Abstract: 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.

Key words: protease, skin collagen, hydroxyproline, colorimetric method, amino acid analyzer method