骨素及骨素酶解液中蛋白质及肽的电泳分离

Electrophoretic Separation of Proteins and Peptides in Bone Extract and Bone Extract Hydrolysates

摘要:

为了分离纯化骨素及其酶解产物中的蛋白质及肽，并比较骨素蛋白与骨蛋白和肌肉蛋白的差异，本文以骨蛋白和肌肉蛋白为参照，采用十二烷基磺酸钠凝胶电泳法（SDS-PAGE）对骨素蛋白及其酶解产物中的肽进行分离。利用不同浓度的分离胶（5%、7.5%、10%、15%）对骨素蛋白、骨蛋白和肌肉蛋白样品进行分离，结果发现，各种浓度的分离胶均可以分离骨蛋白和肌肉蛋白，但是不能分离骨素蛋白。对骨素蛋白酶解液采用N-三（羟甲基）甲基甘氨酸凝胶电泳法分离，能有效分离骨素酶解液中的肽。对SDS-PAGE电泳分离时骨蛋白的迁移比和分离胶的相关性分析发现，骨蛋白的迁移比与分离胶浓度间呈指数相关，且具有极显著的相关性（R2≥0.996，p<0.01）。利用该相关性并结合蛋白质的分子量与迁移比的相关性方程，可以通过蛋白质的分子量选择合理的分离胶浓度，或者通过迁移比预测蛋白质的分子量。

Abstract:

Bone extract (BE) and BE hydrolysate proteins and peptides were isolated and purified, and the differences in BE protein, bone protein, and muscle protein were compared. Bone protein and muscle protein were used as the controls, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate BE protein and BE hydrolyzed peptides. The effect of different running gel concentrations (5%, 7.5%, 10%, 15%) on the separation of BE protein, bone protein, and muscle protein was investigated. The results showed that bone protein and muscle protein were separated successfully, while BE protein was not separated by SDS-PAGE. In contrast, BE hydrolysate peptides were separated by N-Tris-(hydroxymethyl)-methylglycine gel electrophoresis. Analysis of the relationship between the electrophoresis migration ratio of BP and the running gel concentrations for SDS-PAGE revealed an extremely significant (R2≥0.996, p<0.01) correlation between the migration ratio and the running gel concentration. Combining this correlation with the correlation equation of the molecular weight of the proteins and the migration ratio allows us to choose the running gel concentration according to the molecular weight of a protein, or in turn to calculate the molecular weight of a protein based on its migration ratio.