Interaction of a fluorescent probe, CAPIDAN, with human serum albumin

Abstract

► We studied interaction of a fluorescent reporter CAPIDAN with human serum albumin. ► 1 H NMR data show that the aromatic part of CAPIDAN is immersed into albumin. ► Computer modeling shows that CAPIDAN prefers rather polar media. ► Indeed, fluorescence is consistent with polar properties of the drug-binding site I. ► It was suggested that the drug binding site I contains entrapped water. Fluorescent probe CAPIDAN (or “K-35”), N-(p-carboxyphenyl)imide of 4-(dimethylamino)naphthalic acid, is used as a detector of structural changes in human serum albumin in some diseases. In these cases observed changes in its fluorescence can be used to predict disease severity, stage, or outcome as good as or better than routine diagnostic tests. In this work, some aspects of probe's interaction with albumin molecule were studied using optical methods, NMR and computer simulations. It was shown that probe's fluorescence originates mostly from molecules bound to the drug-binding site I. It is suppressed by phenylbuthazone, a marker for this site. 1 H NMR data shows that the aromatic part of CAPIDAN, including its amino group, is immersed into protein. Sensitivity of the interaction to the ionic strength of solution suggests that CAPIDAN's negatively charged carboxyl group localizes at the site/solution interface, near exposed positively charged residues. Positions of the peaks of CAPIDAN's absorption/excitation spectra indicate polar environment around the bound probe and suggests the existence of water molecules entrapped in the pocket. Limited Stokes shift of the fluorescence spectrum and 1 H NMR data provide evidence of hindered motion of bound probe, pocket's polar groups and entrapped water molecules.