Spectroscopic Techniques in the Study of Protein Binding: The Use of 1-Anilino-8-naphthalenesulphonate as a Fluorescent Probe for the Study of the Binding of lophenoxic and lopanoic Acids to Human Serum Albumin

The binding of iopanoic and iophenoxic acids to human serum albumin (HSA) has been studied by spectroscopic techniques. The protein fluorescence of human serum albumin was quenched by the binding of either drug. Analysis of this quenching indicated that iophenoxic acid binds very tightly to at least one site on the albumin molecule so that, at less than a 1:1 ratio of iophenoxic acid to HSA, almost no drug exists free in solution. This provides an explanation for the unusual pharmacokinetics of this drug. The binding of 1-anilino-8-naphthalenesulphonate (ANS) to human serum albumin has been studied and is shown to be consistent with strong binding at one site and weaker binding at three further sites. The fluorescence of ANS bound to HSA was increased by iophenoxic acid and decreased by iopanoic acid. Measurements of fluorescence emission spectra and fluorescence lifetimes indicated that the drug-induced changes in fluorescence were due to changes in the quantum yield of the bound ANS. It is concluded that iophenoxic and iopanoic acids induce different changes in albumin conformation which can be detected by changes in the fluorescence of bound ANS. Iophenoxic acid enhanced the fluorescence of ANS bound to its tight site but displaced ANS from the weaker binding sites. Furthermore, 1 mole of iophenoxic acid displaced more than 1 mole of ANS, indicating that this effect was also related to the drugnduced change in the albumin structure.