A dityrosine-based substrate for a protease assay: Application for the selective assessment of papain and chymopapain activity

Chan Jin Kim, Dong Ik Lee, Chang Ha Lee, Ik Sung Ahn

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Abstract

N,N'-diBoc-dityrosine (DBDY), which was synthesized by the oxidative C-C coupling of 2 N-Boc-l-tyrosine molecules, was conjugated with two isoniazid (INH) molecules. Due to the quenching effect of INH, DBDY-(INH) 2 lacks the fluorescence of DBDY. As such, it was tested for use in the detection of proteases by measuring fluorescence recovery. In this study, serine proteases (chymotrypsin, trypsin, subtilisin, and proteinase K), metalloproteases (thermolysin and carboxypeptidase A, dispase, and collagenase), aspartic proteases (pepsin and aspergillopepsin) and cysteine proteases (papain and chymopapain) were chosen. Reported optimum assay conditions were chosen for each enzyme. Only papain and chymopapain catalyzed the hydrolysis of DBDY-(INH) 2 and led to fluorescence recovery, possibly due to their extensive binding sites and the INH-mediated inhibition of metalloproteases and aspartic proteases.

Original languageEnglish
Pages (from-to)101-107
Number of pages7
JournalAnalytica Chimica Acta
Volume723
DOIs
Publication statusPublished - 2012 Apr 20

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All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy

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