CHYMOTRYPSIN LINKED TO POLY(ETHYLENIMINE) DERIVATIVES - PERTURBATION OF IONIZATION OF ACTIVE-SITE GROUPS

α-Chymotrypsin (ChT) is covalently linked to various poly(ethylenimine) (PEI) derivatives by using a carbodiimide as a coupling reagent. The PEI derivatives contain cationic or anionic microenvironments. In addition, hydrophobic microdomains are created on the PEI backbones in conjunction with the ionic environments. Stabilization of the tertiary structure of ChT by the cross-linking PEI derivatives is reflected by much greater resistance of the PEI-bound ChTs to thermoinactivation. Depending on the structural elements incorporated into the PEI derivatives, the activity of ChTs linked to PEI derivatives in either/both acidic or/and basic pH ranges is much greater than that of native ChT. The nature of microdomains introduced to the globular backbone of PEI affects the pKa values of the active-site groups of ChT sensitively. Cationic microenvironments created on the PEI backbone retards protonation of His-57 more than that of Ile-16. Anionic microenvironments of the PEI backbone stabilize the ammonium ion of Ile-16 more than the imidazolium ion of His-57. Moreover, substrate binding by the active site appears to render His-57 more sensitive to the change in the microenvironments. © 1992.