Fluorescent method for detection of cleaved collagens using O-phthaldialdehyde (OPA)
Fluorescent method for detection of cleaved collagens using O-phthaldialdehyde (OPA)
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DOI：
10.1016/j.jbbm.2007.05.004
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发表时间：
2008-04-24
影响因子：
--
通讯作者：
Villarreal, Francisco J.
中科院分区：
文献类型：
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作者：
Go, Katrina;Horikawa, Yousuke;Villarreal, Francisco J.
胶原蛋白降解的分析仍然是一项重要但繁琐的任务。传统的胶原酰氯衍生化方法已被用于定量胶原损伤。荧光标记试剂已开发，提供的优点，如更大的溶解度在水中和低本底辐射。其中一种试剂是邻苯二醛（OPA）。在这项研究中，我们使用OPA作为检测少量降解胶原蛋白的手段。从皮肤或心脏分离的胶原样品用于OPA偶联到暴露的氨基末端（“乳清”）。利用微量滴度板上的小样本进行实验，以评估增加OPA浓度、改变胶原浓度和基质金属蛋白酶（NIMP）消化的影响。结果表明，在反应10分钟内，OPA可用于检测皮肤或心脏中断裂与未断裂胶原蛋白的相对差异。相对于MMP活性较低的组织，从MNIP活性高的区域获得的心脏样本与OPA荧光增加相关。在这些结果的基础上，我们得出结论，OPA在小组织样品中检测裂解胶原的分析中具有宝贵的实用优势。(c) 2007 Elsevier b.v.版权所有
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.