Biophysical Chemistry 103 (2003) 77–88 0301-4622/03/$ - see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0301-4622 Ž 02 . 00233-8 Net proton charge of b- and k-casein in concentrated aqueous electrolyte solutions M. Cordeschi, L. Di Paola, L. Marrelli*, M. Maschietti Chemical Engineering Department, University of Rome ‘La Sapienza’, Via Eudossiana 18, 00184 Rome, Italy Received 11 April 2002; received in revised form 23 July 2002; accepted 25 July 2002 Abstract Titration experiments have been carried out in order to measure the net proton charge of b- and k-casein in NaCl solutions at 0.1 M and 1 M salt concentrations, at 4 8C, in the pH range between 5.5 and 10.5. Experimental data are compared with model values calculated through pK ’s of titrable groups neglecting the electrostatic perturbation a term (DpK ) in order to evaluate the magnitude of the error caused by this approximation and to delimit its a effectiveness. At both ionic strengths, the agreement is good for k-casein in the pH range w5.5, 9.5x, while errors of up to 2 charges are observed for b-casein in the same range. These deviations are likely to be caused by strong electrostatic effects induced by the high density of negative charges of b-casein 1–21 peptide. In order to account for these electrostatic effects, the net proton charge on this peptide is evaluated through a model based on the counterion condensation theory developed for the titration of polyelectrolytes with different types of ionizable groups.  2002 Elsevier Science B.V. All rights reserved. Keywords: b-casein; k-casein; Net proton charge; Potentiometric titration; Counterion condensation theory 1. Introduction Experimental and theoretical information about hydrogen ion binding to protein molecules is very useful both from a fundamental and a practical point of view. Electrostatic interactions play a key role in determining the behavior of solutions con- taining large ionic biomolecules, such as proteins in aqueous electrolyte solutions. For example, the balance between repulsive electrostatic and attrac- *Corresponding author. Tel.: q39-06-4458-5608; fax: q39- 06-4827-453. E-mail address: marrelli@ingchim.ing.uniroma1.it (L. Marrelli). tive van der Waals forces controls numerous phe- nomena, such as protein aggregation and stability of aggregates or their precipitation. Furthermore, the net proton charge allows differences between like proteins to be emphasized or the occurrence of denaturation to be easily detected. Finally, reliable and useful molecular–thermodynamic models can be developed on the basis of interac- tions between salt ions and proteins w1x and between protein molecules. For example, the net proton charge at different ionic strengths is required to predict binding of other ions (i.e. chloride, sodium, etc.) to protein molecules in terms of the electric double layer theory w2x.