Reversible inactivation of trypsin by anhydrous formic acid.

The inactivation of lysozyme and ribonuclease by anhydrous formic acid has been shown by Josefsson and Edman (1, 2) to be reversed by adjustment of an aqueous solution of the inactivated enzyme to pH 6.0 to 8.5. Reactivation was accompanied by an uptake of base, approximately equivalent to the number of serine and threonine residues in the proteins. With both proteins an increase in the positive electrophoretic mobility was observed, and it was postulated that the action of anhydrous formic acid was to bring about an Nto 0-acyl migration at the hydroxyamino acid residues of the proteins. Such a migration should result in the generation of primary amine groups equivalent to the number of residues of serine and threonine involved in the shift. In the case of lysozyme there was an increase in amino nitrogen content as measured by the Van Slyke method, but the yields were poor and no dinitrophenylserine could be detected after reaction at pH 5.0 with fluorodinitrobenzene. In the case of ribonuclease, attempts to demonstrate an increase in the amino nitrogen content of the protein failed completely. Other observations (3) included an increase in the sedimentation coefficient and viscosity of ribonuclease but no change in the optical rotatory properties of the enzyme. Because of the high content of serine and threonine in trypsin (40 and 11 residues, respectively (4)), it was of considerable interest to extend the investigation of the action of anhydrous formic acid to this enzyme. As with lysozyme and ribonuclease, trypsin was inactivated and could be regenerated by incubation at alkaline pH, and the reactivation was accompanied by an uptake of base. However, the physical and chemical properties of the inactivated enzyme do not support the hypothesis of an Nto 0-acyl shift produced by formic acid.