Hydrophobic affinity chromatography of human thrombin

Hydrophobic affinity chromatography on p-chlorobenzylamido-agarose (p-CBA-agarose) was used to characterize various modified forms of human thrombin. Native alpha-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 to p-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 to p-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 chloromethylketone. The presence of either hirudin or heparin blocked the binding of thrombin to p-CBA-agarose but dansyl-arginine-N-(3-ethyl-1,5-pentanediyl)amide had no effect. These results indicate that p-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.