Protein binding of piroxicam studied by means of affinity chromatography and circular dichroism

The protein binding of piroxicam, a widely used non-steroidal anti-inflammatory drug has been investigated by high-performance liquid affinity chromatography, with phenylbutazone and diazepam used as markers for binding-site characterization, and by circular dichroism titration. It was found that piroxicam binds to high-affinity phenylbutazone-binding sites and to high-affinity diazepam-binding sites. No binding to the low-affinity sites of either marker was established. High values of the primary (high-affinity) binding constants corresponding to both types of binding site were obtained by means of a mathematical method cited in the literature. The circular dichroic spectra of piroxicam were studied at a given albumin concentration and various drug concentrations. A new Cotton effect was observed and was ascribed to the binding of piroxicam to the protein molecule. The values of differential molar ellipticity (delta theta) were treated by a new mathematical procedure for analysis of the data obtained. A high affinity constant was calculated for one class of binding site. Its value is in good agreement with the values obtained by affinity chromatography. These results reveal that circular dichroism is an acceptable method for investigation of protein binding.