Thrombin binding properties of insoluble modified polystyrene: Part II.
Antithrombin III (AT III) inhibits thrombin via an arginine-serine interaction. Insoluble polystyrene resins grafted with arginyl methyl ester have been synthesized, and their interaction with thrombin tested. One of these resins was selected for its high affinity for thrombin. In this paper we report the characteristics of this thrombin resin interaction. Using this substituted polystyrene resin as a support for affinity chromatography, we have compared the binding of thrombin with that of other proteins (prothrombin, Factor IXa, trypsin and AT III). It was found that 0.7 mg of highly purified human thrombin (2,100 U/mg) was bound to 1 g of resin. This could only be eluted at high ionic strength (1.5 M) and the amidolytic and clotting activities of the eluted thrombin remained unchanged. The binding of thrombin to the resin involves the active site of the enzyme but also other residues since, when DIP thrombin was used, the inactive enzyme could be eluted at lower ionic strength (1.0 M). This resin seems to be specific for thrombin because it does not bind the other serine-proteases (trypsin or Factor IXa), prothrombin (the inactive precursor of thrombin) or AT III. The arginyl residues of the resin are important for the specificity of the interaction with Factor IIa since prolyl residues are totally ineffective. Chromatography performed on such a resin is a very efficient method of purifying thrombin, and may be very useful for the removal of thrombin as a contaminant of plasma protein fractions.
Fischer
A M
AM
Département d'Hématologie, C.H.U. Necker-Enfants Malades, Paris, France.
Tapon-Bretaudiere
J
J
Bros
A
A
Boisson
C
C
Gulino
D
D
Jozefonvicz
J
J
D000990:Antithrombin III / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity; D015949:Factor IXa; D006801:Humans; D011137:Polystyrenes / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D012995:Solubility; D013917:Thrombin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D011137:Polystyrenes; D000990:Antithrombin III; D011516:Prothrombin; D012697:Serine Endopeptidases; D015949:Factor IXa; D014357:Trypsin; D013917:Thrombin