Thermodynamic analysis of heparin binding to human antithrombin.
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.
DeLauder
S
S
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, Rockville, MD.
Schwarz
F P
FP
Williams
J C
JC
Atha
D H
DH
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
D000990:Antithrombin III; D006493:Heparin