Crystalline Trypsin Iv. Reversibility of the Inactivation and Denaturation of Trypsin by Heat

I t was noted by Mellanby and Wooley (1) that trypsin solutions in dilute acid could be heated nearly to boiling with very little loss in activity, and this observation was confirmed by Eddie (2). At temperatures below 40°C., on the other hand, the enzyme is more stable near pH 4 or 5 than it is in acid solution. This latter result has also been obtained by the writer (3) and by Pace (4). In the course of the preparation of crystalline trypsin by Kunitz and the writer (5) it was found that the further purification proceeded the more heat-stable the preparation became. The final crystalline material may be heated to boiling in dilute solution over the whole range of acidity between pH 1 and pH 7 with little or no loss in activity and apparently without the formation of any denatured protein. This behavior is remarkable, since in general heating denatures proteins and inactivates enzymes. The result is, however, somewhat analogous to that in the case of serum albumin. Spiegel-Adolf (6) found that serum albumin also could be heated in certain pH ranges without the appearance of any denatured protein in the solution after it had been cooled. Spiegel-Adolf, and Anson and Mirsky (7) also showed that the denaturation of serum albumin is easily reversible and it is probable that the fact that no denatured protein is found in the solution after cooling is due to the reformation of native from denatured protein on cooling. I t has also been found in the case of crystalline pepsin (8) that the activity is lost when the protein is denatured and regained when the denaturation is reversed. The possibility therefore exists in the case of trypsin that inactivation of the enzyme and denaturation of the protein occur, as 323