Titration Study of Acetylated Lysozyme

Abstract

To study the interaction between carboxyl groups and amino groups in native lysozyme [EC 3. 2. 1. 17], and to identify the positions and the pK values of the abnormal carboxyl groups, N-acetylated lysozyine was prepared. The acetylation did not affect the molecular shape of the enzyme, but changed six amino groups to a non-ionizable form, leaving one amino group free; this was determined to be Lys 33. In addition, pH titration of the acetylated lysozyme in 0.2 or 0.02_M KCl aqueous solution indicated fewer titratable groups with pK_int of 7.8 or 10.4 compared with the native protein, though the number of titratable carboxyl groups was not affected by the acetylation. From the pH titration results and structural considerations, the untitratable carboxyl groups were suggested to be Asp 48, Asp 66, and Asp 87.
On the other hand, spectrophotometric titration in 0.2_M KCl showed that all three tyrosine residues are titratable in the acetylated protein, although an abnormal tyrosine residue exists in the native state. Tyr 20 was suggested to be untitratable in the pH range of 8-12.6.