The Prototropic Behavior of alpha-Chymotrypsin and Chymotrysinogen in Water and Formaldehyde. An Examinatioin of Reactivity of the Amino Groups in Fromaldehyde

1 A careful study of the ionic behavior of α-chymotrypsin and chymotrypsinogen has shown that all the α- and ɛ-amino groups of these proteins are capable of their normal ionization. 2 The ionic difference between chymotrypsinogen and α-chymotrypsin is that expected from the known route of activation and these may be attributed to two C-terminal carboxyl groups (pK′ 3.8 ± 0.2) and two N-terminal α-amino groups (pK′ 7.8 ± 0.2). 3 The two N-terminal amino groups react with formaldehyde to form hydroxymethyl derivatives with pK′≃ 5. 4 All the amino groups of the proteins examined react quantitatively with formaldehyde to form hydroxymethyl derivatives and this reaction appears to be complete in 15–30 sec. 5 α-Chymotrypsin is catalytically active in 3.1 M formaldehyde with: (a) diminution of the catalytic rate, (b) retention of all active sites, and (c) a bell-shaped kcat/Kmvs. pH profile similar to that obtained in water. These data coupled with the present findings appear to be inconsistent with the postulated involvement of the α-amino group of the N-terminal isoleucine residue in the catalytic activity of α-chymotrypsin.