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.
Lundblad
R L
RL
Dental Research Center, School of Dentistry, University of North Carolina, Chapel Hill 27599-7455.
Tsai
J
J
Wu
H F
HF
Jenzano
J W
JW
White
G C
GC
Connolly
T M
TM
D000097:Acetonitriles; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D002846:Chromatography, Affinity / Q000379:methods*; D003619:Dansyl Compounds / Q000494:pharmacology; D004146:Dioxanes; D005340:Fibrinogen / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013917:Thrombin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000097:Acetonitriles; D000590:Amino Acid Chloromethyl Ketones; D003619:Dansyl Compounds; D004146:Dioxanes; C055180:dansylglutamyl-glycyl-arginyl chloromethyl ester; D006639:Histidine; D011732:Pyridoxal Phosphate; D005340:Fibrinogen; D006493:Heparin; D013917:Thrombin; C025223:1,4-dioxane; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone; C032159:acetonitrile