A novel, sensitive method for detecting protease inhibitors by using fluorescent protease substrates in gels is described. The protease inhibitors were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels containing a copolymerized peptide substrate, namely 4-methyl-coumaryl-7-amide (MCA). As the incorporated substrates in the gel, Boc-Phe Ser-Arg-MCA was used for trypsin, Suc-Ala-Ala-Pro-Phe-MCA for α-chymotrypsin, and Z-Phe-Arg-MCA for papain. After electrophoresis, washing and incubating the gel with the target protease solutions allowed the substrate to be cleaved by the protease, and the release of the fluorescent 7 amino-4 methyl-coumarin (AMC), which was detected under a UV transilluminator. The uncleaved peptide-MCA substrate remained where the inhibitors were present, and was visualized as dark blue bands on the light-green fluorescent background gel. This new method offers several advantages over other previous methods including: (i) greatly increased sensitivity can be achieved in a shorter period of time, which may be useful for discovering new protease inhibitors in small amounts of crude material; (ii) the procedure is quite simple and quick since the incubation period is very short and no time is needed for staining and destaining steps; (iii) since these probes using substrate specificity/target proteases, they are excellent tools for detection and discrimination of unknown protease inhibitors for various target proteases. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Le Q.T., Ohashi A., Hirose S., Katunuma N.

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Từ khóa : 4 methylcoumaryl 7 amide; amino acid derivative; aprotinin; benzyloxycarbonylphenylalanylarginine 4 methylcoumaryl 7 amide; chymotrypsin A; chymotrypsin inhibitor; coumarin derivative; cystatin C; fluorescent dye; papain; polypeptide; proteinase; proteinase inhibitor; serine proteinase inhibitor; soybean trypsin inhibitor; succinylalanylalanylprolylphenylalanine 4 methylcoumaryl 7 amide; tert butyloxycarbonylphenylalanylserylarginine 4 methylcoumaryl 7 amide; trypsin; unclassified drug; article; drug determination; drug discrimination; electrophoresis; enzymatic assay; enzyme specificity; enzyme substrate; fluorescence; protein degradation; reverse zymography; zymography; Animals; Chymotrypsin; Coumarins; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Intestinal Mucosa; Intestine, Small; Protease Inhibitors; Sensitivity and Specificity; Swine; Trypsin Inhibitors