A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen.
A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen.
复制标题
DOI：
10.1016/0003-2697(85)90642-6
复制
发表时间：
1985-07
影响因子：
2.9
通讯作者：
D. Dean;J. Woessner
中科院分区：
文献类型：
--
作者：
D. Dean;J. Woessner
通过与从大鼠皮肤提取并用[2-3H]乙酸酐标记的可溶性、不含端肽的胶原蛋白一起孵育来测定胶原酶。胶原蛋白被胶原酶裂解，产生的片段用胰蛋白酶和胰凝乳蛋白酶消化。通过用三氯乙酸沉淀来回收未消化的胶原蛋白，将其收集在玻璃纤维过滤器上，并通过液体闪烁光谱法进行定量。该程序结合了 Cawston 和 Barrett（T. E. Cawston 和 A. J. Barrett, 1979, Anal. Biochem.99, 340–345）和 Ryhänen 等人的特点。 （L. Ryhänen 等人，1982，Collagen Rel. Res.2, 117–130）方法。第一种方法提供了一种制备大量均匀底物的简单方法，而第二种方法通过去除标记的端肽来增加测定的特异性。该测定具有重复性，并且与时间和酶浓度呈线性关系。它比 Cawston 和 Barrett 方法灵敏约 10 倍，可以轻松检测 1–8 mU 胶原酶（1 单位等于 30°C 下/分钟裂解 1 μg 胶原酶）。该底物对弹性蛋白酶、胰蛋白酶和胰凝乳蛋白酶具有抗性，并能被细菌胶原酶完全降解。胶原酶是迄今为止发现的唯一能裂解底物的组织金属蛋白酶。
Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T. E. Cawston and A. J. Barrett, 1979, Anal. Biochem.99, 340–345) and the Ryhänen et al. (L. Ryhänen et al., 1982, Collagen Rel. Res.2, 117–130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10× more sensitive than the Cawston and Barrett method and can readily detect 1–8 mU collagenase (1 unit equals 1 μg collagen cleaved/min at 30°C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate.