Precipitation of Proteolytic Enzymes of Bacillus Proteus by Azine and Azonium Bases.

Abstract

That there is a definite chemical union between a proteolytic enzyme and an azine or azonum base was first suggested by Robertson, 1 who observed that if a drop of a saturated solution of safranine was added to a solution of trypsin a flocculent precipitate was formed which gradually settled to the bottom of the tube.

Holzberg 2 tested the proteolytic activity of this safranine-trypsin precipitate and found that it hydrolized casein, thus proving that the enzyme was precipitated by the safranine. Marston 3 made a study of the chemical nature of this compound. He found that the proteolytic enzyme trypsin was completely precipitated from its solution by safranine, and also by several other compounds which were water soluble and contained the azine nucleus, such as neutral violet, neutral red and the more complex indulines.

Marston states that the type of union between the protease and the azine base in a direct combination of the enzyme with the azine nitrogen, the acid nature or property of the enzyme being responsible for this combination. As evidence of this union he says that with the reduced leuko compound of safranine where the nitrogen bonds are all satisfied with hydrogen, no precipitation of the proteolytic enzyme takes place. The results of the work quoted above suggested the possibility of purifying, and studying the chemical nature and action of bacterial proteases.

With this in view a series of experiments were begun. Bacillus proteus was selected as the organism to use as it excretes a very active proteolytic enzyme. A culture of this organism was inoculated into sugar-free nutrient broth, and incubated for five days. At the end of this period the broth culture was filtered through Berkefeld filters to remove the organisms. The clear filtrate consisting of the broth, plus the soluble products of the bacteria, was first tested for the presence of the enzyme.