Inactivation of human thrombin by water-soluble carbodiimides. The essential carboxyl has a pKa of 5.6 and is one other than Asp-189.
Human thrombin is inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) by a second order process. A plot of the pseudo-first order rate constant for inactivation by 20 mM EDC at different pH values from 4.2 to 7.7 at 25 degrees C shows that this inactivation is due to the modification of a protonated carboxyl with a pKa of 5.6. The rate of inactivation by EDC at pH 6.0 is reduced, but not eliminated by saturating levels of the competitive inhibitor dansyl-L-arginine N-(3-ethyl-1,5-pentanediyl)amide, suggesting that the essential carboxyl modified is one other than Asp-189 in the substrate specificity pocket of thrombin. Complete inactivation by 14C-EAC correlates with the incorporation of approximately 2.5-3 EACs per thrombin.
Borders
C L
CL
Department of Chemistry, College of Wooster, OH 44691.
Chan
V W
VW
Miner
L A
LA
Weerasuriya
Y M
YM
D001224:Aspartic Acid; D002234:Carbodiimides / Q000494:pharmacology*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors*
D002234:Carbodiimides; D001224:Aspartic Acid; C044568:1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide; D013917:Thrombin; D005022:Ethyldimethylaminopropyl Carbodiimide