THERMODYNAMICS OF CHARGED OLIGOPEPTIDE HEPARIN INTERACTIONS

To better understand the electrostatic component of the interaction be tween proteins and the polyanion heparin, we have investigated the the rmodynamics of heparin binding to positively charged oligopeptides con taining lysine or arginine and tryptophan (KWK-CO2 and RWR-CO2). The b inding of these peptides to heparin is accompanied by an enhancement o f the peptide tryptophan fluorescence, and we have used this to determ ine equilibrium binding constants. The extent of fluorescence enhancem ent is similar for both peptides, suggesting that the tryptophan inter action is similar for both. Titrations of these peptides with a series of simple salts suggest that this fluorescence enhancement is due to the interaction of tryptophan with sulfate moieties on the heparin. Eq uilibrium association constants, K-obs (M(-1)), for each peptide bindi ng to heparin were measured as a function of temperature and monovalen t salt concentration in the limit of low peptide binding density. At p H 6.0, 25 degrees C, 20 mM KCH3CO2, K-obs = 3.2 (+/-0.3) x 10(3) M(-1) for KWK-CO2 binding, whereas K-obs = 4.5 (+/-0.5) x 10(3) M(-1) for R WR-CO2. However, the dependence of K-obs on KCH3CO2 concentration is t he same for both oligopeptides, each of which possesses a net charge o f +2 at pH 6.0. The logarithm of K-obs is a linear function of the log arithm of [KCR(3)CO(2)] over the range from 12 mM less than or equal t o KCH3CO2 less than or equal to 30 mM (pH 6.0, 25 degrees C), with (a log K-obs/partial derivative log [KCH3CO2]) = -2.0 +/- 0.3, indicating that similar to 2 ions are released per bound peptide upon formation of the complex. The van't Hoff Delta H-obs(o) for each peptide-heparin interaction is independent of [KCH3CO2], with Delta H-obs(o) = -1 +/- 1.5 kcal/mol for KWK-CO2, and Delta H-obs(o) = -3.5 +/- 1.5 kcal/mol for RWR-CO2. Comparison of these results with similar studies of the b inding of these same peptides to single-stranded polynucleotides indic ates that binding of these peptides to heparin at low salt concentrati ons is largely driven by the favorable increase in entropy resulting f rom the release of ions, presumably K+ from the heparin. The results f rom these model peptide studies are compared with similar studies of p rotein-heparin interactions.