Isoelectric focusing (IEF) and band detection with fluorogenic protease substrates.
In a previous paper, combined dye histochemistry and analytical isoelectric focusing (IEF) of supernatants from organ homogenates have been shown to yield good results for the detection of protease isoenzymes. Difficulties arise when the protease to be studied is partially or completely inhibited by diazonium salts and when synthetic peptide substrates different from 4-methoxy-2-naphthylamine (MNA) amino acids and peptides are to be used. In this paper a technique is described in which cellulose acetate foils impregnated with MNA, 7-amino-4-methyl-coumarin (AMC) and 7-amino-4-trifluoromethylcoumarin (AFC) substrates are overlaid on electrophoresis strips after IEF. After incubation, the foils are viewed with an UV-lamp and photographed. The MNA, AMC and AFC peptides are equally suitable for fluorescence band detection. Using this technique, occasionally protease isoenzymes are found which are more sensitive towards diazonium salts, e.g. aminopeptidase A and M. Sometimes it is also possible to detect thiolproteases which is not the case when employing dye histochemistry.
D000626:Aminopeptidases / Q000032:analysis; D000818:Animals; D018826:CD13 Antigens; D002863:Chromogenic Compounds; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D043384:Glutamyl Aminopeptidase; D006651:Histocytochemistry; D006868:Hydrolysis; D007525:Isoelectric Focusing / Q000379:methods; D007527:Isoenzymes / Q000032:analysis* / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D010447:Peptide Hydrolases / Q000032:analysis* / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013363:Submandibular Gland / Q000201:enzymology
D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D007527:Isoenzymes; D010450:Endopeptidases; D010447:Peptide Hydrolases; D000626:Aminopeptidases; D018826:CD13 Antigens; D043384:Glutamyl Aminopeptidase; D003546:Cysteine Endopeptidases