DETERMINATION OF THE PK(A) VALUES OF TITRATABLE GROUPS OF AN ANTIGEN-ANTIBODY COMPLEX, HYHEL-5-HEN EGG LYSOZYME

The titration behavior of the ionizable residues of the HyHEL-5-hen eg g lysozyme complex and its individual components has been studied usin g continuum electrostatic calculations. Several residues of HyHEL-5 ha d pK(a) values shifted away from model values for isolated residues by more than three pH units. Shifts away from the model values were smal ler for the residues of hen egg lysozyme. A moderate variation in the pK(a) values of the titratable groups was observed upon increase of th e ionic strength from 0 to 100 mM, amounting to 1-2 pH units in most e ases, Under physiological conditions, the net charge of HyHEL-5 was op posite that for hen egg lysozyme. Several residues, including those in volved in the Arg-Glu salt bridges that have been proposed to be impor tant in antibody-antigen binding, had pK(a) values that were changed s ignificantly upon binding. The main titration event upon antibody-anti gen binding appears to be loss of a proton from residue GluH50 of the Fv molecule. The limitations of our calculation methods and the role t hey might play in the design of antibodies for use in assays, sensors and separations are discussed.