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

A number of methods have been described for the estimation of peptic activity, many of which are accurate and convenient for comparative experiments but none of which give results which are accurately reproducible with different preparations of the protein used. This variation is due to the difficulty involved in obtaining reproducible protein preparations and keeping them unchanged. Hemoglobin has been chosen as the protein substrata for the estimation of pepsin because it is easily prepared in large quantities, because it can be stored in solution for a long time without change, because it is rapidly digested, and because the rate at which it is digested by a given pepsin solution does not vary from one hemoglobin preparation to another. Not only is hemoglobin a reproducible protein which can be brought to reproducible conditions but the conditions of digestion have been so chosen that reasonable variations in these conditions have little effect on the amount of digestion. In general the procedure used to estimate pepsin is this. A pepsin solution is added to acidified hemoglobin. After 5 minutes trichloracetic acid is added. The resulting precipitate which contains all the pigment and all the undigested hemoglobin is faltered off. The filtrate contains an amount of digested hemoglobin which is a measure of the amount of pepsin used. This digested hemoglobin is estimated by the blue color it gives with the phenol reagent, which reacts with tyrosine, tryptophane and cysteine groups, tyrosine being used as a standard. The intensity of this color is proportional to the amount of enzyme used and to the time of digestion.

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A method is described for determining the diffusion coefficient of solutes by determining the rate of passage of the solute through a thin porous membrane between two solutions of different concentration. The method has been used to determine the diffusion coefficient of carbon monoxide hemoglobin. This was found to be 0.0420 ± 0.0005 cm.2 per day at 5°C. The molecular weight of carbon monoxide hemoglobin calculated by means of Einstein's equation from this quantity is 68,600 ± 1,000.

An account of previous work on cathepsin may be found in the monograph of Pozzi (1935).In the hemoglobin method for the estimation of protelnase, denatured hemoglobin is digested and the digestion products not precipitable by trichloracefic acid are estimated colorimetrically. Hemoglobin, unlike commercial casein, edestin, and gelatin, is a reproducible substrate, Only the very first stages of digestion are measured by the tfichloracetic acid method, for only a small amount of digestion is needed to make hemoglobin not precipitable by trichloracetic acid. Thus, only true proteinase is estimated by the hemoglobin method.Almost all the estimations of proteinase recorded in the literature have been made with substrates that are not reproducible. As a result, the values of proteinase activity obtained in different laboratories, or in the same laboratory with different batches of substrate, are not quantitatively comparable. Furthermore, most of the values of proteinase activity recorded, in particular those obtained by the Willst~tter school, were obtained by methods which estimate not proteinase activity alone but the activity of proteinase plus other proteolytic enzymes. As a result, the values obtained by different investigators are not even qualitatively comparable. The hemoglobin-trichloracetic acid method estimates proteinase alone, yields reproducible results, and has now been applied to the four known types of proteinase, pepsin (Anson and Mirsky (1932-33)), trypsin (Anson and Mirsky (1933-34)), papain (Anson (1936-37)), and cathepsin. It would seem desirable that if some other method is used, either independent evidence be given that the method estimates proteinase 565