Rapid Procedure for the Quantitative Estimation of Antitrypsin

Gates, 1 Gilman and Cowgill, 2 and Pickford and Dorris, 3 have found that photographic film furnishes a useful substratum for the titration of proteolytic enzymes, since the gelatin layer, which is readily digested by pepsin or trypsin, is sufficiently uniform in thickness and consistency to permit quantitative tests. The release of the silver from an exposed (opaque) film, through the progressive proteolysis of the gelatin, causes a proportionate decrease in density, and finally, when digestion is complete, the film becomes entirely transparent.

We have utilized this principle in devising a new technique for the quantitative titration of the antitryptic power of serum or other fluids. At the same time, we have modified and simplified the procedures followed by the authors mentioned. We have avoided the use of the colorimeter for estimating changes in the density of the film, as advocated by Gilman and Cowgill, and instead take for the end-point the film showing complete transparency to the naked eye, thus measuring digestion of the gelatin only, without reference to an arbitrary standard of initial density. After determining by experiment the influence of various factors on the reaction, we are able to outline a definite procedure, which permits the exact reproduction of results at will, and the expression of the findings in simple figures. Further, we have provided for the preservation of the films themselves as a permanent record.

The following is the procedure now recommended.

Eastman Process film is fully exposed to light, and developed for 5 minutes in D-72 developer. It is then cleared and fixed in freshly prepared hypo solution, containing the usual proportion of Fl—a hardener, for 15 minutes. After thorough washing and drying, the film is cut into discs 1/4 inch in diameter with a hand punch.

A stock 2.0% solution of Fairchild's trypsin is made in a borax-boric acid, salt, buffer solution, pH 7.3, prepared according to the directions of Palitzsch, as given by Clark. 4 One gm. of the powdered trypsin is stirred into 50 cc. of the buffer solution, and the mixture allowed to stand at room temperature, with frequent shaking of the flask, for 15 minutes. It is then filtered into a sterile, screw-top bottle, and 0.1 cc. of a 1:1000 aqueous solution of merthiolate is added. Kept in the refrigerator, this stock solution loses potency gradually, but remains sufficiently active for at least 10 days.