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Inactivation of human thrombin by water‐soluble carbodiimides
Author(s) -
Borders C.L.,
Chan Vivien W.F.,
Miner Lydia A.,
Weerasuriya Yohan M.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)81123-8
Subject(s) - thrombin , chemistry , thrombin generation , biophysics , medicine , biology , platelet
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°C shows that this inactivation is due to the modification of a protonated carboxyl with a p K a 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 14 C‐EAC correlates with the incorporation of approximately 2.5–3 EACs per thrombin.