The Estimation of Papain with Hemoglobin

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. Definition of Activity [P. U.]Hb.--One unit of activity is defined as the formation per minute at 35.5°C. in 6 ml. of the standard digestion 59