Heparin exerts its anticoagulant effects through its interaction with antithrombin (AT), leading to the inhibition of thrombin, a major player in the coagulation cascade. AT exists in equilibrium between a latent and an active state. In latent AT, the reactive centre loop (RCL) of AT is inserted into the central b-sheet (A-Sheet), forming a six-stranded A-Sheet. In active AT, residues P10-P14 of the RCL of AT are inserted into the A-sheet, retaining inhibitory activity. We have produced a molecule of covalently linked AT and heparin (ATH). Intrinsic fluorescence studies of ATH have shown that the AT is permanently activated in the covalent molecule. This study was designed to examine degree of the insertion of the RCL into the A-sheet of ATH. The tetradecapeptide P1-P14 was used to compete with the RCL of AT for insertion into the A-sheet. Peptides corresponding to the RCL residues P7-14, P3-P14 and P1-P14 of human AT were radiolabelled using reductive alkylation with formaldehyde + [3H]-NaBH4. Varying concentrations of radiolabelled peptide (0-3000 nM) were incubated with 1000 nM AT in the presence or absence of unfractionated heparin (UFH), and with ATH in 0.05 M Tris HCl buffer (pH 7.3) at 37°C. A 24-hour equilibrium dialysis against 0.05 M Tris HCl buffer followed. The amount of peptide bound to the AT or ATH was quantified following dialysis using liquid scintillation counting of samples taken from inside and outside the dialysis tubing. Results were expressed as bound vs. free peptide in moles. No binding to AT in the presence or absence of UFH, nor to ATH was seen using the P7-P14 peptide, while some binding to AT was seen for both the P3-P14 and P1-P14 peptides. There was a trend towards increased binding with AT in the presence of UFH over AT alone or ATH with the P3-P14 peptide. Binding to AT was lost in the presence of UFH, and no binding was seen with ATH in the P1-P14 condition, consistent with the literature. Furthermore, binding to AT was greatly reduced compared to that seen with the P3-P14 peptide. These data suggest that binding of heparin creates a conformational change in AT preventing the insertion of the RCL peptide into the molecule. Furthermore, results with the P3-P14 peptide may infer that it is the P1-P2 region that is most affected by the conformational change. This is consistent with the AT being in a partially activated conformation in the covalently linked ATH molecule.