基于弹性蛋白-RBB底物的弹性蛋白酶活力检测方法研究

Elastase Activity Assay Method Using Elastin-RBB as Substrate

以Remazol brilliant blue R染色牛颈韧带弹性蛋白粉,制成染色弹性蛋白。底物稳定性好,在较宽pH范围内染料不易洗脱。以弹性蛋白-RBB为底物,建立了一种能在较宽pH范围内测定制革蛋白酶的弹性蛋白水解活力方法。确定酶活测定条件:反应时间为30min,温度为40℃,基于5mg底物量,待测酶液中弹性蛋白酶活在90～160U/mL范围内,水解液的吸光度与酶的浓度呈良好的线性关系。方法重线性较好。几种常用制革蛋白酶在不同pH下测定的结果表明,一些细菌蛋白酶显示出较高的弹性蛋白酶活力,最适作用pH值在9左右。

A kind of stained elastin （Elastin- RBB） was derived from elastin powder labeled covalently with Remazol brilliant Blue R. Elastin powder was extracted by hot alkaline process from bovine ligamentum nuchae. Elastin - RBB was stable and it is difficult to elute off the dyestuff from the substrate, and the absorption spectrum of the enzymatic hydrolysates of the substrate al- most has no change at a rather broad pH range. A suitable and sensitive method for determining the elastase activity at the broad spectrum of pH, based on Elastin - RBB substrate was established. The assay conditions are that the reaction time is 30min, the temperature is 40℃. The relationship between the enzyme concentration and the absorbance is linear when the elastase activity of the protease solution to be determined was in the range of 90 - 160U/mL, based on 5mg Elastin- RBB substrate. The reproducibility of the proposed method is also good. Several leather proteases were chosen to investigate their impact on elastin under different pH values. The results indicate that some bacterial proteases show higher elastolytic activity against elastin and the optimum pH of the elastase is at around pH 9.0.