Isolation, purification and partial characterization of novel fluorophores from aging human insoluble collagen-rich tissue.
Isolation, purification and partial characterization of novel fluorophores from aging human insoluble collagen-rich tissue.
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DOI：
10.3109/03008208909016816
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发表时间：
1989
影响因子：
2.9
通讯作者：
D. Sell;V. Monnier
中科院分区：
文献类型：
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作者：
D. Sell;V. Monnier
胶原蛋白随着衰老和糖尿病而经历以变黄、荧光和交联为特征的进行性布朗宁，其原因仍未阐明。作为理解胶原蛋白在老化中不溶解的机制的第一步，从人硬脑膜的不溶性部分中分离出主要的荧光团/发色团，并对其光谱特性进行表征。不溶性胶原蛋白的高分子量胰蛋白酶肽通过连续酶消化裂解，然后通过凝胶过滤色谱法分离成高（HMW）和低分子量（LMW）级分。通过纸层析和反相色谱（HPLC）进一步分离LMW。从LMW中获得了两个荧光峰（昵称为P和M），其UV最大值分别位于325和350 nm处，激发/荧光最大值分别位于335/385和360/460 nm处。荧光团M是硼氢化物还原和不稳定的酸水解，而P保持不受影响。大量的荧光团M和吡啶啉被发现在高度交联的HMW馏分后，剩余的彻底蛋白水解消化。荧光团P和M是从不溶性硬脑膜的胰蛋白酶消化物中回收的主要荧光团。M的荧光光谱表明亚氨基丙烯型构型，其可能是由胶原蛋白的非酶促布朗宁引起的，例如，葡萄糖或丙二醛，作为脂质过氧化的结果。荧光团P的光谱和化学性质令人想起，但不相同的吡啶鎓交联。这些荧光团的结构解析预计将提供重要的洞察细胞外基质的老化过程。
Collagen undergoes progressive browning with aging and diabetes characterized by yellowing, fluorescence and crosslinking, the cause of which remains unelucidated. As an initial step towards understanding the mechanism(s) of insolubilization of collagen in aging, the major fluorophores/chromophores from the insoluble fraction of human dura mater were isolated and their spectroscopic properties were characterized. High molecular weight tryptic peptides of insoluble collagen were cleaved by sequential enzymatic digestion followed by separation into high (HMW) and low molecular weight (LMW) fractions by gel filtration chromatography. LMW was further separated by paper and reverse phase chromatography (HPLC). Two fluorescent peaks, nicknamed P and M, were obtained from LMW which had UV maxima at 325 and 350 nm and excitation/fluorescence maxima at 335/385 and 360/460 nm, respectively. Fluorophore M was borohydride reducible and unstable to acid-hydrolysis, while P remained unaffected. Large quantities of fluorophore M and pyridinoline were found in the highly crosslinked HMW fraction remaining following exhaustive proteolytic digestion. Fluorophore P and M were the major fluorophores recovered from the tryptic digest of insoluble dura mater. Fluorescence spectra of M suggest an iminopropene type of configuration which could result from nonenzymatic browning of collagen with, e.g., glucose or malonyldialdehyde, as a result of lipid peroxidation. Spectroscopic and chemical properties of fluorophore P were reminiscent but not identical with those of pyridinium crosslinks. Structure elucidation of these fluorophores is expected to provide important insight into the aging processes of the extracellular matrix.