title: Thermodynamic analysis of heparin binding to human antithrombin.
abstract: The binding of heparin to human antithrombin III (ATIII) was investigated by titration calorimetry (TC) and differential scanning calorimetry (DSC). TC measurements of homogeneous high-affinity pentasaccharide and octasaccharide fragments of heparin in 0.02 M phosphate buffer and 0.15 M sodium chloride (pH 7.3) yielded binding constants of (7.1 +/- 1.3) x 10(5) M-1 and (6.7 +/- 1.2) x 10(6) M-1, respectively, and corresponding binding enthalpies of -48.3 +/- 0.7 and -54.4 +/- 5.4 kJ mol-1. The binding enthalpy of heparin in phosphate buffer (0.02 M, 0.15 M NaCl, pH 7.3) was estimated from TC measurements to be -55 +/- 10 kJ mol-1, while the enthalpy in Tris buffer (0.02 M, 0.15 M NaCl, pH 7.3) was -18 +/- 2 kJ mol-1. The heparin-binding affinity was shown by fluorescence measurements not to change under these conditions. The 3-fold lower binding enthalpy in Tris can be attributed to the transfer of a proton from the buffer to the heparin-ATIII complex. DSC measurements of the ATIII unfolding transition exhibited a sharp denaturation peak at 329 +/- 1 K with a van 't Hoff enthalpy of 951 +/- 89 kJ mol-1, based on a two-state transition model and a much broader transition from 333 to 366 K. The transition peak at 329 K accounted for 9-18% of the total ATIII. At sub-saturate heparin concentrations, the lower temperature peak became bimodal with the appearance of a second transition peak at 336 K. At saturate heparin concentration only the 336 K peak was observed. This supports a two domain model of ATIII folding in which the lower stability domain (329 K) binds and is stabilized by heparin.
authors.affiliation: Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, Rockville, MD.
authors.forename: S
authors.initials: S
authors.lastname: DeLauder
authors.forename: F P
authors.initials: FP
authors.lastname: Schwarz
authors.forename: J C
authors.initials: JC
authors.lastname: Williams
authors.forename: D H
authors.initials: DH
authors.lastname: Atha
mesh_terms: D000818:Animals; D000990:Antithrombin III / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D002152:Calorimetry, Differential Scanning; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008433:Mathematics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: D000990:Antithrombin III; D006493:Heparin