离子交换色谱法对肌肉中钙激活酶分离纯化的研究

Quantification of Calpains activities by ion-exchange chromatography

试验通过离子交换色谱法对肌肉中钙激活酶(Calpain)进行定量分析研究,确定了Calpain粗提液制备工艺。对Calpain粗提液进行全波长紫外扫描说明276～280nm处有一个吸收高峰。应用DEAE离子交换层析对Calpain进行纯化,用0～500mmol·L-1NaClTris-HCl进行离子强度线性梯度洗脱,在280nm波长下测定洗脱液的光吸收值,洗脱得3个蛋白峰,μ-Calpain和m-Calpain分别在NaCl为130～180mmol·L-1和260～300mmol·L-1时洗脱下来,钙激活酶抑制蛋白(Calpastatin)在NaCl为70～110mmol·L-1时洗脱下来。Calpain分子量为110000u,通过电泳试验表明,本检测结果提取液正好在分子量约为110000u处有一条清晰的带,说明所得提取物为Cal-pain,而且纯度较高。

A improved method for quantificational determination of Calpains activities was developed by ion-exchange chromatography. On the basis of repeated experiments, we had found a satisfactory way of Calpain crude extract processing. The Calpain crude extract was treated with ultraviolet scanning, which indicates there was an absorption peak in the range from 276 to 280 nm. To purify Calpain-Calpastatin system, first crude extract was treated with DEAE-ion exchange chromatography and then with ion strength linear gradient elution by NaCl with the concentration ranged from 0 to 500 mmol·L-1. The absorbance was tested at 280 nm. There were 3 absorbent peaks. μ-Calpain and m-Calpain were eluted when the concentration of NaCl in the range from 130 to 180 mmol·L-1 and from 260 to 300 mmol·L-1 respectively, while Calpastatin do so in the range from 70 to 110 mmol·L-1. It had been well documented the molecular weight of Calpain is 110 000 u. From SDS-PAGE we knew, there was a clear band when the molecular weight was at 110 000, which indicated the extract was calpain with high purity.