Hydrophobic affinity chromatography of human thrombin. Arch Biochem Biophys 1993 Apr;302(1):109-12
Date
04/01/1993Pubmed ID
8470887DOI
10.1006/abbi.1993.1187Scopus ID
2-s2.0-0027295888 (requires institutional sign-in at Scopus site) 4 CitationsAbstract
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.
Author List
Lundblad RL, Tsai J, Wu HF, Jenzano JW, White GC 2nd, Connolly TMAuthor
Gilbert C. White MD Professor in the Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AcetonitrilesAmino Acid Chloromethyl Ketones
Amino Acid Sequence
Binding Sites
Chromatography, Affinity
Dansyl Compounds
Dioxanes
Fibrinogen
Heparin
Histidine
Humans
Molecular Sequence Data
Pyridoxal Phosphate
Thrombin
