胶原纤维的酶解液中羟脯氨酸含量测定方法的优化

Optimization of Method for Determination of Hydroxyproline in Enzymatic Hydrolysate of Collagen Fibers

针对文献报道的测定蛋白水解液中羟脯氨酸含量的氯胺T显色法(比色法)中的操作条件不一致的问题,对该方法的关键条件进行了优化。用优化后的方法和氨基酸分析仪法(AA法)对系列皮粉和皮块蛋白酶水解液中的羟脯氨酸的含量进行了测定和比较。结果表明:优化后比色法的最适测定条件是:氧化时间5 min、显色时间15 min、消解时间4 h、样品pH 6.0～6.5;优化后的比色法的测定结果具有较好的重复性(RSD小于1.17%);比色法与AA法测定相同样品的结果比值在0.97～1.09之间,测定结果基本一致。与AA法相比,比色法具有快速、简便、成本低、易实现等优点,可以用于表征酶对胶原纤维的作用性能。

In order to solve the problem of inconsistent operating conditions in the chloramine T colorimetric method (colorimetric method) for the determination of hydroxyproline (Hypro) in protein hydrolysate reported in literatures, the key conditions of the method were optimized. Then the contents of Hypro in hydrolysis solutions of different proteases toward hide powder and skin were measured with the optimized method, and compared with amino acid analyzer method (AA).The results showed that the optimal conditions of the optimized colorimetric method were oxidation time of 5 min, color development time of 15 min, digestion time of 4 h, and pH of 6.0-6.5 after digestion. The results of the colorimetric method showed good repeatability (RSD less than 1.17%). The ratio of measured values from the two methods for the same sample was between 0.97 and 1.09. Compared with AA method, colorimetric method has the advantages of rapid, simple, low cost and easy realization in ordinary laboratory, which can be used to characterize the function of enzyme on collagen fiber.