DETERMINATION OF BINDING CONSTANTS OF SERUM ALBUMIN FOR PENICILLIN

¹ Medical Service, Veterans Administration Hospital, Minneapolis, Minnesota

Free, active penicillins (benzylpenicillin, oxacillin, methicillin, nafcillin) and cephaloridine were measured in the presence of human and bovine serum albumin by means of a photometric assay of growth inhibition of Staphylococcus aureus. The decrease in antibiotic activity in the presence of albumin was assumed to reflect the antibiotic binding to the protein which other workers have demonstrated by direct physicochemical means and by the diminution of the concentration of free, active antibiotic. The percent of penicillin bound or inactivated was calcuated from the decreased antimicrobial activity in the presence of albumin. In the concentration range near therapeutic dosages, this percent antibiotic bound was independent of the antibiotic concentration and resembled a logarithmic function of the albumin concentration. Binding constants for bovine serum albumin were calculated from a simple equation for chemical equilibrium giving these values for the primary binding site: nafcillin, 3.1 X 10⁴ M^-1; oxacillin, 4.7 x 10³ M^-1; methicillin, 1.1x 10³ M^-1; benzylpenicilhin, 12 X l0⁴ M^-1 and cephaloridine, 0. These values for human serum albumin were: nafcillin, 12 X 10⁴ M^-1; methicillin, 9 x 10³ M^-1; benzylpenicillin, 1.1 x 10³ M^-1 and cephaloridine, 0. The similarity of these values with those obtained by others in the absence of bacteria supports the assumption that bound antibiotic is unavailable for bacteriostasis.