title: Binding of bovine serum albumin to heparin determined by turbidimetric titration and frontal analysis continuous capillary electrophoresis.
abstract: The association of proteins with glycosaminoglycans is a subject of growing interest, but few techniques exist for elucidating this interaction quantitatively. Here we demonstrate the application of capillary electrophoresis to the system of serum albumin (SA) and heparin (Hp). These two species form soluble complexes, the interaction increasing with reduction in pH and/or ionic strength (I). The acid-base property of Hp was characterized by potentiometric titration of ion-exchanged Hp. Conditions for complex formation with SA were qualitatively determined by turbidimetry, which revealed points of incipient binding (pH(c)) and phase separation (pH(phi)), both of which depend on I. At pH > pH(phi), i.e., prior to phase separation, frontal analysis continuous capillary electrophoresis was used to measure the concentration of free protein and to determine the protein-HP binding isotherm. The binding isotherms were well fit by the McGhee-von Hippel model to yield quantitative binding information in the form of intrinsic binding constants (K(obs)) and binding site size (n). The strong increase in K(obs) with decrease of pH or I could be explained on the basis of electrostatic interactions, considering the effects of protein charge heterogeneity. The value of n, independent of pH, was rationalized on the basis of size considerations. The implications of these findings for clinical applications of Hp and for its physiological behavior are discussed.
authors.affiliation: Department of Chemistry, Indiana University-Purdue University, Indianapolis, IN 46202, USA.
authors.forename: T
authors.initials: T
authors.lastname: Hattori
authors.forename: K
authors.initials: K
authors.lastname: Kimura
authors.forename: E
authors.initials: E
authors.lastname: Seyrek
authors.forename: P L
authors.initials: PL
authors.lastname: Dubin
mesh_terms: D001665:Binding Sites; D019075:Electrophoresis, Capillary / Q000379:methods; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009391:Nephelometry and Turbidimetry; D009994:Osmolar Concentration; D011199:Potentiometry; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D055672:Static Electricity; D013329:Structure-Activity Relationship
chemical_list: D012710:Serum Albumin, Bovine; D006493:Heparin