Sensitive Method to Identify and Characterize Proteinases In Situ after SDS-PAGE

• Published in: BioTechniques Vol. 29; no. 4; pp. 1108 - 1113
• Main Authors: Williams, Jennifer, McGrath, William J, Mangel, Walter F
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.11.2000; Eaton
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cattle; Diverse techniques; Electrophoresis, Polyacrylamide Gel; Endopeptidases - analysis; Endopeptidases - chemistry; Endopeptidases - metabolism; Enzymes and enzyme inhibitors; Fluorescence; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Fundamental and applied biological sciences. Psychology; Hydrolases; Image Processing, Computer-Assisted; Molecular and cellular biology; Molecular Structure; Molecular Weight; Peptides - chemistry; Peptides - metabolism; Protease Inhibitors - analysis; Protease Inhibitors - metabolism; Protein Denaturation; Rhodamines - chemistry; Rhodamines - metabolism; Sensitivity and Specificity; Substrate Specificity; Trypsin - analysis; Trypsin - chemistry; Trypsin - metabolism; Peptidases; Sensitivity; Fluorogenic substrate; Enzyme; Gel electrophoresis; In situ; Hydrolases; Identification; Method; Quantitative analysis
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/00295rr07

Cells and body fluids contain numerous, different proteinases; to identify and characterize them are both important and difficult tasks. Especially difficult to identify and characterize are highly specific proteinases. Here, we present an extremely sensitive and quantitative method to characterize proteinases fractionated by SDS-PAGE that cleave specific rhodamine-based fluorogenic substrates. To test the sensitivity of the technique, we used trypsin as our model system. Filter paper impregnated with rhodamine-based fluorogenic substrates was placed on a gel, and bands of fluorescence originating from specific proteinases were visualized in real time. The method is very sensitive; picogram amounts of trypsin can be detected. The method should be very general, in that even proteinases whose substrates require amino acids C-terminal to the cleavage site may be identified and characterized. The results allow one to obtain not only information on the substrate specificity of a specific enzyme but also information about its molecular weight.