Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay

Journal Article · Wed May 01 00:00:00 EDT 1991 · Analytical Biochemistry; (United States)

DOI:https://doi.org/10.1016/0003-2697(91)90248-R· OSTI ID:5229943

Jean, F; Basak, A; Chretien, M; Lazure, C ^[1]

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1. J. A. De Seve Laboratory of Molecular Neuroendocrinology, Clinical Research Institute of Montreal (Affiliated with l'Universite de Montreal), Quebec (Canada)

A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human {alpha} 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.

• OSTI ID: 5229943
• Journal Information: Analytical Biochemistry; (United States), Vol. 194:2; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PEPTIDE HYDROLASES
RADIOENZYMATIC ASSAY
AMINO ACID SEQUENCE
CLEAVAGE
ENZYME ACTIVITY
IODINE ISOTOPES
LIQUID COLUMN CHROMATOGRAPHY
PEPTIDES
SPECIFICITY
SUBSTRATES
TRACER TECHNIQUES
CHROMATOGRAPHY
CRYSTAL STRUCTURE
ENZYMES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
MICROSTRUCTURE
MOLECULAR STRUCTURE
ORGANIC COMPOUNDS
PROTEINS
SEPARATION PROCESSES
550201* - Biochemistry- Tracer Techniques