Complexation of fluoroquinolone antibiotics with human serum albumin: A fluorescence quenching study

Mechanism of interaction and detailed physico-chemical characterization of the binding of four fluoroquinolones: levofloxacin, sparfloxacin, ciprofloxacin HCl and enrofloxacin with human serum albumin has been studied at physiological pH (7.4) using fluorescence spectroscopic technique. The stoichiometry of interaction was found to be 1:1 for all the drugs used. The association constants for the interaction were of the order of 10⁴ in most cases. At low drug:protein ratios, a significant fraction of the added drug was bound. The predominant interactions involved are hydrogen bonding and Van der Waal’s interactions in the case of levofloxacin, hydrophobic interactions in the case of ciprofloxacin hydrochloride and enrofloxacin and hydrogen bonding, hydrophobic and electrostatic interactions in the case of sparfloxacin.The drug binding region did not coincide with that of the hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS). From the displacement of site-specific probes and site-marker drugs, it was concluded that ciprofloxacin hydrochloride is site II-specific while enrofloxacin is a site I-specific drug. Levofloxacin binds at both site I and site II with equal affinity. Sparfloxacin had higher affinity for site II than site I. It is also possible that sparfloxacin binds at the interface between site I and site II. Stern-Volmer analysis of the data showed that the quenching mechanism is predominantly collisional for the binding of ciprofloxacin HCl and enrofloxacin while both static and collisional quenching mechanisms are operative in the case of levofloxacin and sparfloxacin. High magnitude of the rate constant for quenching showed that the process is not entirely diffusion controlled. Circular dichroism (CD) spectroscopic studies showed that the presence of drugs did not cause any major changes in the secondary structure of HSA.