Binding of bovine serum albumin to heparin determined by turbidimetric titration and frontal analysis continuous capillary electrophoresis

The association of proteins with glycosaminoglycans is a subject of growing interest, but few techniques exist for elucidating this interaction quanti tatively. Here we demonstrate the application of capillary electrophoresis to the system of serum albumin (SA) and heparin (Hp). These two species for m soluble complexes, the interaction increasing with reduction in pH and/or ionic strength (I). The acid-base property of Hp was characterized by pote ntiometric titration of ion-exchanged Hp. Conditions for complex formation with SA were qualitatively determined by turbidimetry, which revealed point s of incipient binding (pH,) and phase separation (pH(phi)), both of which depend on I. At pH > pHb(phi), i.e., prior to phase separation, frontal ana lysis continuous capillary electrophoresis was used to measure the concentr ation 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 qu antitative binding information in the form of intrinsic binding constants ( K-obs) and binding site size (n). The strong increase in K-obs with decreas e of pH or I could be explained on the basis of electrostatic interactions, considering the effects of protein charge heterogeneity. The value of n, i ndependent 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. (C) 2001 Academic Press.