Physiological studies of exocrine pancreatic secretion in conscious rats. 3. Communication. Separation and determination of enzymes by gel-electrophoresis

Abstract

1. A method is described for the electrophoretic separation and following quantitative determination of the different protein fractions of pancreatic juice. 2. A polyacrylamide (PAA) gel-electrophoresis is performed in two systems: one anionic at pH = 9.0 with 7.5% PAA and a cationic one at pH = 5.0 with 10% PAA. 3. After about two hours of separation using a current of 3 and 4 mA respectively, the gels are stained with Amido-black 10B after fixation of the proteins with acetic acid. 4. The colour in excess is removed with acetic acid. 5. The coloured bands are isolated by cutting. 6. Evaluation of the colour by NaOH and determination of the colour intensity by photometry. 7. Evaluation of the colour extinction as relative percentages of the different protein and enzyme fractions. (Extinction of total dye = 100%) 8. There are at least 13 bands in the cationic system and about 7 additional ones in the anionic system distinctly separated, 12 of them could be identified using enzymatic methods. 9. Evidence is given that the colour intensity has a linear relationship to enzyme activity. 10. The validity of the presented method is discussed and compared with other electrophoretic methods.