title: Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin.
abstract: This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. Alpha-chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of alpha-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 degrees C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.
mesh_terms: D000215:Acylation; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D003911:Dextrans; D004227:Dithionite; D004795:Enzyme Stability; D005362:Ficoll; D006358:Hot Temperature; D007700:Kinetics; D011489:Protein Denaturation; D013499:Surface Properties; D013774:Tetranitromethane; D014357:Trypsin / Q000378:metabolism
chemical_list: D003911:Dextrans; D004227:Dithionite; D005362:Ficoll; D002918:Chymotrypsin; D014357:Trypsin; D013774:Tetranitromethane