A Method of Proteolytic Enzyme Titration.

Abstract

In 1906 Müller and Jochmann 1 proposed a simple method of determining the presence of proteolytic enzymes in biological fluids by erosion of the surface of a Loeffler serum plate under a drop of the fluid. Later this technique was modified by the use of a gelatin plate dyed with carmine, but at best it was a + or − method, probably because the substrate was not sufficiently uniform to permit quantitative estimates of enzyme action.

If a photographic plate, or film, is fogged on both sides by equal exposures, is developed, fixed, washed, and dried, it presents a gelatin surface and texture of sufficient uniformity for quantitative tests. Proteolytic enzymes erode the surface and free the included silver, so that the light transmission of the exposed area increases as digestion proceeds, and can be estimated relatively by colorimetry, or directly by a calibrated thermopile and galvanometer. The method is as follows:

Small paraffined chambers are filled with trypsin or pepsin solution so that the meniscus projects above the rim. Face down over each chamber is placed a small square of the photographic plate, which has been previously brought to the proper pH concentration in phosphate buffer solution and allowed to dry without rinsing. Due to the inverted position of the gelatin layer the products of proteolysis and the released silver fall away from the surface, and interfere less with the progress of the reaction than as if they accumulated there. Temperature is carefully controlled, and plates are removed at timed intervals, rinsed quickly in cold water, immersed in a bath of the opposite reaction to arrest enzyme action, and dried rapidly before a fan.