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Detection of proteolytic activity by fluorescent zymogram in-gel assays.
Biotechniques. 2000 Jun; 28(6):1166-8, 1170, 1172-3.B

Abstract

Proteases are involved in the regulation of many biological functions. This study describes a novel method for detecting protease activity by fluorescent zymogram in-gel protease assays, using SDS polyacrylamide gels copolymerized with a peptide-MCA (4-methyl-coumaryl-7-amide) substrate. This method allows simultaneous determination of protease cleavage specificity and molecular weight. Trypsin was electrophoresed in SDS polyacrylamide gels copolymerized with Boc-Gln-Ala-Arg-MCA, the gel was then incubated in assay buffer, and trypsin cleavage of the peptide-MCA substrate generated fluorescent AMC (7-amino-4-methyl-coumarin), which was subsequently detected under UV transillumination. Chymotrypsin activity was detected in gels copolymerized with Suc-Ala-Ala-Pro-Phe-MCA substrate. Selective detection of these proteases was demonstrated by the absence of trypsin activity in gels containing the chymotrypsin substrate, and the lack of chymotrypsin activity in gels containing the trypsin substrate. Detection of proteolytic activity from secretory vesicles of adrenal medulla (chromaffin granules) was observed with the trypsin substrate, Z-Phe-Arg-MCA, but not with the chymotrypsin substrate. Overall, this sensitive fluorescent zymogram in-gel protease assay method can be used for rapid determination of protease cleavage specificity and enzyme molecular weight in biological samples. This assay should be useful for many research disciplines investigating the role of the many proteases that control cellular functions.

Links

Publisher Full Text
future-science.com
future-science.com

Authors+Show Affiliations

Yasothornsrikul S
University of California, San Diego, La Jolla, USA.
Hook VY
No affiliation info available

MeSH

ChymotrypsinElectrophoresis, Polyacrylamide GelEndopeptidasesFluorescenceTrypsin

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10868282

Citation

Yasothornsrikul, S, and V Y. Hook. "Detection of Proteolytic Activity By Fluorescent Zymogram In-gel Assays." BioTechniques, vol. 28, no. 6, 2000, pp. 1166-8, 1170, 1172-3.
Yasothornsrikul S, Hook VY. Detection of proteolytic activity by fluorescent zymogram in-gel assays. Biotechniques. 2000;28(6):1166-8, 1170, 1172-3.
Yasothornsrikul, S., & Hook, V. Y. (2000). Detection of proteolytic activity by fluorescent zymogram in-gel assays. BioTechniques, 28(6), 1166-8, 1170, 1172-3.
Yasothornsrikul S, Hook VY. Detection of Proteolytic Activity By Fluorescent Zymogram In-gel Assays. Biotechniques. 2000;28(6):1166-8, 1170, 1172-3. PubMed PMID: 10868282.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection of proteolytic activity by fluorescent zymogram in-gel assays. AU - Yasothornsrikul,S, AU - Hook,V Y, PY - 2000/6/27/pubmed PY - 2000/10/21/medline PY - 2000/6/27/entrez SP - 1166-8, 1170, 1172-3 JF - BioTechniques JO - Biotechniques VL - 28 IS - 6 N2 - Proteases are involved in the regulation of many biological functions. This study describes a novel method for detecting protease activity by fluorescent zymogram in-gel protease assays, using SDS polyacrylamide gels copolymerized with a peptide-MCA (4-methyl-coumaryl-7-amide) substrate. This method allows simultaneous determination of protease cleavage specificity and molecular weight. Trypsin was electrophoresed in SDS polyacrylamide gels copolymerized with Boc-Gln-Ala-Arg-MCA, the gel was then incubated in assay buffer, and trypsin cleavage of the peptide-MCA substrate generated fluorescent AMC (7-amino-4-methyl-coumarin), which was subsequently detected under UV transillumination. Chymotrypsin activity was detected in gels copolymerized with Suc-Ala-Ala-Pro-Phe-MCA substrate. Selective detection of these proteases was demonstrated by the absence of trypsin activity in gels containing the chymotrypsin substrate, and the lack of chymotrypsin activity in gels containing the trypsin substrate. Detection of proteolytic activity from secretory vesicles of adrenal medulla (chromaffin granules) was observed with the trypsin substrate, Z-Phe-Arg-MCA, but not with the chymotrypsin substrate. Overall, this sensitive fluorescent zymogram in-gel protease assay method can be used for rapid determination of protease cleavage specificity and enzyme molecular weight in biological samples. This assay should be useful for many research disciplines investigating the role of the many proteases that control cellular functions. SN - 0736-6205 UR - https://www.unboundmedicine.com/medline/citation/10868282/Detection_of_proteolytic_activity_by_fluorescent_zymogram_in_gel_assays_ L2 - https://www.future-science.com/doi/10.2144/00286st07?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -