Hydrophobic affinity chromatography of human thrombin.
Hydrophobic affinity chromatography of human thrombin.
复制标题
人凝血酶的疏水亲和层析。
DOI：
10.1006/abbi.1993.1187
复制
发表时间：
1993
影响因子：
3.9
通讯作者：
Connolly,TM
中科院分区：
文献类型：
--
作者：
Lundblad,RL;Tsai,J;Wu,HF;Jenzano,JW;White2nd,GC;Connolly,TM
采用疏水亲和层析法对不同修饰形式的人凝血酶进行了亲和层析。天然α-凝血酶与柱子紧密结合，用乙腈或1，4-二氧六环洗脱，而催化失活的凝血酶前2不与基质结合。5‘-磷酸吡哆醛的定点化学修饰使纤维蛋白原凝血活性丧失至少80%，但不影响凝血酶TOP-CBA琼脂糖凝胶的结合。用5-磷酸吡哆醛修饰凝血酶被认为发生在纤维蛋白原结合部位和肝素结合部位。相反，用H-D-苯丙氨基-L-脯氨基-L-精氨酸氯甲酮或丹磺酰-L-谷氨酰-甘氨酰-L-精氨酸氯甲酮修饰活性部位组氨酸可消除凝血酶顶部-CBA琼脂糖凝胶的结合，但不能与对甲苯磺酰-L-赖氨酸氯甲酮结合。水飞蓟素或肝素均可阻断凝血酶TOP-CBA-琼脂糖凝胶的结合，而丹氨酰-精氨酸-N-(3-乙基-1，5-戊二基)酰胺无此作用。这些结果表明，p-CBA琼脂糖能与酶活性部位以外的凝血酶结合，在表征通过蛋白质工程或化学修饰获得的定点修饰凝血酶方面具有一定的应用价值。
Hydrophobic affinity chromatography onp-chlorobenzylamido-agarose (p-CBA-agarose) was used to characterize various modified forms of human thrombin. Native α-thrombin bound tightly to the column and was eluted with either acetonitrile or 1,4-dioxane, while the catalytically inactive prethrombin 2 did not bind to the matrix. Site-specific chemical modification with pyridoxal 5′-phosphate resulted in the loss of at least 80% of fibrinogen clotting activity but did not influence the binding of thrombin top-CBA agarose. Modification of thrombin with pyridoxal 5-phosphate is thought to occur at the fibrinogen-binding site and the heparin-binding site. In contrast, binding of thrombin top-CBA agarose was eliminated by modification of the active site histidine using either H-D-phenylalanyl-L-prolyl-L-arginine chloromethylketone or dansyl-L-glutamyl-glycyl-L-arginine chloromethylketone but not with tosyl-L-lysine chioromethylketone. The presence of either hirudin or heparin blocked the binding of thrombin top-CBA-agarose but dansyl-arginine-N- (3-ethyl-1,5-pentanediyl)amide had no effect. These results indicate thatp-CBA agarose binds to thrombin outside of the enzyme active site and its use should be valuable in characterizing site-specific modified thrombins obtained by either protein engineering or chemical modification.