一种凝血酶亲和色谱介质的制备方法

Preparation of thrombin affinity chromatography media

对凝血酶-琼脂糖亲和色谱介质的制备方法进行了研究。首先使用凝血酶和溴化氰活化的琼脂糖制备凝血酶-琼脂糖亲和色谱介质,然后用生色底物法考察亲和色谱介质上凝血酶的活性,以凝血酶活性为指标对最佳偶联条件进行了优化。结果表明最佳条件为使用pH8.3的Na2CO3-NaHCO3溶液(含0.5 mol/L NaCl)为缓冲溶液,凝血酶用量为每1 g色谱介质加入凝血酶200 U,室温反应10 h。在最佳条件下所制备的色谱介质有较好的稳定性,在4℃条件下存放40天,亲和介质上的凝血酶活性仍有70.6%保留。该亲和色谱介质可广泛用于含凝血酶抑制剂的天然药物筛选和分离纯化。

The preparation of thrombin affinity chromatography media is described in this paper. First, the thrombin affinity chromatography media was prepared through thrombin cou- pled with cyanogen bromide activated agarose. And then the enzyme activity of thrombin on the coupled media was measured by chromogenic substrate method. The conditions were con- firmed based on the enzyme activity of thrombin on the coupled media. The optimum condi- tions were as follows： the use of Na2CO3-NaHCQ （containing 0.5 mol/L NaC1） solution of pH 8.3 as the reaction solution, 200 U thrombin per 1 g agarose, and reaction for l0 h at room temperature. The prepared affinity chromatography media had good stability, on which 70.6% of the enzyme activity of thrombin existed after 40 days＇ storage. The thrombin affinity chro- matography media can be widely applied in the screening and purification of thrombin inhibitors in natural products.