Analytical Biochemistry 295, 158–167 (2001) doi:10.1006/abio.2001.5129, available online at http://www.idealibrary.com on Binding of Bovine Serum Albumin to Heparin Determined by Turbidimetric Titration and Frontal Analysis Continuous Capillary Electrophoresis Toshiaki Hattori,1 Kozue Kimura, Emek Seyrek, and Paul L. Dubin2 Department of Chemistry, Indiana University–Purdue University, Indianapolis, Indiana 46202 Received October 23, 2000; published online July 19, 2001 The binding of proteins to proteoglycans (PGs)3 is an The association of proteins with glycosaminogly- important biological phenomenon. For example, the cans is a subject of growing interest, but few tech- interaction between chondroitin sulfate or keratan sul- niques exist for elucidating this interaction quantita- fate proteoglycans and collagen fibrils in the cartilage tively. Here we demonstrate the application of matrix defines the final shape of the tissue and pro- capillary electrophoresis to the system of serum albu- vides the ability to withstand compressive load (1). min (SA) and heparin (Hp). These two species form Heparan sulfate proteoglycans (HSPGs) interact with soluble complexes, the interaction increasing with re- fibronectin–collagen complexes in plasma membranes duction in pH and/or ionic strength (I). The acid–base to assist cell adhesion (1). HSPGs in the glomerular property of Hp was characterized by potentiometric basement membrane (GBM) also provide selective fil- titration of ion-exchanged Hp. Conditions for complex tration of proteins (1, 2). These interactions appear to formation with SA were qualitatively determined by have a strong electrostatic component, arising from the turbidimetry, which revealed points of incipient bind- interactions between charges on the proteins and the ing (pHc) and phase separation (pH␾), both of which intense negative charge on glycosaminoglycans (GAGs) depend on I. At pH > pH␾, i.e., prior to phase separa- in PGs. tion, frontal analysis continuous capillary electro- GAGs are highly diversified sulfated and carboxy- phoresis was used to measure the concentration of lated linear polysaccharides that constitute the major free protein and to determine the protein–HP binding components of PGs. The backbone of the extracellular isotherm. The binding isotherms were well fit by the matrix PG aggregate is a high-molecular-weight (ca. McGhee–von Hippel model to yield quantitative bind- 2 ϫ 106) hyaluronic acid, to which are bound a large ing information in the form of intrinsic binding con- number of core proteins; each core protein, in turn, is stants (Kobs) and binding site size (n). The strong in- covalently bound to numerous GAGs (3–5). Many of the crease in Kobs with decrease of pH or I could be roles of PGs in extracellular signaling appear to de- explained on the basis of electrostatic interactions, pend on the interaction between GAGs and other mac- considering the effects of protein charge heterogene- romolecules, in particular proteins (3, 6). Conse- ity. The value of n, independent of pH, was rational- quently, the study of GAG–protein interactions can ized on the basis of size considerations. The implica- facilitate a physical understanding of important biolog- tions of these findings for clinical applications of Hp ical phenomena. and for its physiological behavior are discussed. © 2001 Many techniques have been used to investigate the Academic Press interaction between GAGs and proteins. For example, the formation of complexes has been detected using fluorescence (7, 8) and UV (8) absorption. Diffusion 3 Abbreviations used: PGs, proteoglycans; HSPGs, heparin sulfate 1 Current address: Research Center for Chemometrics, Toyohashi proteoglycans; GBM, glomerular basement membrane; GAGs, gly- University of Technology, Toyohashi, Japan 441-8580. coaminoglycans; CE, capillary electrophoresis; Hp, heparin; FACCE, 2 To whom correspondence should be addressed. Fax: (317) 274- frontal analysis continuous capillary electrophoresis; BSA, bovine 6879. E-mail: [email protected]. serum albumin; SEC, size-exclusion chromatography. 158 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ELECTROPHORETIC DETERMINATION OF SERUM ALBUMIN–HEPARIN BINDING 159 coefficients and sizes of complexes have been measured gand and the complex can then be fit to appropriate using light scattering (9, 10). Protein structural binding isotherms to yield binding constants and changes as a result of complexation between GAGs and binding site size, two parameters that are very use- proteins have been observed by circular dichroism ful in elucidation of the binding mechanism. More- measurement (CD) (11, 12) and NMR (13), and the over, sample requirements are smaller, and the mea- binding sites of proteins and GAGs have been identi- surement time is short. FACCE thus appears to be a fied by X-ray crystallography of complexes between simple, rapid, economical, precise, and reproducible proteins and GAG oligomers (14). Complex mobilities method for quantitative characterization of protein– have been measured by capillary electrophoresis (CE) polyelectrolyte binding. (15). Previous studies with FACCE focused on combina- Although numerous methods have been used to ex- tions of proteins and synthetic polyelectrolytes (21). amine protein–GAG complexes, few techniques are ca- Here we are interested in developing FACCE method- pable of quantitatively determining an important vari- ology for combinations of proteins and biological poly- able: the GAG–protein complex formation constant. electrolytes. While the ultimate goal is to apply this These potentiometric, spectroscopic, or chromato- technique to physiological cognates, our purpose here graphic methods all pose respective difficulties. For is to develop the methodology, as opposed to addressing example, the heparin(Hp)–protamine binding constant a specific biochemical question. To do this, we have was determined using a Hp-sensitive electrode after chosen bovine serum albumin (BSA) as the protein and neutralization of the complex (16), but the mechanism heparin (Hp) as the biological polyelectrolyte. of this electrode response is not well understood, and The interaction of Hp and Hp-like GAGs with pro- other bulky anions e.g., free proteins of negative net teins is clearly of considerable interest. Hp–protein charge, can act as interferents in this assay. Quantita- interactions regulate biological processes as diverse tion of complexation has been carried out using fluo- as complement activation, coagulation, cell adhe- rescence (17–19), but this approach depends on a large sion, angiogenesis, platelet aggregation, lipolysis, change in absorption before and after complexation. and smooth muscle proliferation. Hp regulates an- While affinity chromatography has been used to mea- giogenesis, the growth of new capillary blood vessels, sure the dissociation constant (20), large amounts of by interacting with peptide growth factors (27). It sample are required and the dissociation constant can has an anticoagulant effect in coagulation, where be perturbed by the immobilization of protein or GAG. binding to a plasma Hp cofactor is required for its Frontal analysis continuous capillary electro- activity (28); consequently, Hp is often used during phoresis (FACCE) (21) is a new technique that has surgical procedures or as an anticoagulant for been demonstrated as a way to obtain precise and reliable binding data for protein–polyelectrolyte thrombosis. Hp also has a direct aggregatory effect complexes. This technique resembles other capillary on platelets and lipoproteins; the interaction with electrophoresis methods, namely the Hummel– the platelets causes platelet proaggregating and po- Dreyer method, conventional frontal analysis tentiating effects (29), while the binding of Hp to method, affinity capillary electrophoresis, and va- lipoprotein leads to aggregation or fusion of the lipo- cancy peak and vacancy affinity capillary electro- protein during lipolysis (30). Hp is also responsible phoresis; however, each of these measurements of for the antiproliferative activity in smooth muscle quantitative binding parameters poses some prob- proliferation (31). While these diverse and complex lems (22). Because the perturbation of the binding biological roles are not fully understood, they clearly equilibria intrinsic to conventional frontal analysis involve the binding of many proteins. method is overcome by continuous sampling, FACCE Serum albumin is not a biochemical cognate of Hp, can be performed even when the mobility of the but it is the most abundant protein in the body and in complex is not equal to the mobility of ligand or particular is present at high concentration for intrave- acceptor. Affinity capillary electrophoresis is useful nously administered Hp. Because the molecular weight for determining the dissociation constant of pro- of serum albumin is large (even in the absence of mul- tein–Hp binding (23–26), but the resulting constant timerization), complex formation of serum albumin is not regarded as a true quantitative binding pa- with polymers is easily observed by many methods rameter when multiple ligands bind to the acceptor based on scattering or electrical mobility (32). The (22). On the other hand, FACCE may be used even in structure of serum albumin is well understood and its multiple complexation equilibria to measure the con- properties, particularly its ionization behavior, are centration of free ligand because its concentration is thoroughly