Biochimica et Biophysica Acta, 756 (1983) 49-55 49 Elsevier Biomedical Press BBA21386 EXPERIMENTAL TESTS OF CHARGE CONSERVATION IN MACROMOLECULAR INTERACTIONS CHRISTOPHER L. FORD and DONALD J. WINZOR * Department of Biochemistry, University of Queensland, St. Lucia, Queensland 4067 (Australia) (Received September 1st, 1982) Key words: Protein valence," Charge conservation," Ultrafiltration; Albumin-dye interacion; Chymotrypsin; Equilbrium dialysis A combination of equilibrium dialysis and ultrafiltration has been used to demonstrate the conservation of charge in the interaction between bovine sermn albumin and methyl orange in Tris-HCI buffer, pH 7.4, I = 0.05 M; and also in the dimerization of ct-chymotrypsin in acetate/chloride buffer, pH 3.9, I--- 0.11 M, containing various concentrations of indole (0-10 mM) in order to displace the equilibrium position towards monomer. In the former study the magnitude of the negative charge on the albumin was shown to increase linearly with the number of molecules of methyl orange hound to the protein, the observed slope (0.96 ± 0.08) of this relationship being in excellent agreement with that predicted on the basis of charge conservation for attachment of the univalent, negatively charged methyl orange ligand. In the study of ct-chymotrypsin, the net charge (expressed per monomeric enzyme unit) was + 10 in solutions in which the mole fraction of monomer varied between 0.47 and 0.88, the extent of this range having been established by means of constituent association equilibrium constants obtained from sedimentation equilibrium studies. Introduction Studies of macromolecular interactions fre- quently entail the assumption being made that net charge is conserved. For example, an electro- phoretic characterization of interactions between proteins and small charged ligands [1-4] is based on the concept of a constant incremental change in protein mobility with each successive ligand at- tachment. Conservation of charge is an assump- tion inherent in the use of sedimentation equi- librium distributions to characterize the self-as- sociation of charged macromolecules [5]. We also note that allowance for thermodynamic nonideal- ity in terms of composition-dependent activity coeficients [6-8] requires an estimate to be made of the charge on each macromolecular species pre- * To whom correspondence should be addressed. sent, and that these estimates are made by ap- propriate summations of the reactant valences [8]. Since there are likely to be many interactions in which charge is not conserved, it becomes im- portant to have available methodology for estab- lishing the validity or othe~'ise of adopting the concept of charge conservation for the particular interacting system being studied. In the present investigation, advantage is taken of a recently reported method for estimating the net charge of a macromolecule [9] to test the validity of assuming charge conservation. Its appli- cation to the bovine serum albumin/methyl orange system (pH 7.4, I = 0.05 M) is used to verify the concept for this experimental situation involving protein-ligand interactions, and its application to a-chymotrypsin used to illustrate conservation of charge in the dimerization of this enzyme at pH 3.9, I--0.11 M. 0304-4165/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers