Complexes of bilirubin with proteins.

Complexes of bilirubin with chymotrypsin, lysozyme and apomyoglobin in neutral aqueous solution are characterized by circular dichroism spectra in the visible region. These are analogous to previously investigated bilirubin-serum albumin complexes. Ferriprotoporphyrin IX displaces bilirubin from its apomyoglobin complex.

Relative affinities of bilirubin for serum albumins from different species.

Relative equilibrium constants ("affinity ratios") of complexes of bilirubin with a molar excess of charcoal-treated serum albumins from different species (human, bovine, rabbit and chicken) in aqueous solution, were estimated by circular dichroism measurements in the visible region at 26-27 degrees C, pH 7.4, and in the presence of 0.1 M NaCl. By variation of the mol ratios of the components of pairs of different bilirubin-serum albumin complexes showing circular dichroic bands of opposite sign, the apparent association constants of complexes of bilirubin with either human or chicken albumin were found to be greater by factors between 6 and 17 than those of bovine or rabbit albumins. The usefulness in the determination of affinity ratios is illustrated by the evaluation of single equilibrium constants of systems of high-ligand affinity from those of relatively lower affinity, the latter of which are more readily amendable to direct experimental measurement.

Optical properties and structure of tetrapyrroles. A report of a symposium held at the University of Konstanz, FRG, August 12-17, 1984.

Many basic life processes such as oxygen transport, electron transport, photosynthesis and plant development, are mediated by tetrapyrroles. It has long been recognized that optical and other physicochemical properties of tetrapyrroles are highly important not only for characterization of the various relevant systems but also to provide a key role in the understanding of their structural and functional aspects. The symposium described below was devoted mainly to recent advances in the optical and molecular properties of biologically important systems, involving both cyclic and open-chain tetrapyrroles. The topics presented and discussed ranged from physics and chemistry to plant biology and medicine. This interdisciplinary character of the meeting conforms with previous symposia also held in Konstanz: Protein-Ligand Interactions (1974) and Transport by Proteins (1978) [see FEBS Lett. (1975) 54, 1-4, and FEBS Lett. (1979) 103, 1-4]. In order to stimulate exchange of ideas, new experimental approaches and techniques, emphasis was placed on the discussions following each presentation, reports of which are also included in the book published. Preprints of the papers presented were distributed some time prior to the symposium. The symposium was mainly supported by the Stiftung Volkswagenwerk and the University of Konstanz. The meeting was based on 29 invited lectures which were divided into four sections.

Further evidence for the interaction of the antimalarial drug amodiaquine with ferriprotoporphyrin IX.

Evidence for complex formation of the antimalarial drug amodiaquine (AD) with ferriprotoporphyrin IX (FP) in aqueous medium is presented, in addition to previous preliminary data. A mole ratio of one between the complex components is determined for the insoluble complex at pH 6.7-6.8. Mössbauer data obtained at pH 7-8 and at higher concentrations in the millimolar range confirm the interactions existing between the complex components. These data are considered to aid in removing previous objections to a mechanism of antimalarial action involving complexes of FP with AD and related drugs.

Investigations of B- and beta-hematin.

The preparation from ferriprotoporphyrin IX (FP) in aqueous acid medium of the related pigments beta- and B-hematin [see G. Blauer and M. Akkawi, Biochem. Mol. Biol. Int. 35, 231 (1995)] is presented under different conditions. Both pigments are characterized by infrared spectra which differ in the range of 1600-1700 cm-1 in their strong bands with absorption peaks measured at 1648 +/- 2 cm-1 for B-hematin and at 1663 +/- 1 cm-1 for beta-hematin. The pH dependence of B-hematin formation at 37 degrees C and at different concentrations of acetic acid and FP exhibits a maximum yield near pH 4. The formation of beta-hematin at 70 degrees C shows high yield in 6 M acetic acid or in the presence of 0.028 M trichloroacetate at pH 4.6. The dependence of the yield of the pigments on the time and temperature of incubation, concentration of FP, and the presence of different electrolytes was investigated. Both B- and beta-hematin are either insoluble or very slightly soluble in different solvents at room temperature, and appear to dissociate into regular FP in strongly alkaline aqueous medium. In the presence of different quinoline-based drugs, the formation of both B- and beta-hematin at pH 4-5 is inhibited. Under certain conditions, the effect of added carboxylic acids on pigment formation is suggested to be due, at least in part, to the prevention of initial hydrogen bonding among FP carboxyl groups. For both B- and beta-hematin, branched and cyclic macromolecular structures are proposed involving linkages between an FP iron and a side-chain carboxylate group of another FP, in addition to hydrogen bonds between FP carboxyl groups. B- and beta-hematin are assumed to differ in molecular weight and the extent of bond formation. Possible mechanisms for beta-hematin production from B-hematin and certain relations between the synthetic pigments and the malaria pigment are suggested.

Optical properties of complexes of antimalarial drugs with ferriprotoporphyrin IX in aqueous medium. I. The system ferriprotoporphyrin IX-quinine.

Circular dichroism (CD) and light-absorption spectra of the system ferriprotoporphyrin IX-(-)-quinine as measured at 26 to 27 degrees C in dilute aqueous solutions of both pH 7.4 and 11.5 are reported. The CD spectra changed significantly with time during measurements extending for many days. By CD titrations, a predominant mole ratio of 1:1 in the complex is indicated. Sedimentation velocity and viscosity measurements, carried out under similar conditions, suggest the formation of large and specific aggregates at both pH 7.4 and 11.5-12. The large CD bands observed in the Soret region indicate chiral interactions between FP molecules arrayed within aggregates of FP-Q complexes. The observed time dependence of the ellipticities is considered to be due to dynamic changes in the steric arrangement of interacting units within the aggregates.

Optical properties of complexes of antimalarial drugs with ferriprotoporphyrin IX in aqueous medium. II. The system ferriprotoporphyrin IX-quinidine.

Light absorption and circular dichroism (CD) were recorded at 26 to 27 degrees C in dilute aqueous solutions (10(-4) M) of ferriprotoporphyrin IX (FP) in the presence of (+)-quinidine. In contrast to the appearance of relatively small and positive CD bands between pH 7 and 10, two bands of opposite sign, having unusually large molar ellipticities of the order of 10(6) deg X cm2 X dmol-1 FP were observed in the Soret region near 400 nm at pH 11.0-11.5. This unique complex A was formed only slowly over periods of hours or days at 26 degrees C. By lowering the pH of Complex A below 10, under certain conditions, an "enantiometric" mirror-image CD spectrum, with all bands having opposite sign to Complex A, was obtained in the range of 650 to 300 nm (Complex B), while the light-absorption spectra of Complexes A and B were similar. The formation of Complex A was inhibited at mole fractions of FP greater than 0.5. Also, this complex was not measurably formed at low salt concentrations. Ultracentrifugation measurements of the complex solutions indicated the presence of very high aggregates. Possible interpretations of the optical properties observed are based on interactions between FP molecules, which are assumed to be arrayed chirally within aggregates of FP with quinidine. A comparison between quinine and quinidine complexes of FP is presented.

Interaction of ferriprotoporphyrin IX with the antimalarials amodiaquine and halofantrine.

Light-absorption spectra in the range of 350 to 650 nm of ferriprotoporphyrin IX (ferriheme), both in the presence and absence of either amodiaquine or halofantrine, have been compared in aqueous solution (pH 7.4) at room temperature. In the presence of the drugs, a distinct absorption maximum in the region of 600 to 610 nm, a shoulder near 490 to 500 nm, a maximum around 393 nm and a shoulder near 370 nm have been recorded. The spectral characteristics observed differ significantly from those of free ferriheme under similar conditions and indicate complex formation between ferriheme and either amodiaquine or halofantrine. Analogous absorption spectra were also described previously for complexes of ferriheme with either chloroquine, quinine or quinidine. The present results are considered to be consistent with a receptor action of ferriheme suggested previously and therefore remove recent objections to this hypothesis.

B-hematin.

Very rapid production in vitro of a particular type of hematin (B-hematin) with high yield at physiological pH and temperature is presently reported, using a simple chemical system including an organic acid in the absence of any enzyme. B-hematin is characterized by infrared spectra and solubility properties. The antimalarial drug chloroquine practically prevents formation of this hematin under the conditions used. It has recently been suggested that the purified malaria pigment hemozoin is identical with beta-hematin, a form of ferriprotoporphyrin IX sparingly soluble in different solvents which is prepared under non-physiological conditions. Relations between B- and beta-hematin and possible mechanisms of hemozoin formation and antimalarial drug action, are discussed.

On the preparation of beta-haematin.

Synthetic beta-haematin is considered to be identical, or at least very similar to, purified malaria pigment. Methods for its preparation use ferriprotoporphyrin IX at acid pH in the presence of acetic acid at different concentrations and degrees of ionization, elevated temperatures and long reaction times. Here we show that certain widely used reaction conditions, involving very high concentrations of acetic acid/acetate mixtures, do not produce substantial amounts of polymeric beta-haematin on immediate isolation of the reaction products, but only during prolonged drying of the products at 37 degrees C after washing with water. Alternative, more convenient methods of preparation of pure beta-haematin are suggested.

Alcohol-water as a novel medium for beta-hematin preparation.

It is shown that alcohol-water mixtures (e.g., 43 wt% ethanol) provide a suitable medium for the efficient production in high yield of beta-hematin from ferriprotoporphyrin IX (FP), as measured by infrared spectroscopy. Previous and present light-absorption data of FP, obtained under specific acid conditions in an ethanol-water medium, suggest the presence of FP monomers, which are considered to promote the reaction leading to beta-hematin. Other aspects of the mechanism of reaction are discussed.