FORMOL TITRATION OF ALPHA-CHYMOTRYPSIN CHYMOTRYPSINOGEN AND MODEL COMPOUNDS IN 4.8 M GUANIDINE HYDROCHLORIDE

Titration of model compounds in 4.8 M guanidine 0.15 M KCl at 20° show that pK′ values of imidazoyl, phenolic, and carboxyl groups are shifted only slightly. Amino group ionizations are affected most, varying ±0.3 unit. The useful range of this system is from pH 1.5 to 12.0, in which all the normally ionizing groups of proteins can be titrated. Form-aldehyde titrations may also be performed to obtain additional information about the amino groups of proteins. In guanidine/formaldehyde, the pK′ of the hydroxymethylamino derivatives are shifted to lower values than in water. When chymotrypsinogen and α-chymotrypsin titrations were performed, the data indicated 36 ionizing groups in chymotrypsinogen, 15 of which are amino groups. Analysis of the difference between the ionic behavior of these proteins shows that α-chymotrypsin has two additional groups with pK′ 3 and two with pK′ 7.5 in guanidine. The groups with pK′ 7.5 are shifted to pK′ 4.2 in 3.1 M formaldehyde, which is indicative of the behavior of α-amino groups. The comparison of the Sørensen titration of α-chymotrypsin in water or guanidine indicates that all the amino groups of the protein react with formaldehyde in the native or denatured form. © 1968.