PMID- 22545408
OWN - NLM
STAT- MEDLINE
DCOM- 20120518
LR  - 20161125
IS  - 1934-578X (Print)
IS  - 1555-9475 (Linking)
VI  - 7
IP  - 3
DP  - 2012 Mar
TI  - Monitoring stepwise proteolytic degradation of peptides by supramolecular domino 
      tandem assays and mass spectrometry for trypsin and leucine aminopeptidase.
PG  - 343-8
AB  - A label-free optical detection method has been designed that allows direct 
      monitoring of enzymatic peptide digestion in vitro. The method is based on the 
      addition of a reporter pair, composed of the macrocyclic host cucurbit[7]uril 
      (CB7) and the fluorescent dye acridine orange (AO), to detect the proteolytic 
      degradation of peptides. The enzymatic activity of trypsin and leucine 
      aminopeptidase (LAP) was investigated using H-LSRFSWGA-OH as a substrate. The 
      substrate as well as the intermediary and final products (i.e., H-FSWGA-OH and 
      phenylalanine) formed during its enzymatic hydrolysis differ in their binding 
      affinity to the receptor CB7, which results in varying degrees of dye 
      displacement and, therefore, different fluorescence intensities. CB7 showed a 
      relatively weak binding constant of K approximately 10(4) M(-1) with the 
      substrate, a relatively strong binding constant of K > or = 10(6) M(-1) with 
      H-FSWGA-OH (which is a final product formed by trypsin digestion and the 
      intermediary product formed during the enzymatic activity of LAP), and a moderate 
      binding constant of K < or = 10(5) M(-1) with phenylalanine. Owing to this 
      differential binding affinity of CB7 with the substrate and the corresponding 
      products, the digestion of a peptide by trypsin was followed as a decrease in 
      fluorescence signal, while the complete degradation of the peptide by LAP was 
      monitored as a decrease and a subsequent increase in fluorescence signal. The 
      k(cat)/K(M) value for trypsin (2.0 x 10(7) min(-1) M(-1)) was derived from the 
      change in fluorescence signal with time. Additionally, the complete degradation 
      of the peptide by LAP was also followed by mass spectrometry. The use of a 
      supramolecular sensing ensemble (macrocyclic host and dye) as a fluorescent 
      reporter pair gives this method the flexibility to adapt for monitoring the 
      stepwise degradation of different biologically relevant peptides by other 
      proteases.
FAU - Ghale, Garima
AU  - Ghale G
AD  - School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 
      Bremen, Germany.
FAU - Kuhnert, Nikolai
AU  - Kuhnert N
FAU - Nau, Werner M
AU  - Nau WM
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Nat Prod Commun
JT  - Natural product communications
JID - 101477873
RN  - 0 (Bridged-Ring Compounds)
RN  - 0 (Imidazoles)
RN  - 0 (Peptides)
RN  - 0 (cucurbit(7)uril)
RN  - EC 3.4.11.1 (Leucyl Aminopeptidase)
RN  - EC 3.4.21.4 (Trypsin)
RN  - F30N4O6XVV (Acridine Orange)
SB  - IM
MH  - Acridine Orange/*chemistry
MH  - Bridged-Ring Compounds/*chemistry
MH  - Imidazoles/*chemistry
MH  - Leucyl Aminopeptidase/*metabolism
MH  - Mass Spectrometry
MH  - Peptide Mapping
MH  - Peptides/*metabolism
MH  - Trypsin/*metabolism
EDAT- 2012/05/02 06:00
MHDA- 2012/05/19 06:00
CRDT- 2012/05/02 06:00
PHST- 2012/05/02 06:00 [entrez]
PHST- 2012/05/02 06:00 [pubmed]
PHST- 2012/05/19 06:00 [medline]
PST - ppublish
SO  - Nat Prod Commun. 2012 Mar;7(3):343-8.