Nonspecific electrostatic binding characteristics of the heparin-antithrombin interaction.
We evaluated the role of nonspecific electrostatic binding in the interaction of antithrombin (AT) with heparin (Hp), a paradigmatic protein-glycosaminoglycan (GAG) system. To do so, we obtained the ionic-strength dependence of the binding constant, since a common feature in protein-polyelectrolyte systems is a maximum in affinity in the ionic strength range 10 mM <I<30 mM (Seyrek et al, Biomacromolecules 2003, 4, 273-282). Because this feature is seen for both synthetic and biological polyelectrolytes, and because the value of I(max) correlates with protein size and charge asymmetry through the Debye length (Seyrek et al, Biomacromolecules 2003, 4, 273-282), this behavior appears to be a signature of non-specific electrostatic protein-polyelectrolyte binding. Binding of AT to both standard (14 kDa) Hp and partially degraded (5 kDa) low molecular weight heparin (LMWH) exhibited this same behavior. Capillary electrophoresis (CZE) of Hp and LMWH yielded electropherograms whose remarkable breadth revealed the enormous heterogeneity of average charge density among the innumerable molecular species of Hp and LMWH. These distributions were somewhat reduced after affinity chromatography (AC) fractionation, indicating that the high-affinity fraction was generally depleted of the lower-charge species. Size-exclusion chromatography coupled with Electrospray Mass Spectrometry confirmed lower levels of sulfation for the lower affinity fractions. Comparisons of LMWH with Dermatan sulfate (DS) by CZE and AC suggested a correlation between the relative absence of very highly charged components in DS and its weaker binding to AT. These findings point to a significant role of the charge density of GAG chains in their affinity for AT.
Seyrek
Emek
E
Department of Chemistry, Indiana University Purdue University Indianapolis, 402 N. Blackford St. Indianapolis, IN 46202, USA.
Dubin
Paul L
PL
Henriksen
Jens
J
D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D000991:Antithrombins / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding; D012680:Sensitivity and Specificity; D055672:Static Electricity; D013552:Swine
D000925:Anticoagulants; D000991:Antithrombins; D006493:Heparin