STUDIES OF METHODS FOR MEASURING THE PROTEOLYTIC ACTIVITY OF PAPAIN

Abstract

The fresh latex obtained from the green fruit of Carica papaya L. contains powerful proteolytic enzymes, which are used in the form of the dried whole latex, called papain.Since the utility of papain depends upon its enzyme content, various methods have been employed to measure its proteolytic activity.Unfortunately, they have not all given comparable results.Papain, along with a method for its assay, was recently admitted to the 8th edition of the National Formulary and thus became an official drug in 1946.It was soon found that the official method of assay gave results that were different from those obtained by other methods employed in these laboratories, so that either a new method of assay for the proteolytic activity of papain had to be developed or means found for reconciling the results of existing methods.In all methods, papain is allowed to digest a protein or peptide and the extent of such digestion is then measured.The methods can be divided into three main groups, depending on the manner of measuring the amount of digestion, i.e., those depending upon the measurement of undigested residues, those depending upon the measurement of liberated amino groups, and miscellaneous methods based on the measurement of such things as the optical rotation of digests, the length of time required to clot a standard milk solution, the color produced by certain reagents on filtered digests, the increase of soluble solids, etc.In the first group belong such methods as the National Formulary method,' which is based on the volume of undigested residue left after digesting a suspension of powdered dry beef.This method is probably derived from the prior work of Graber?Adams: and Unwin,4 who reported the use of both fresh and dried beef as the protein substrate.Maher and Wirthb proposed an improvement of the N.F.method.Rippetoes and Heyl, et aZ.7 employed heat-coagulated egg albumin, the former measuring the volume of undigested residue and the latter weighing the undigested protein after washing and drying.Pratt* used condensed skimmed milk as his substrate and weighed the amount of protein precipitated by CuSOd and acetic acid solutions.Siewertg reported a method similar to the U.S.P. method for pancreatin:O in which tubes containing casein solution are incubated with varying amounts of papain.Alcoholic acetic acid solution is added and those tubes developing precipitates are distinguished from those remaining clear.The method presented in the Methods of Analysis of the A.O.A.C." belongs in the second group.It employs a casein solution bufTered with citrate a t pH 5. The liberated amino1* groups are titrated with 0.1 N alcoholic KOH after the addition of large volumes of alcohol to the digest.Bergmann and co-workers'g-'b made extensive use of synthetic peptides as substrates for studying the proteolytic activity of papain, the extent of