胰蛋白酶消化法测定牛胶原蛋白三股螺旋结构的含量

本研究建立了一种测定胶原蛋白的三股螺旋结构含量的方法。该方法通过使用柱前衍生高效液相色谱(HPLC)法表征经胰蛋白酶酶解后胶原蛋白羟脯氨酸(Hyp)质量浓度的变化,进而对胶原蛋白的三股螺旋结构进行定量。探讨了不同的酶解时间(0~48h)、酶与底物的比例(1∶100、1∶50和1∶20)和温度(20、25、30、37℃)对明胶降解率的影响。获得了酶解的最佳条件——当胰蛋白酶与底物的比例为1∶50时,25℃酶解3h。使用该方法对明胶胶原蛋白混合液检测,结果表明,该方法能灵敏(RSD<10%)的测定胶原蛋白三股螺旋结构的含量。该方法不仅可用于生物组织研究领域,也可用于胶原蛋白食品、保健品和组织工程产品质量的评价。

Proportion of the content of bovine collagen triple helix by trypsin digestion

We established a quantitative method to determine the content of the triple helix conformation of collagen.This method based on using High Performance Liquid Chromatography (HPLC) coupled with precolumn derivatization assay to determine the variation of hydroxyproline (Hyp) content subsequent to digestion with trypsin.It was evaluated the effect of digestion time (0~48 h),enzyme/substrate ratios (1 ∶100,1 ∶50 and1 ∶20) and temperature (20,25,30 and37 ℃) on the degradation of gelatin.The optimal enzymatic hydrolysis conditions were obtained with1 ∶50 ratio of trypsin/substrate,being agitated at25 ℃ for3 h.Application of the method to determine mixture of gelatin and collagen solution showed that the method was precise(<10% RSD) for determining the content of triple helix conformation.This method is useful not only in the field of native tissue research,but also the quality assessment of collagen food,health food and bioengineered tissues.