Thermodynamics of Charged Oligopeptide - Heparin Interactionst

To better understand the electrostatic component of the interaction between proteins and the polyanion heparin, we have investigated the thermodynamics of heparin binding to positively charged oligopeptides containing lysine or arginine and tryptophan (KWK-C02 and RWR-(202). The binding of these peptides to heparin is accompanied by an enhancement of the peptide tryptophan fluorescence, and we have used this to determine equilibrium binding constants. The extent of fluorescence enhancement is similar for both peptides, suggesting that the tryptophan interaction 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. Equilibrium association constants, Kobs (M-l), for each peptide binding to heparin were measured as a function of temperature and monovalent salt concentration in the limit of low peptide binding density. At pH 6.0, 25 "C, 20 mM KCH3C02, Kobs = 3.2 (f0.3) x lo3 M-' for KWK-C02 binding, whereas Kobs = 4.5 (310.5) x lo3 M-' for RWR-CO2. However, the dependence of Kobs on KCH3C02 concentration is the same for both oligopeptides, each of which possesses a net charge of f 2 at pH 6.0. The logarithm of Kobs is a linear function of the logarithm of [KCH3C02] over the range from 12 mM I KCH3C02 I 30 mM (pH 6.0, 25 "C), with (8 log KobA8 log [KCH3C02]) = -2.0 f 0.3, indicating that -2 ions are released per bound peptide upon formation of the complex. The van't Hoff Awobs for each peptide-heparin interaction is independent of [KCH3C021, with A H o o b s = -1 f 1.5 kcdmol for KWK-C02, and AH'obs = -3.5 f 1.5 kcal/mol for RWRCO2. Comparison of these results with similar studies of the binding of these same peptides to singlestranded polynucleotides indicates that binding of these peptides to heparin at low salt concentrations is largely driven by the favorable increase in entropy resulting from the release of ions, presumably K+ from the heparin. The results from these model peptide studies are compared with similar studies of protein-heparin interactions. The sulfated polysaccharide heparin interacts with a number of proteins involved in a variety of biological processes including cell growth and differentiation (Hassel et al., 1986) and control of blood coagulation (Bjork et al., 1989). Among these proteins are fibroblast growth factors (FGF) (Rapraeger et al., 1991; Yayon et al., 1991; Thompson et al., 1994), thrombospondin (Dixit et al., 1984; Guo et al., 1992), thrombin (Olson et al., 1991), and antithrombin (Olson & Bjork, 1991). Since heparin is a polyanion, one expects that some component of its free energy of interaction with proteins will be electrostatic. In fact, since heparin is a highly charged linear polyelectrolyte, counterions (e.g., Na+, K+, Mg2+) will bind to heparin in order to reduce its net charge density (Oosawa, 1971; Manning, 1969), and this has been observed experimentally (Delville & Laszlo, 1983). As a result, when a positively charged ligand or a protein with a positively charged heparin binding site binds to heparin, some fraction of the heparin charge will be neutralized resulting in the displacement of some of the heparin-bound counterions in a manner similar to what has been observed This work was supported in part by NIH Grants GM39062 and GM30498. * Address correspondence to this author at Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, 660 S. Euclid Ave., St. Louis, MO 63110. * In partial fulfillment for the Ph.D. degree in Biochemistry at Texas A&M University, College Station, TX 77843-2128. Current address: Department of Biology, Washington University, St. Louis, MO 63130. @ Abstract published in Advance ACS Abstracts, February 15, 1995. 0006-2960/95/0434-2908$09.00/0 for the binding of charged ligands to linear nucleic acids (Record et al., 1976b, 1978,1991; Mascotti & Lohman, 1990, 1992, 1993; Lohman & Mascotti, 1992a). Of course, for proteins binding to heparin, the salt dependence of the equilibrium constant will generally also be influenced by contributions from ion release or uptake by the protein. Recent studies probing the effects of salt concentration on the equilibrium binding of several proteins to heparin have demonstrated these polyelectrolyte effects. Olson et al. (1991) have measured the equilibrium constant for thrombin binding to heparin as a function of NaCl concentration and temperature and by analogy with ligand-polynucleotide interactions conclude that this interaction is stabilized significantly by nonspecific electrostatic interactions and that a major component of the favorable binding free energy change results from the displacement of cations (Na+) from heparin. Similar effects of salt concentration have been demonstrated for the equilibrium binding of heparin to antithrombin I11 (Olson & Bjork, 1991), mucus proteinase inhibitor (Faller et al., 1992), and basic fibroblast growth factor (bFGF) (Thompson et al., 1994), although the magnitudes of these salt effects differ for each interaction indicating different extents of ion release. Studies of the equilibrium binding of simple positively charged peptides to DNA and RNA have proven to be very useful in probing the contributions of electrostatic interactions and the role of counterion release in stabilizing protein