Reverse zymography using fluorogenic substrates for protease inhibitor detection.
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 alpha-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.
Le
Quang T
QT
Tokushima Bunri University, Institute for Health Sciences, 180 Nishihamabouji, Yamashiro-cho, Tokushima City, Tokushima 770-8514, Japan.
Ohashi
Atsushi
A
Hirose
Sayu
S
Katunuma
Nobuhiko
N
D000818:Animals; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D003374:Coumarins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes; D007413:Intestinal Mucosa / Q000737:chemistry; D007421:Intestine, Small / Q000737:chemistry; D011480:Protease Inhibitors / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013552:Swine; D014361:Trypsin Inhibitors / Q000302:isolation & purification
C502573:4-methyl-coumaryl-7-amide; D003374:Coumarins; D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; D002918:Chymotrypsin