Enzymoblotting: A method for localizing proteinases and their zymogens using para-nitroanilide substrates after agarose gel electrophoresis and transfer to nitrocellulose

• Published in: Analytical biochemistry Vol. 152; no. 2; pp. 239 - 244
• Main Authors: Ohlsson, Bertil G., Weström, Björn R., Karlsson, Börje W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.1986
• Subjects: Aniline Compounds; Animals; Cattle; Chymotrypsin - isolation & purification; Collodion; Electrophoresis, Agar Gel; Endopeptidases - isolation & purification; gel electrophoresis; gel electrophoresis,proteins; methodology; Methods; Pancreas - enzymology; Pancreatic Elastase - isolation & purification; para-nitroanilide substrates; protein blotting; proteinase; proteinases; Swine; swine pancreas; Trypsin - isolation & purification; zymogens; zymogens; gel electrophoresis,proteins; para-nitroanilide substrates; protein blotting; proteinases; swine pancreas
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(86)90404-5

A method-enzymoblotting-was developed for localizing various enzymes after electrophoretic separation, transfer to nitrocellulose, and incubation with specific substrates. As an application, the proteinases porcine trypsin (EC 3.4.21.4), bovine chymotrypsin (EC 3.4.21.1), porcine elastase (EC 3.4.22.11), and their zymogen forms from porcine pancreas homogenate were analyzed utilizing specific p-nitroanilide substrates. After agarose gel electrophoresis, transfer of the separated proteinases to a nitrocellulose membrane was performed by capillary diffusion for 30 min. After airdrying of the nitrocellulose membrane, it was incubated in the appropriate substrate solution for 60 min. N-α-Benzoyl- dl-arginine- para-nitroanilide HCl was used as a substrate for trypsin, N-benzoyl- l-tyrosine- para-nitroanilide and succinyl- l-phenylalanine- para-nitroanilide for chymotrypsin, and N-succinyl- l-alanyl- l-alanyl- l-alanine- para-nitroanilide for elastase. p-Nitroaniline, the product thus obtained, was diazotized with N-(1-naphthyl)ethylenediamine to a red azo dye, visible at the site of the proteinases on the nitrocellulose membrane. The results could be preserved at −18°C. Zymogen forms of the pancreas proteinases were detected in a similar manner. They were converted to active proteinases in situ on the nitrocellulose membrane after preincubating the nitrocellulose membrane in the activation enzymes enteropeptidase or trypsin.