Inactivation of human thrombin by water‐soluble carbodiimides

FEBS Letters

Miner, Lydia A.¹  Borders, C.L.¹  Weerasuriya, Yohan M.¹  Chan, Vivien W.F.¹ 

[1] Department of Chemistry, The College of Wooster, Wooster, OH 44691, USA

关键词: Thrombin;    Carboxyl;    essential;    Carbodiimide;    EDC;    EAC;    DAPA;    EDC;    1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;    EAC;    1-ethyl-3-(4-azonia-4;    4-dimethylpentyl)carboiimide iodide;    DAPA;    dansyl-L-arginine N-(3-ethyl-1;    5-pentanediyl)-amide;    Mes;    2-(N-morpholino)ethanesulfonic acid;    Hepes;    N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid;    S-2238;    tosyl-D-phenylalanyl-D-pipecolyl-L-arginine-p-nitroanilide;    k′;    the pseudo-first order rate constant for inactivation of thrombin by ECD or EAC under a given set of conditions;    K 50;    the DAPA concentration at which thrombin is half-saturated;   

DOI  :  10.1016/0014-5793(89)81123-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 pK _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 ¹⁴C-EAC correlates with the incorporation of approximately 2.5–3 EACs per thrombin.

【 授权许可】

Unknown