Characterization of TAMRA- and biotin-conjugated peptide arrays for on-chip matrix metalloproteinase activity assay

Deok Hoon Kong, Mahendra Prasad Bhatt, Seung-Taek Lee, Young Myeong Kim, Kwon Soo Ha

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Peptide arrays have been widely used for high-throughput determination of protease activities in cells and tissues because specific peptides have high binding affinity for the active site of enzymes. Designing peptide substrate probes for enzyme activity assays have been considered to be important; however, the significance of its reporter tag for detecting enzymatic reactions is relatively underestimated. Thus, we investigated the effect of the reporter tag of peptide substrate probes on on-chip protease activity assays. We optimized and characterized proteolytic activity assay of matrix metalloproteinase-3 using direct and indirect substrate probes, tetramethyl-6-carboxyrhodamine (TAMRA)- and biotin-conjugated peptide arrays, respectively. Proteolytic activity assays using both substrate probes demonstrated similar sensitivity, ratio of maximal to minimal FI, IC50 of GM6001, and inter-array reproducibility. However, biotin-conjugated substrate arrays showed a wider dynamic range than TAMRA-conjugated substrate arrays. Thus, this comparative study provides a wealth of information for developing optimal probes necessary for effective analysis of enzyme activity and kinetics.

Original languageEnglish
Pages (from-to)307-313
Number of pages7
JournalBiochip Journal
Volume6
Issue number4
DOIs
Publication statusPublished - 2012 Dec 1

    Fingerprint

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Electrical and Electronic Engineering

Cite this