Journal
JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS
Volume 70, Issue 6, Pages 878-882Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbbm.2007.05.004
Keywords
proteases; matrix metalloproteinases; myocardium; extracellular matrix
Funding
- NHLBI NIH HHS [HL-80049, R01 HL067922-04, R01 HL067922, R01 HL043617, HL-43617, R01 HL043617-12] Funding Source: Medline
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Analysis of collagen degradation remains an important but cumbersome task. Traditional methods with dansyl chloride derivatization of collagen have been used to quantify collagen damage. Fluorescent labeling reagents have been developed that offer advantages such as greater solubility in water and low background emission. One such reagent is o-phthalaldehyde (OPA). In this study, we used OPA as a means of detecting small amounts of degraded collagen. Collagen samples isolated from skin or heart were used for OPA conjugation to exposed amino termini (opalation). Experiments utilizing small samples aliquoted in microtiter plates were performed to evaluate effects of increasing concentrations of OPA, varying concentrations of collagen, and effects of matrix metalloproteinase (NIMP) digestion. Results indicate that within 10 min of reaction, OPA can be used to detect relative differences in cleaved vs. uncleaved collagen from skin or heart. Heart samples obtained from regions of high MNIP activity correlated with increased OPA fluorescence relative to tissue with lower MMP activity. On the basis of these results, we conclude that OPA has valuable practical advantages for analytical use in detecting cleaved collagen in small tissue samples. (c) 2007 Elsevier B. V. All rights reserved.
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