Non-specific influence of chemical modification upon the properties of antithrombin III:modification of carboxyl groups.
The ability of antithrombin III to inhibit thrombin was observed to be rapidly inactivated upon specific modification of carboxyl groups. The loss of activity, upon treatment with nitrotyrosyl ester in the presence of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate, was concomitant to the incorporation of 2 moles of nitrotyrosine per mole of inhibitor indicating the modification of only two carboxyl groups. Moreover, the modification occurred with loss, also, of the ability of the native protein to bind tightly to heparin. The modified antithrombin III retained a reduced affinity for heparin (eluting at 0.3M NaCl from heparin Agarose) and was observed to be a competitive inhibitor of the heparin-dependent rate of inhibition of thrombin by native antithrombin III. However, FAB-MS (fast atom bombardment mass spectroscopy) analysis of digests of modified material gave no indication that modification was localized to specific Asp or Glu residues. It is concluded that the loss of activity is due to deleterious change in conformation during modification. These findings, together with our previous report upon tryptophan modification of antithrombin III [1] suggest that the nature of the molecule is such that considerable care must be taken in interpretation of results when investigating the structure/function relationships of this protein by chemical modification.
Scully
M F
MF
Thrombosis Research Institute, Chelsea, London.
Kakkar
V V
VV
Goodwin
C A
CA
D000990:Antithrombin III / Q000031:analogs & derivatives* / Q000494:pharmacology; D003028:CME-Carbodiimide / Q000031:analogs & derivatives*; D002234:Carbodiimides; D002846:Chromatography, Affinity; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013917:Thrombin / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000031:analogs & derivatives*
D002234:Carbodiimides; C074111:N-cyclohexyl-N'-2-morpholinoethyl-carbodiimide-methyl-4-toluolsulfonate; D003028:CME-Carbodiimide; C002744:3-nitrotyrosine; D014443:Tyrosine; D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin