Reactivation of enzymes irreversibly denatured at elevated temperature. Trypsin and alpha-chymotrypsin covalently immobilized on Sepharose 4B and in polyacrylamide gel.
Trypsin (EC 3.4.21.4) and chymotrypsin (EC  3.4.21.2) covalently immobilized on Sepharose or in polyacrylamide gel has been irreversibly denatured at 70--90 degrees C and then reactivated in an almost 100% yield. Thermoinactivated enzyme is first made to unfold under the action of urea with S-S bonds being simultaneously reduced and then made to refold (under the optimal conditions for the thiol-disulfide exchange) into its native conformation. It is demonstrated that the 'irreversible monomolecular thermoinactivation-reactivation' cycle can be repeated many times. The contribution of various mechanisms to thermoinactivation of the enzymes is discussed. Based on the data obtained, the irreversible thermoinactivation of enzymes under investigation should be ascribed only to changes in their secondary and teritary structures; the primary structure is not likely to be affected.
D002918:Chymotrypsin / Q000378:metabolism*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006358:Hot Temperature; D011489:Protein Denaturation; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*
D004800:Enzymes, Immobilized; D013438:Sulfhydryl Compounds; D002918:Chymotrypsin; D014357:Trypsin