The Estimation of Pepsin, Trypsin, Papain, and Cathepsin with Hemoglobin

In the hemoglobin method for the estimation of proteinase, denatured hemoglobin is digested under standard conditions, the undigested hemoglobin is precipitated with trichloracetic acid, and the amount of unprecipitated protein split products, which is a measure of the amount of proteinase present, is estimated with the phenol reagent which gives a blue color with tyrosine and tryptophane. Hemoglobin, unlike casein and gelatin, is a reproducible substrate. Different batches of hemoglobin are digested at the same rate by a given proteinasesolution. Even when peptidase is present in addition to proteinase, the formation of products not precipitable by trichloracetic acid is due so far as is known to proteinase alone. Hemoglobin methods have been described for pepsin (Anson and Mirsky, 1932), trypsin (Anson and Mirsky, 1933), papain (Anson, 1937a), and cathepsin (Anson, 1937 b). Since the methods were first worked out several minor errors have been corrected, the preparation of the hemoglobin substrates has been simplified, and the estimation procedures have been standardized. Rather than point out the numerous changes which have been made I have considered it simpler and more useful to describe completely the procedures as they are now used in this laboratory. To avoid confusion about results already published no radical changes have been made. The pepsin, trypsin, and papain substrates are the same in composition as those originally described. The cathepsin substrate now contains 0.001 M ammonium sulfate which increases the rate of digestion. In the case of the estimation of pepsin the procedure for the estimation 79