Chloroquine interaction with ferric uroporphyrin in solution

Abstract

It is estimated that, in 1979, 80% of the world's population was at risk to Plasmodium infection (malaria), primarily in the developing nations [1]. Chemotherapeutic agents useful in treatment include the quinine based drugs and the quinoline derivatives such as chloroquine [2, 3], although their molecular basis of action is not presently well understood. In patients undergoing malaria chemotherapy the drug is found in association with hemozoin pigments in the erythrocyte. Hemozoin is an aggregate of precipitated heme and denatured hemoglobin and this finding has stimulated our interest in the possibility that the antimalarial efficacy originated from association with heme complexes. Fitch et al. [4] have, in fact, suggested that erythrocyte localized protohemin IX is the putative receptor of chloroquine. In this report we present some of our initial results concerning the interaction of chloroquine with iron porphyrins in solution. Urohemin was chosen due to its high solubility in aqueous solutions and because recent raman and nuclear magnetic resonance (NMR) work has allowed us to thoroughly characterize its solution dynamics [5]. Experimental Urohemin was purchased from Porphyrin Products, Logan, Utah and was further purified by column chromatography. Chloroquine (Sigma) was used without further purification. Titrations of urohemin with the drug were carried out at pH 6 (unbuffered) employing a Cary 219 ultraviolet-visible spectrometer. pH was monitored throughout the experiment. Results and Discussion The data of Figs. 1 and 2 reveal that chloroquine does indeed associate with urohemin in solution. Changes in the optical spectrum (Fig. 1) occur, which may be used in an attempt to quantitate equilibrium behavior (Fig.2). The result of adding chloroquine to a solution of urohemin monomer (Fig. 1) is a spectrum in which the Soret intensity is lost, characteristic of the dimer (and higher order aggregates). However, unlike the ▪ ▪ dimer spectrum chloroquine binding induces a shift in the Soret maximum to longer wavelengths. Attempts to characterize the chloroquine–urohemin association by standard uv-visible methods [6] revealed that this system cannot be characterized by a single equilibrium process. This is shown by the deviation from linearity of the data in Fig. 2 [6, 7]. This data is the result of successive additions of chloroquine to a solution of monomer urohemin. This process produced standard appearing difference absorption spectra with two isosbestic points between 350 and 500 nm. The fact that linearity in Fig. 2 is not observed suggests that multiple equilibria are present in this system. Further work on malaria drug interactions with free hemins and heme proteins is in progress in our laboratories.