An accurate, quantitative assay for collagenase activity based on the synergistic hydrolysis of collagen.
An accurate, quantitative assay for collagenase activity based on the synergistic hydrolysis of collagen.
复制标题
基于胶原蛋白协同水解的胶原酶活性的准确定量测定。
DOI：
10.1016/0003-2697(82)90330-x
复制
发表时间：
1982
影响因子：
2.9
通讯作者：
Bond,MD
中科院分区：
文献类型：
--
作者：
VanWart,HE;Bond,MD
基于嗜热菌蛋白酶和胶原酶对可溶性[14CH3]胶原的协同水解作用，开发了一种准确定量的胶原酶测定法。嗜热菌蛋白酶本身不会以明显的速率水解胶原蛋白。然而，当存在于在35°C下进行的胶原酶测定中时，其通过将由真正的胶原酶产生的大胶原片段水解成小得多的片段而协同地帮助胶原蛋白的消化。这些较小的消化产物比由初级裂解产生的较大片段更可溶，并且在未消化的胶原蛋白用磷钨酸沉淀后，它们保留在溶液中。由于该测定测量的是不可沉淀的14 C的出现，因此嗜热菌蛋白酶的二次消化大大增加了其灵敏度。此外，与在不存在嗜热菌蛋白酶的情况下进行时相比，该测定显示出在更大百分比的完全水解下呈线性的初始速率和与更宽范围的胶原酶浓度成比例的活性。此外，由于嗜热菌蛋白酶消除了污染蛋白酶的协同效应，因此它给出了不纯样品的真实胶原酶活性的准确阅读。该测定的一个重要特征是它能够计算“协同比”，所述“协同比”定义为在嗜热菌蛋白酶和胶原酶两者存在下观察到的速率除以各自单独获得的速率之和。该比率反映了胶原酶的特异性程度，因此提供了关于该酶的额外信息。
An accurate and quantitative collagenase assay has been developed which is based on the synergistic hydrolysis of soluble [14CH3]collagen by thermolysin and collagenase. Thermolysin itself does not hydrolyze collagen at an appreciable rate. However, when present in collagenase assays carried out at 35°C, it synergistically assists in the digestion of collagen by hydrolyzing the large collagen fragments produced by true collagenases into much smaller fragments. These smaller digestion products are much more soluble than the larger fragments produced by the primary cleavages and they remain in solution after the undigested collagen is precipitated with phosphotungstic acid. Since the assay measures the appearance of nonprecipitable14C, this secondary digestion by thermolysin greatly increases its sensitivity. Furthermore, the assay exhibits initial rates that are linear over a greater percentage of full hydrolysis and activities that are proportional to a wider range of collagenase concentrations than when carried out in the absence of thermolysin. In addition, it gives an accurate reading of the true collagenase activity of impure samples, since thermolysin levels out the synergistic effects of contaminating proteinases. An important feature of the assay is that it enables the calculation of a “synergistic ratio” defined as the rate observed in the presence of both thermolysin and collagenase divided by the sum of the rates obtained for each alone. This ratio reflects the degree of specificity of the collagenase and, thus, provides additional information about the enzyme.