Is surface patch binding between proteins symmetric about isoelectric pH?

Abstract

Surface selective patch binding (SPB) interaction occurring between two protein molecules, bovine serum albumin (BSA) and gelatin B (GB), both having same isoelectric pH (pI ≈ 5) and identical pH-zeta potential profile, was systematically examined. BSA : GB mixing ratio r was varied in the range 0.16–2.00 and ionic strength was varied in the range 0–10 mM, which yielded optimum binding ratio r = 1. The binding profiles produced asymmetric bell-like curves with clearly identifiable pairs of transition pHs: onset of intermolecular interaction, formation of soluble complexes and coalescence of the soluble complexes occurring at pH_c1,2, pH_φ1,2 and pH_m respectively. Since pH_m could be approached from either lower or higher side of pI, these profiles yielded pairs of pH_c and pH_φ values. In fact, we found (pH_c2 − pI) > (pH_c1 − pI), which clearly indicated that initiation of intermolecular associative interaction was not symmetric about pI (pI = pH_m for r ≤ 1, an observation not reported hitherto. Secondly, (pH_φ2 − pI) ≈ (pH_φ1 − pI) implied that the pH at which soluble complexes formed (pH_φ) was always located symmetrically about pH_m, irrespective of the binding ratio. Higher binding affinity determined from higher value of pH_c2 was confirmed from size measurement results. The change in the turbidity maximum Δτ could be correlated as Δτ ∼ I^1/2 implying electrostatic screening of SPB with increase in ionic strength (I). This interaction was modelled using a linear combination of attractive and repulsive electrostatic forces which revealed considerable screening of the interaction potential U, consistent with aforesaid experimental data; ΔU ∼ I^1/2. Further, it is concluded that intermolecular binding in protein–polyampholyte systems is qualitatively different from that in protein–polyelectrolyte class.