equilibrium constant between the native and denatured lysozyme in the presence of 3.84 M GuCl is proportional to the 1.7 th power of the hydrogen ion concentration ([H⁺]). The acid denaturation of lysozyme in the presence of GuCl obeyed a reversible first order reaction kinetics. The apparent rate constant, k, in the presence of 3.84 M GuCl was expressed by k=k₁′[H⁺¹²+K₂′/[H⁺]⁰⁸. The results obtained here suggest that carboxyl groups which have a pK value of 3.2 and interact strongly with tryptophan residues play an important role in the stabilization of the lysozyme molecule.