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目的探讨不同方法对于测定具有抗2型糖尿病活性的海洋寡聚甘露糖醛酸铬配合物(HS203)分子量测定的影响。方法采用聚丙烯酰胺凝胶电泳(PAGE),高效凝胶渗透色谱(HPGPC)及高效凝胶渗透色谱与多角度激光光散射器联用(HPGPC-MALLS)法分别测定HS203的分子量,并考察了不同色谱柱以及不同分子量对照品的选择对于HS203分子量测定结果的影响。结果 PAGE法表明HS203的聚合度为5~20之间,通过计算得出HS203的重均分子量范围在2 400~2 600Da。在HPGPC分析过程中,不同色谱柱对于HS203分子量测定结果影响不大,采用右旋糖酐作为对照品测得分子量为6 000~7 100Da,明显大于PAGE结果,采用结构相似的自制对照品测定结果为2 900~3 100Da,与PAGE结果较为接近。采用HPGPCMALLS法测定结果约为3 000Da,也与PAGE法所测结果相近。结论不同测定方法测得的HS203分子量结果不同。PAGE法能直观地观察到HS203聚合度的分布情况,并能真实反映分子量大小。HPGPC法应用普遍,操作方面,但对照品的选择对HPGPC法测定分子量十分重要,必须选用与待测样品结构相似的对照品。HPGPC-MALLS法测定糖类化合物分子量无需对照品且结果可靠,但该法需要专用且昂贵的仪器。
Objective To investigate the influence of different methods on the determination of the molecular weight of marine oligo-mannuronate chromium complex (HS203) with anti-type 2 diabetes activity. Methods The molecular weight of HS203 was determined by polyacrylamide gel electrophoresis (PAGE), high performance gel permeation chromatography (HPGPC), high performance gel permeation chromatography coupled with multi-angle laser light scattering (HPGPC-MALLS) Different columns and different molecular weight control selection of HS203 molecular weight determination results. Results PAGE showed that the degree of polymerization of HS203 was between 5 and 20, and the weight average molecular weight of HS203 was calculated to be between 2 400 and 2 600 Da. In the HPGPC analysis, different columns have little effect on the molecular weight determination of HS203. The dextran was used as a reference substance to determine the molecular weight of 6 000-7 100 Da, which was significantly larger than that of PAGE. The result of the self-made control with similar structure was 2 900 ~ 3 100Da, which is closer to PAGE. The result of HPGPCMALLS assay was about 3 000 Da, which was similar to that of PAGE. Conclusions The results of molecular weight measurement of HS203 by different assay methods are different. PAGE method can visually observe the degree of polymerization of HS203 distribution, and can truly reflect the size of the molecular weight. The application of HPGPC method is universal and operational. However, the choice of reference substance is very important for the determination of molecular weight by HPGPC method. The reference substance of similar structure to the sample to be tested must be selected. The HPGPC-MALLS method determines the molecular weight of carbohydrates without reference substance and is reliable, but the method requires specialized and expensive instruments.