Effects of Ionic Strength and Temperature on the Ionization of the Catalytic Groups, Asp 52 and Glu 35, in Hen Lysozyme1

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Abstract
The pH difference spectra of intact hen lysozyme [EC 3.2.1.17], Asp 52-esterified lysozyme, and Glu 35-Trp 108-esterified lysozyme were measured at various ionic strengths and temperatures. The pH dependence of the extinction difference at 301.5 nm was well interpreted in terms of the ionization of the catalytic groups, Asp 52 and Glu 35, which interact electrostatically. The macroscopic pK values of Asp 52 and Glu 35 were determined to be 3.4 and 6.1, respectively, at 0.1 ionic strength and 25°C. Regarding the ionization of the catalytic groups as that of a dibasic acid and assuming that the microscopic ionization constant of Glu 35 of intact lysozyme in which Asp 52 has been protonated (pk1,35 is equal to the ionization constant of Glu 35 in Asp 52-esterified lysozyme, we estimated the four microconstants for the ionization of Asp 52 and Glu 35 in intact lysozyme. The microconstants for the first ionization of Asp 52 (pk1,52) and Glu 35 (pk1,35) were more sensitive to ionic strength of the medium than those for the second ionization of Asp 52 (pk2,62) and Glu 35 (pk2,35. The increase in (pk1,52) and (pk1,35) with an increase in ionic strength was interpreted in terms of the electrostatic interaction between the positively charged lysozyme molecule and a proton which is going to bind to Asp 52 or Glu 35, and the smaller increase in pk and (pk1,52) and (pk2,35) in terms of an additional electrostatic interaction between negatively charged catalytic carboxylate ions. The pk values for the lysozyme molecule with zero net charge at zero ionic strength which were obtained by correcting for these electrostatic interactions were 4.7-5.1 for Asp 52 and 6.1-6.5 for Glu 35. The absolute values of ΔH° of the ionization for Asp 52 and Glu 35 were less than 0.5 kcal mol−1. The ΔS° values were -16 and -2O cal deg−1⋅mol−1 for the first and second ionizations of Asp 52, respectively, and -24 and -28 cal deg−1⋅mol−1 for the first and second ionizations of Glu 35, respectively, at 0.1 ionic strength and 25°C.