Published: August 11, 2011 r2011 American Chemical Society 11597 dx.doi.org/10.1021/la2024605 | Langmuir 2011, 27, 11597–11604 ARTICLE pubs.acs.org/Langmuir Measurements and Theoretical Interpretation of Points of Zero Charge/Potential of BSA Protein Andrea Salis,* ,† Mathias Bostr€om,* ,†,‡ Luca Medda, † Francesca Cugia, † Brajesh Barse, † Drew F. Parsons, ‡ Barry W. Ninham, ‡ and Maura Monduzzi † † Department of Chemical Science, University of Cagliari-CSGI and CNBS, Cittadella Universitaria, S.S. 554 bivio Sestu, 09042- Monserrato (CA), Italy ‡ Research School of Physical Sciences and Engineering, Australian National University, Canberra, 0200 Australia 1. INTRODUCTION The net electrical charge of proteins is a parameter that strongly affects their physicochemical behavior in living organ- isms. Protein surfaces in an aqueous medium naturally charge to form an electrical double layer. The most common surface charge-determining ions are H + and OH  . In this case, the net surface charge is affected by the pH of the medium in which the protein is dispersed. Of particular importance is the pH value at which the protein surface is electrically neutral. At this pH, the electric repulsion between proteins is minimal. Hence, they can easily coagulate and precipitate. Protein precipitation can also be induced by the addition of salts through the “salting out” phenomenon, following a Hofmeister series. 1 In all cases, electrolytes (acids, bases, and salts) are responsible for either protein stabilization or precipitation because they strongly affect forces between colloidal particles. 2 Knowledge of these phenomena can help biochemists interested in protein purification. Moreover and intriguingly, protein aggregation/ precipitation seems to play a role in several neurodegenerative disorders, such as Alzheimer's disease, prion disease, Parkinson's disease, and amyotrophic lateral sclerosis. 3 The pH value at which protein electrical neutrality occurs is termed either as the isoionic point (IIP) or, alternatively, as the isoelectric point (IEP). The conceptual difference between these two points is in principle known, but they are often used interchangeably without too much thought on the subtleties associated with them. This is the matter we explore here. It leads to a useful and rigorous demarcation. The isoionic point is defined as the pH value at which a zwitterionic molecule has an equal number of positive and negative charges and no adsorbed ionic species. 4 The isoelectric point, instead, is the pH value at which the zeta potential (or surface potential), equivalent to the net charge of the molecule including bound ions, is zero. Thus, the isoelectric and isoionic points should, in principle, coincide when the concentration of electro- lytes is zero. The two points can be determined with different kinds of experimental measurements. The isoelectric point is measured by electrokinetical methods. It is determined by the value of pH at which the protein molecule remains stationary in an electrical Received: June 29, 2011 Revised: August 10, 2011 ABSTRACT: The points of zero charge/potential of proteins depend not only on pH but also on how they are measured. They depend also on background salt solution type and concentration. The protein isoelectric point (IEP) is determined by electro- kinetical measurements, whereas the isoionic point (IIP) is determined by potentiometric titrations. Here we use potentio- metric titration and zeta potential (ζ) measurements at different NaCl concentrations to study systematically the effect of ionic strength on the IEP and IIP of bovine serum albumin (BSA) aqueous solutions. It is found that high ionic strengths produce a shift of both points toward lower (IEP) and higher (IIP) pH values. This result was already reported more than 60 years ago. At that time, the only available theory was the purely electrostatic DebyeH€uckel theory. It was not able to predict the opposite trends of IIP and IEP with ionic strength increase. Here, we extend that theory to admit both electrostatic and nonelectrostatic (NES) dispersion interactions. The use of a modified Poisson Boltzmann equation for a simple model system (a charge regulated spherical colloidal particle in NaCl salt solutions), that includes these ion specific interactions, allows us to explain the opposite trends observed for isoelectric point (zero zeta potential) and isoionic point (zero protein charge) of BSA. At higher concentrations, an excess of the anion (with stronger NES interactions than the cation) is adsorbed at the surface due to an attractive ionic NES potential. This makes the potential relatively more negative. Consequently, the IEP is pushed toward lower pH. But the charge regulation condition means that the surface charge becomes relatively more positive as the surface potential becomes more negative. Consequently, the IIP (measuring charge) shifts toward higher pH as concentration increases, in the opposite direction from the IEP (measuring potential).