PMID- 21513303
OWN - NLM
STAT- MEDLINE
DCOM- 20110830
LR  - 20110511
IS  - 1520-5126 (Electronic)
IS  - 0002-7863 (Linking)
VI  - 133
IP  - 19
DP  - 2011 May 18
TI  - Determining protease substrate selectivity and inhibition by label-free 
      supramolecular tandem enzyme assays.
PG  - 7528-35
LID - 10.1021/ja2013467 [doi]
AB  - An analytical method has been developed for the continuous monitoring of protease 
      activity on unlabeled peptides in real time by fluorescence spectroscopy. The 
      assay is enabled by a reporter pair comprising the macrocycle cucurbit[7]uril 
      (CB7) and the fluorescent dye acridine orange (AO). CB7 functions by selectively 
      recognizing N-terminal phenylalanine residues as they are produced during the 
      enzymatic cleavage of enkephalin-type peptides by the metalloendopeptidase 
      thermolysin. The substrate peptides (e.g., Thr-Gly-Ala-Phe-Met-NH(2)) bind to CB7 
      with moderately high affinity (K  approximately  10(4) M(-1)), while their cleavage products 
      (e.g., Phe-Met-NH(2)) bind very tightly (K > 10(6) M(-1)). AO signals the 
      reaction upon its selective displacement from the macrocycle by the high affinity 
      product of proteolysis. The resulting supramolecular tandem enzyme assay 
      effectively measures the kinetics of thermolysin, including the accurate 
      determination of sequence specificity (Ser and Gly instead of Ala), 
      stereospecificity (d-Ala instead of l-Ala), endo- versus exopeptidase activity 
      (indicated by differences in absolute fluorescence response), and sensitivity to 
      terminal charges (-CONH(2) vs -COOH). The capability of the tandem assay to 
      measure protease inhibition constants was demonstrated on phosphoramidon as a 
      known inhibitor to afford an inhibition constant of (17.8 +/- 0.4) nM. This robust 
      and label-free approach to the study of protease activity and inhibition should 
      be transferable to other endo- and exopeptidases that afford products with 
      N-terminal aromatic amino acids.
CI  - (c) 2011 American Chemical Society
FAU - Ghale, Garima
AU  - Ghale G
AD  - School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 
      D-28759 Bremen, Germany.
FAU - Ramalingam, Vijayakumar
AU  - Ramalingam V
FAU - Urbach, Adam R
AU  - Urbach AR
FAU - Nau, Werner M
AU  - Nau WM
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20110422
PL  - United States
TA  - J Am Chem Soc
JT  - Journal of the American Chemical Society
JID - 7503056
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Protease Inhibitors)
RN  - EC 3.4.- (Peptide Hydrolases)
SB  - IM
MH  - Enzyme Assays/methods
MH  - Fluorescent Dyes/*chemistry
MH  - Hydrophobic and Hydrophilic Interactions
MH  - Kinetics
MH  - Molecular Structure
MH  - Peptide Hydrolases/*chemistry
MH  - Protease Inhibitors/*chemistry
MH  - Substrate Specificity
EDAT- 2011/04/26 06:00
MHDA- 2011/08/31 06:00
CRDT- 2011/04/26 06:00
PHST- 2011/04/26 06:00 [entrez]
PHST- 2011/04/26 06:00 [pubmed]
PHST- 2011/08/31 06:00 [medline]
AID - 10.1021/ja2013467 [doi]
PST - ppublish
SO  - J Am Chem Soc. 2011 May 18;133(19):7528-35. doi: 10.1021/ja2013467. Epub 2011 Apr 
      22.