170 Biochimiea etBiophysicaActa, 645 (1981) 170-176 Elsevier/North-Holland Biomedical Press BBA 79304 SURFACE POTENTIAL OF PHOSPHATIDYLSERINE MONOLAYERS II. DIVALENT AND MONOVALENT ION BINDING SHINPEIOHKI a and ROBERT KURLAND a,b a Departments of Biophysical Sciences and b Chemistry, State University of New York at Buffalo, Buffalo, NY 14214 (U.S.A.) (Received December 5th, 1980) Key words: Surface potential; Phosphatidylserine monolayer; Ion binding Ion binding constants for phosphatidylserine membranes have been derived from the variation of the surface po- tential of phosphatidylserine monolayers with divalent cation concentrations in the presence of various mono- valent salts in the aqueous subphase. The observed surface potential data for the monolayers, analyzed by use of the Gouy-Chapman diffuse potential theory, together with a simple binding reaction formula, yield, for Ca 2+, Mg2+, Na÷ and (Me)4N + binding constant values of 30 M-1, 10 M-1, 0.6 M-~ and 0.05 M-1, respectively. The effect of pH on surface potential of phosphatidylserine monolayers was found to be dependent upon ionic species other than H+ in the subphase solution. The distinction between apparent and intrinsic dissociation constants of H÷ for biomolecules was made in terms of ion binding due to other ions at the same site as for H÷ in biomolecules. Introduction In a previous study [1], we attempted to deter- mine the association constants for binding of various divalent ions to phosphatidylserine membrane sur- faces by measuring the surface potential of the mono- molecular film. However, in the calculation of the binding constants, we assumed that monovalent ions did not bind to the phosphatidylserine polar groups. Consequently, the values for binding constants of divalent cations thus derived were too small. Since then, it has been shown that monovalent ions do bind to model membranes of phosphatidyl- serine [2-5]. Therefore, the previously obtained binding constants of divalent ions [1] should be reevaluated. Although the monovalent ions bind to phosphatidylserine membranes more weakly than do divalent ions, the effects of monovalent ions are important in the interpretation of such phenomena as membrane aggregation and fusion of phospholipid systems in different ionic environments. In this work we describe surface potential mea- surements for phosphatidylserine monolayers and the dependence of this potential on the concentration of divalent ions in the presence of different monovalent ionic salts at various ionic strengths. The data are analyzed to obtain the binding constants of divalent as well as monovalent ions. We also interpret the dif- ference in phosphatidylserine monolayer pH-surface potential (AAV) curves obtained for the two sub- phase salt solutions (NaC1 and tetramethylammonium chloride, (Me)4NC1) in terms of the different binding constants obtained from the former experiments for Na ÷ and tetramethylammonium ion ((Me)4N+). Finally, we discuss the determination of pK a values of biomolecules in a salt solution medium and the importance of taking into account the effect of other cation binding on the evaluation of the dis- sociation constant of H÷ for various biomolecules. Materials and Methods Materials. Phosphatidylserine was extracted from beef brain and purified according to the method of Rouser et al. [6] with minor modifications. Ultra- pure NaC1 (Brinkman Instruments); histidine (D-a- 0 005-2736/81/0000-0000/$02.50 © Elsevier/North-Holland BiomedicalPress