凝胶过滤色谱-多角度激光散射法测定琥珀酰明胶自由氨基修饰度

Determination of Modification Degree of Succinylated Gelatin by Size Exclusion Chromatography Coupled with Multi Angle Laser Light Scatting

琥珀酰明胶作为血浆代用品的临床疗效与其分子量分布和修饰度密切相关。本研究利用高效凝胶过滤色谱-多角度激光散射法(HPSEC-MALLS)并结合2,4,6-三硝基苯磺酸(TNBS)衍生法,探索建立一种测定琥珀酰明胶修饰度的方法。实验利用TNBS将琥珀酰明胶衍生,通过HPSEC-MALLS测定琥珀酰明胶的分子量变化,计算出琥珀酰明胶的修饰度。实验以人血清白蛋白为模型进行了方法验证,结果表明:该方法能较好地表征人血清白蛋白在TNBS衍生过程中的分子量变化,实验值与根据其氨基酸组成计算值一致。实验在HPSEC-MALLS测定琥珀酰明胶及TNBS衍生后琥珀酰明胶分子量及分布的基础上,利用凝胶过滤色谱分子量-保留时间拟合曲线,计算得到琥珀酰明胶的自由氨基修饰度为38%。结果表明利用高效凝胶过滤色谱-多角度激光散射法结合2,4,6-三硝基苯磺酸衍生法测定琥珀酰明胶的修饰度是可行的。

The clinical effectiveness of succinylated gelatin as a plasma substitute depends on its molar mass and free amino modification degree. In this study, a new method was developed to determine the modification degree of succinylated gelatin. Gelatin and succinylated gelatin were derived with trinitrobenzenesulfonic acid （TNBS）. The molar masses of gelatin and succinylated gelatin were determined before and after TNBS-derivation by size exclusion chromatography coupled with multi angle laser light scatting （SEC-MALLS）. The modification degree was obtained by determining the free amino groups of gelatins before and after succinylation. Human serum album in was used as a model protein to validate the method. The determined value was consistent with the calculated value. Actual samples were analyzed with the developed method. The result indicates that the mean modification degree of succinylated gelatins is 38%. This research indicates that size exclusion chromatography multi angle laser light scatting coupled with the trinitrobenzenesulfonic acid derivation is a possible method to determine the modification degree of succinylated gelatin.