FRACTIONATION OF TRYPSIN BY PAPER ELECTROPHORESIS

P work from this laboratoryl-S has demonstrated the heterogeneity of crystalline trypsin by paper and starch column electrophoresis. This article presents an electrophoretic method of greater resolution. Using this technique it was possible to demonstrate in a sample of crystalline trypsin the occurrence of fractions showing different ratios of proteolytic to amidasic activity. In the experiments recorded here a buffer of pyridine acetate of pH 4·9 (0,48 : 0·56 : 98·96 v/v of acetic acid: pyridine: water) was used for electrophoresis in a Durrumtype apparatus. To a strip of Whatman 3 filter paper of 12 x 50 cm, 0·12 ml. of an 8 per cent trypsin solution in 0·001 N hydrochloric acid were applied. An electrical tension of 450 V was used and the experiment was allowed to run for 5 h in the cold room. In these conditions at the end of the experiment the temperature of the buffer was 9° C. The strip, while still wet, was cut in 1-cm wide transversal segments and transferred individually to test-tubes containing 3ml. of a 0·25 M pyridine formate buffer of pH 2·7. This extraction was allowed to continue for about 20 h in the cold room. The extract of each tube was then analysed for protein content, proteolytic activity against azo casein and amidasic activity towards cebenzoyl-L-arginine amide (BAA). The protein content was determined by the FolinLowry4 method as applied to the 'auto analyseI" system·. Proteolytic activity against azo casein was measured by the method of Charney and Tomarelli". The hydrolysis of BAA was followed colorimetrically in the auto analyser'. Chromatography of trypsin in carboxy methyl cellulose was carried out according to the method of Liener8• The trypsin samples used in this work were purchased from the 'Worthington Biochemical Corporation ('Cryst. Trypsin, lyophilized, lot TL 747-58'), Freehold, New Jersey, and from Novo Industri ('Cryst. Trypsin Novo, batch 114-3'), Copenhagen, Denmark. The results obtained by the fractionation of the Worthn~on trypsin are shown in Fig. 1. The presence of three