Inactivation of bovine thrombin by water-soluble carbodiimides: the essential carboxyl group has a pKa of 5.51.
Bovine thrombin is rapidly and completely (greater than 99%) inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in a pseudo-first-order process. A plot of the pseudo-first-order rate constant for inactivation by 20 mM EDC at different pH values from pH 4.0 to 7.7 at 25 degrees C shows that inactivation is critically dependent on the protonated form of an acidic side chain with a pKa of 5.51. Significant protection against inactivation is provided by the competitive inhibitor dansyl-L-arginine N-(3-ethyl-1,5-pentanediyl)amide, suggesting that the essential carboxyl group may be involved in substrate binding. 1-Ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) inactivates thrombin much more rapidly than EDC under the same conditions.
Chan
V W
VW
Department of Chemistry, College of Wooster, Ohio 44691.
Jorgensen
A M
AM
Borders
C L
CL
D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D002417:Cattle; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D007700:Kinetics; D012995:Solubility; D013917:Thrombin / Q000037:antagonists & inhibitors*
D002234:Carbodiimides; D013917:Thrombin; D005022:Ethyldimethylaminopropyl Carbodiimide