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Anal Biochem. 2015 Sep 1;484:75-81. doi: 10.1016/j.ab.2015.05.011. Epub 2015 May 27.

A colorimetric-based amplification system for proteinases including MMP2 and ADAM8.

Author information

1
BioZyme, Apex, NC 27523, USA. Electronic address: moss0610@yahoo.com.
2
Dental Institute, King's College London, London SE1 9RT, United Kingdom; Department of Neurosurgery, Philipps University Marburg, 35033 Marburg, Germany.
3
Department of Neurosurgery, Philipps University Marburg, 35033 Marburg, Germany.
4
BioZyme, Apex, NC 27523, USA.

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.

KEYWORDS:

Amplification; Disintegrin metalloproteinase; Enzyme assay; Matrix metalloproteinase; Proteinase

PMID:
26026386
DOI:
10.1016/j.ab.2015.05.011
[Indexed for MEDLINE]

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