title: A colorimetric-based amplification system for proteinases including MMP2 and ADAM8.
abstract: We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity.
authors.affiliation: BioZyme, Apex, NC 27523, USA. Electronic address: moss0610@yahoo.com.
authors.forename: Marcia L
authors.initials: ML
authors.lastname: Moss
authors.affiliation: Dental Institute, King's College London, London SE1 9RT, United Kingdom; Department of Neurosurgery, Philipps University Marburg, 35033 Marburg, Germany.
authors.forename: Garrit
authors.initials: G
authors.lastname: Koller
authors.affiliation: Department of Neurosurgery, Philipps University Marburg, 35033 Marburg, Germany.
authors.forename: Jörg W
authors.initials: JW
authors.lastname: Bartsch
authors.affiliation: Department of Neurosurgery, Philipps University Marburg, 35033 Marburg, Germany.
authors.forename: Sinja
authors.initials: S
authors.lastname: Rakow
authors.affiliation: Department of Neurosurgery, Philipps University Marburg, 35033 Marburg, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Schlomann
authors.affiliation: BioZyme, Apex, NC 27523, USA.
authors.forename: Fred H
authors.initials: FH
authors.lastname: Rasmussen
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001560:Benzidines / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D004151:Dipeptides / Q000494:pharmacology; D057075:Enzyme Assays / Q000379:methods*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D059346:Magnets / Q000737:chemistry; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism* / Q000652:urine; D061965:Matrix Metalloproteinase Inhibitors / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D008863:Microspheres; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001560:Benzidines; D004151:Dipeptides; D061965:Matrix Metalloproteinase Inhibitors; D008565:Membrane Proteins; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D009942:Organometallic Compounds; D010455:Peptides; C088321:nickel nitrilotriacetic acid; C021758:3,3',5,5'-tetramethylbenzidine; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D051722:ADAM Proteins; C068490:ADAM8 protein, human; D020778:Matrix Metalloproteinase 2; D009571:Nitrilotriacetic Acid
keywords: Amplification; Disintegrin metalloproteinase; Enzyme assay; Matrix metalloproteinase; Proteinase