Thermodynamic Analysis of the Heparin Interaction with a Basic Cyclic Peptide Using Isothermal Titration Calorimetry ² Ronald E. Hileman, ‡ Robert N. Jennings, § and Robert J. Linhardt* ,‡ DiVision of Medicinal and Natural Products Chemistry and Department of Chemical and Biochemical Engineering, UniVersity of Iowa, Iowa City, Iowa 52242, and Scios Inc., Mountain View, California 94043 ReceiVed January 27, 1998; ReVised Manuscript ReceiVed July 7, 1998 ABSTRACT: Brain natriuretic peptide (BNP) was examined as part of a continuing study of the interaction of proteins and peptides with the glycosaminoglycan heparin. BNP was tentatively identified as a heparin- binding protein on the basis of its cyclic structure and the high frequency of the basic amino acid residues, lysine and arginine. Thermodynamic analysis using isothermal titration calorimetry confirmed heparin binding to BNP with a micromolar K d . Surprisingly, despite the high frequency (22%) of basic residues in BNP, only a small portion of the free energy of this interaction resulted from ionic contributions under physiologic conditions. The contribution of polar amino acids, representing 28% of BNP, was next examined in a variety of different buffers. These experiments demonstrated the transfer of five protons from buffer to BNP on heparin binding, suggesting that hydrogen bonding between the polar residues of BNP and heparin is a major factor contributing to the free energy of BNP binding to heparin. Hydrophobic forces apparently play only a small role in binding. Heparin contains few nonpolar functional groups, and a positive change in heat capacity (ΔC p ) 1 kcal/mol) demonstrates the loss of polar residues on BNP-heparin binding. Previous studies (1, 2) performed in our laboratory have identified structural features within peptides that are impor- tant for glycosaminoglycan (GAG) 1 interaction. Specifically, a cyclic peptide structure based on the primary sequence of acidic fibroblast growth factor was found to interact more strongly with the structurally related GAG, heparan sulfate, than a linear peptide having the same sequence. We proposed that the cyclic peptide was structurally similar to the constrained loop structure found in the fibroblast growth factor GAG-binding domain and that the presence of a loop brings the basic amino acids into closer apposition. On the basis of these results, we searched the Swiss PIR, Genbank, and Brookhaven PDB databases for naturally occurring basic cyclic peptides for further studies on the role of loop structures in GAG binding and identified brain natriuretic peptide. Brain natriuretic peptide, or B-type natriuretic peptide (BNP), is a member of a family of hormones that includes atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP). BNP is a 32-amino acid peptide containing a disulfide bond between C 10 and C 26 of the mature peptide forming a 17-residue loop. Natriuretic peptides act as vasodilators and counter the vasoconstriction effects of the renin/angiotensin/aldosterone system (3). BNP is involved in the regulation of salt and water homeostasis (4), blood pressure regulation (5), and vascular remodeling (6). Ex- pression of BNP occurs mainly in the ventricles (6), but it was first isolated from brain (7) as a 134-amino acid precursor that is post-translationally modified to the active form containing 32 residues. BNP is stored in atrial granules (8) and secreted in response to myocardial wall stress (9). BNP’s sequence is highly homologous with that of ANP (10), including the disulfide loop structure. In a previous study (11), ANP was shown to interact with heparin, and when ANP was coadministered with heparin, the extents of both the hypotension and diuretic effects of ANP were reduced in rats. Although no reports have characterized BNP as a GAG-binding peptide, its relatively high level of basic residues (approximately 30 mol % and a pI of 11) and its loop structure suggested it would interact avidly with the GAG heparin. Heparin is a repeating copolymer of idouronic (or glu- curonic) acid and glucosamine. Due to its high degree of sulfation, heparin is described as a polyelectrolyte. The polyelectrolyte theory, previously developed for thermody- namically characterizing DNA-protein interactions, can also be used for characterizing protein-GAG interactions (12). Polyelectrolytes have a high charge density and, therefore, are associated with multiple counterions in solution. Al- though the binding of counterions minimizes the repulsive forces within the polyelectrolyte, this binding increases the ordering of the solution and is, thus, entropically unfavored. ² This work was supported in part by Scios, Inc., and NIH Grants HL52622 and GM38060 to R.J.L. * To whom correspondence should be addressed. Fax: (319) 335- 6634. E-mail: robert-linhardt@uiowa.edu. ‡ University of Iowa. § Scios Inc. 1 Abbreviations: BNP, brain natriuretic peptide; CAPS, 2-(N- cyclohexylamino)-1-propanesulfonic acid; CHES, 2-(N-cyclohexylami- no)ethanesulfonic acid; GAG, glycosaminoglycan; HEPES, N-(2- hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; GlcNp, glucosamine; IdoAp, iduronic acid; ∆UAp, 4-deoxy-R-L-threo-hexenopyranosyluronic acid; S, sulfate; ITC, isothermal titration calorimetry; MES, 2-(N- morpholino)ethanesulfonic acid; S, sulfate; Tris, tris(hydroxymethyl)- aminomethane; Tricine, N-[tris(hydroxymethyl)methyl]glycine. 15231 Biochemistry 1998, 37, 15231-15237 10.1021/bi980212x CCC: $15.00 © 1998 American Chemical Society Published on Web 10/01/1998