牛骨蛋白与多糖的逐级提取及分离方法研究

Extraction and purification of protein and proteoglycan from bovine bone

为确定牛骨中蛋白质和多糖的提取及分离方法,该文在分析骨胶原蛋白和多糖存在状态的基础上,分别利用不同溶剂探讨了牛骨中蛋白质与多糖的逐级提取方法。首先碱性蛋白、酸性蛋白分别被0.6 mol/L KCl和0.5 mol/L K-P(K2HPO4/KH2PO4)溶液提取出来;0.1 mol/L HCl脱钙时溶解出酸性、中性及碱性蛋白,再经过NaOH中和后,酸性蛋白随羟基磷灰石沉淀、中性及碱性蛋白存留在上清液中;其他在胶原纤维上的有机物被0.5 mol/L NaCl和1%Na2CO3分别提取出来。各级提取有机物用SDS-PAGE电泳以及Stains All染色法、阿里新兰染色和CBB染色进行检测。利用DEAE-葡聚糖凝胶离子交换树脂有效地将碱性蛋白、酸性蛋白和多糖分离精制。

To establish a method for extracting and purificating proteins and proteoglycans from bovine bone, the extracting method using different step solutions on the basis of collagen and proteoglycans exiting state was analyzed. First, alkaline and acidic proteins were extracted by 0. 6 mol/L KCl and 0. 5 mol/L K-P （K2HPO4/ KH2PO4） solution, respectively. The 0. 1 mol/L HCl extracts contain acidic and neutral （or alkaline） proteins, and they can alternatively disjoin from extraction by neutralize the solution to pH 7.0, the acidic protein deposit with hydroxyapatite, and the neutral protein in the supernatant. Other non-collagenous proteins, which bound to the collagen fibrils could be extracted by 0. 5 mol/L NaCl and 1% Na2CO2 solution respectively. The acidic and alkaline proteins were detected by SDS-polyaerylamide gel eleetrophoresis, Stains All staining, Alcine blue staining and CBB staining. The alkaline protein, acidic protein and proteoglycans were effectively purified from each fraction by using DEAE-sephacel column.