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Reverse zymography using fluorogenic substrates for protease inhibitor detection.
Electrophoresis. 2005 Mar; 26(6):1038-45.E

Abstract

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.

Authors+Show Affiliations

Le QT
Tokushima Bunri University, Institute for Health Sciences, 180 Nishihamabouji, Yamashiro-cho, Tokushima City, Tokushima 770-8514, Japan.
Ohashi A
No affiliation info available
Hirose S
No affiliation info available
Katunuma N
No affiliation info available

MeSH

AnimalsChymotrypsinCoumarinsElectrophoresis, Polyacrylamide GelFluorescent DyesIntestinal MucosaIntestine, SmallProtease InhibitorsSensitivity and SpecificitySwineTrypsin Inhibitors

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15669004

Citation

Le, Quang T., et al. "Reverse Zymography Using Fluorogenic Substrates for Protease Inhibitor Detection." Electrophoresis, vol. 26, no. 6, 2005, pp. 1038-45.
Le QT, Ohashi A, Hirose S, et al. Reverse zymography using fluorogenic substrates for protease inhibitor detection. Electrophoresis. 2005;26(6):1038-45.
Le, Q. T., Ohashi, A., Hirose, S., & Katunuma, N. (2005). Reverse zymography using fluorogenic substrates for protease inhibitor detection. Electrophoresis, 26(6), 1038-45.
Le QT, et al. Reverse Zymography Using Fluorogenic Substrates for Protease Inhibitor Detection. Electrophoresis. 2005;26(6):1038-45. PubMed PMID: 15669004.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Reverse zymography using fluorogenic substrates for protease inhibitor detection. AU - Le,Quang T, AU - Ohashi,Atsushi, AU - Hirose,Sayu, AU - Katunuma,Nobuhiko, PY - 2005/1/26/pubmed PY - 2005/8/6/medline PY - 2005/1/26/entrez SP - 1038 EP - 45 JF - Electrophoresis JO - Electrophoresis VL - 26 IS - 6 N2 - 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. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/15669004/Reverse_zymography_using_fluorogenic_substrates_for_protease_inhibitor_detection_ DB - PRIME DP - Unbound Medicine ER -