Identification by Integrated Computer Modeling and Light Scattering Studies of an Electrostatic Serum Albumin-Hyaluronic Acid Binding Site

Abstract

Dynamic light scattering and turbidimetry, carried out on solutions of hyaluronic acid (HA) and bovine or human serum albumin (SA) at fixed ionic strength (I), revealed a critical pH corresponding to the onset of HA-SA soluble complex formation. Subsequent reduction of pH below pH c , corresponding to an increase in protein net positive charge, results in phase separation of the complex. The sensitivity of pH c to I indicated the primacy of electrostatic interactions in this process. Since pH c was always above the pK a of HA, these effects could be attributed to the influence of protein charge. The electrostatic potential around HSA was modeled using DelPhi (MSI) under pH, I conditions corresponding to incipient binding, phase separation, and noninteraction. At all incipient binding conditions (i.e., pH c , at varying I), an identical region of positive potential 5 Å from the protein van der Waals surface appeared. This unique domain intensified with a decrease in pH or I (corresponding to stronger binding), and diminished with an increase in pH or I (i.e., at noninteracting conditions). The size and low curvature of this domain could readily accommodate a 12 nm (decamer) sequence of HA. Simple electrostatic considerations indicate an electrostatic binding energy for the formation of this complex of ca. 1 kT, consistent with the condition of incipient complex formation. We suggest that such weak electrostatic binding may characterize nonspecific interactions for other proteingylcosaminoglycan pairs.

Effects of Polyelectrolyte Chain Stiffness, Charge Mobility, and Charge Sequences on Binding to Proteins and Micelles

“…48 Z c . BSA in the vicinity of Z ) 0 exhibits a relatively flat positive domain (see Figure 8) that accommodates a 5-nm length of the locally stiff polyanion HA; 23 further increase in chain stiffness does not impede binding to this protein domain. For small spherical micelles, the effect of polymer stiffness is obviously more important.…”

“…In so doing, we sought to identify possible binding sites for DMF25 on the protein at the critical binding conditions. The criteria established for identifying the electrostatic binding site of a protein were previously put forward 23 as follows: (1) the binding site location and size must be the same at all critical binding conditions along the phase boundary, (2) the binding domain must become appropriately larger or smaller upon pH adjustment, and (3) the mean potential ψ on or within the binding domain should satisfy the relationship zψ ≈ kT (i.e., close to thermal energy), where z is the number of polyelectrolyte charges binding cooperatively. For our purposes, we focus on the first two of these criteria.…”

“…22 The exact location of this electrostatic binding site is not a priori known, nor is it constant over the entire I range, although Grymonpré et al suggested criteria by which it might be identified. 23 The question of a positive electrostatic binding site encompassed within a globally negative protein is certainly of biological interest, as anionic biopolyelectrolytes such as GAGs do bind to proteins with pI < 7, i.e., proteins with net negative charge under physiological conditions. 24 But the nonuniformity of protein charge distributions obscures the relevance of eq 1 to polyelectrolyte-protein interactions, despite the tantalizing finding for at least one polyelectrolyte-protein system, of a linear dependence on I 1/2 of the net protein charge at the onset of binding, corresponding to eq 1, with b ) 1.…”

Complex coacervation of silk fibroin and hyaluronic acid

“…This variation was seen within the pH range of 4.5-6. The possibility of such variations above IEP of the protein, on the wrong side of the IEP, was attributed to the existence of a local protein domain forming a charge patch with an effective charge opposite in sign to net protein charge [30]. Around pH 3.5 a strong increase in system turbidity was observed and this point was generally symbolized as pH φ , coacervate formation pH [1][2][3][4][5][6].…”

Effect of Heparin on Protein Aggregation: Inhibition versus Promotion

“…31 The "charge patch", representing the spatial domain in which some sequence of the heparin chain resides in its bound state, does not correspond to the protein surface but to a region of highly positive electrostatic potential, 60 best displayed in the DelPhi images of Figure 5.…”