Simple spectrophotometric assay for measuring protein binding of penem antibiotics to human serum

The binding of antibiotics to plasma (serum) proteins through hydrogen bonding can significantly influence the biological characteristics of these drugs. A rapid spectrophotometric assay has been developed that measures the level of free (unbound) penem antibiotic in serum ultrafiltrates. Whole human serum was adjusted to a standard concentration of antibiotic and then filtered by centrifugation through a Centrifree (Amicon Corp., Lexington, Mass.) filter that retained greater than 99.9% of serum protein. The degree of penem protein binding was determined spectrophotometrically by measuring the level of unbound drug in the ultrafiltrate at 322 nm. At this wavelength, no interfering absorption from residual protein was detected in the ultrafiltrate, and penem absorption was linear over a wide concentration range. The method gave protein-binding values comparable to those obtained by a high-pressure liquid chromatography assay but was more rapid, since it did not require solvent extraction and high-pressure liquid chromatography calibration procedures. The spectrophotometric assay has been used to assay over 100 penems to determine the structure-activity relationships that are involved with the high serum protein binding of these agents. As with penicillins and some cephalosporins, the nonpolar nature of the penem side chain at the C-2 position strongly influenced the degree of penem binding to serum proteins.