Pyridoxylation of essential lysines in the heparin-binding site of antithrombin III.
Pyridoxal 5'-phosphate was used to selectively modify lysine residues on antithrombin III. Pyridoxal 5'-phosphate was incubated with antithrombin, and the Schiff base was reduced with sodium borohydride. A maximum of 3-4 pyridoxal 5'-phosphate groups per antithrombin molecule are bound at high concentrations of pyridoxal 5'-phosphate. A linear decrease in heparin cofactor activity was observed with the incorporation of the first three pyridoxyl phosphate groups. Modification in the presence of added heparin decreased the extent of labeling by 1-2 mol of pyridoxyl phosphate per mol of antithrombin and protected against the loss of heparin cofactor activity. To further examine the role of lysine residues in the interaction of antithrombin III with heparin, the protein was singly labeled with pyridoxyl phosphate and affinity fractionated on heparin-agarose. Three species, each containing approximately one pyridoxyl phosphate group per antithrombin, were obtained. Two of these derivatives had reduced affinity for heparin-agarose, exhibited decreased heparin cofactor activity, and did not show enhanced tryptophan fluorescence upon addition of heparin. The third derivatives possessed high affinity for heparin-agarose, retained native-like heparin cofactor activity, and when titrated with heparin gave a 30-35% increase in tryptophan fluorescence. Fluorescence emission spectra of the phosphopyridoxyl-antithrombin derivative that does not bind to heparin-agarose indicated fluorescence energy transfer from tryptophan to the bound phosphopyridoxyl moiety. These results indicate that 1-2 lysines are essential to heparin bonding and that these residues may be located near to the critical tryptophan (Blackburn, M. N., and Sibley, C. C. (1980) J. Biol. Chem. 255, 824-826) in the heparin-binding domain of antithrombin III.
D000990:Antithrombin III / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D011732:Pyridoxal Phosphate; D000990:Antithrombin III; D006493:Heparin; D008239:Lysine