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Modification of a calcein‐based assay for monitoring proteolytic activity in tissue suffusate
Author(s) -
Mikel Kyle,
Bingaman Susan,
Huxley Virginia H.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.927.5
Subject(s) - calcein , proteolysis , chemistry , proteolytic enzymes , chromatography , biochemistry , microbiology and biotechnology , enzyme , biology , membrane
This investigation was aimed at developing an assay to characterize protein degradation in effluent collected from tissues on which microvascular permeability studies were being conducted (Bingaman S., FASEBJ: 20, 2006). In particular, we were measuring protein clearance from venules and assaying total protein in the effluent. The assumption we wanted to test was whether the protein levels reflected intact material, whether proteolysis was occurring, and whether the treatments which altered protein clearance reflected changes in proeolytic activity. The approach we used was based on the quenching of calcein (Ex 450 nm, Em 530 nm) by Cu(II) and the ability of proteolytic products (amino acids and peptides) to coordinate with the Cu(II), removing it from the calcein, allowing it to fluoresce (Dean, K., Biorganic & Medicinal Chem Letters, 2003). After optimization of assay conditions, samples collected from representative leak index experiments (Bingaman S., FASEBJ: 20, 2006) were tested. The results showed a clear difference between samples with respect to the distribution of proteins and small peptides under both control and test conditions. Therefore, it can be concluded that this assay can be used to screen for proteolytic activity in the microvasculature. Supported by NIH HLO78816, NASA NN04GA50G, AHA 0615515Z (RS), NIHCO6RRO17353 and Missouri Academy