Reduction of ferriprotoporphyrin IX to the ferroderivative by copoly(4-vinylpyridine-N-(p-vinylbenzyl)-3-carbamoyl-1,4-dihydropyridine) in dimethyl sulfoxide

The copolymer which has both ligand sites (4-vinylpyridine) and redox sites (N-(p-vinylbenzyl)-3-carbamoyl-1,4-dihydropyridine) was synthesized by the dithionite reduction of the copoly(4-vinylpyridine-N-(p-vinylbenzyl)-3-carbamoylpyridinium chloride) and the reduction of a central ferric-iron of ferriprotoporphyrin IX by the above-described copolymer was studied spectrophotometrically in dimethyl sulfoxide. The rate of the reduction by the copolymer was much faster than by N-benzyl-3-carbamoyl-1,4-dihydropyridine. This acceleration by the copolymer could be explained by the intramolecular reduction of ferriprotoporphyrin IX which was coordinated by the pyridine residue of the copolymer.     

Exchange rates of pyridine on ferro- and ferriprotoporphyrin (IX) dimethylester in chloroform.

Nuclear magnetic resonance line-widths data have been used to determine the rate of solvent exchange from the first coordination sphere of ferro-and ferriprotoporphyrin(IX) dimethylester (Fe-PPD) in pyridine/chloroform. The average values of kinetic parameters for pyridine (PY) exchange indicate an SN2 mechanism tor Fe(III)-PPD(ΔH^&;#; = 36 kJ · mol⁻¹ ; ΔS^&;#; = −53 J·mol⁻¹K⁻¹; T_M(298 K) = 0.07 msec) and an SNI mechanism for Fe(II)-PPD (ΔH^&;#; = 67 kJ·mol⁻¹; ΔS^&;#; = 42 J · mol⁻¹K⁻¹; T_M(298 K) = 0.06 msec). Parallel to the accelerated ligand exchange rate at rising temperatures a redistribution of the electrons causing a transition of the metal porphyrin from the low-spin state to the high-spin state is observed. Enthalpy and entropy of the thermodynamic equilibrium between low- and high-spin Fe-PPD have been determined from experimental values of the average magnetic moment. A mean lifetime of low-spin Fe(III)-PPD was estimated from line. widths changes (T_L→H(298 K)≈ 20 msec) and the corresponding activation parameters have been obtained (ΔH^&;#;_L→H(298 K) = 26 kJ · mol⁻¹; ΔS^&;#;_L→H(298K) = −125 J · mol⁻¹K⁻¹).     

Oxygenation of cobalt(II) protoporphyrin IX dimethyl ester bound to copolymer of 4-vinylpyridine and styrene

The polymeric cobalt(II) porphyrin complexes were prepared from cobalt(II) protoporphyrin IX dimethyl ester(Co(II)P) and copolymers of 4-vinylpyridine and styrene(PSP), and their binding ability of molecular oxygen was studied in toluene solution. The five- and six-coordinate structure of CoP-PSP complexes were confirmed by esr spectra. The esr parameters for the CoP-PSP complexes were not affected by the molecular weight and the vinylpyridine-unit content of PSP-ligand. The 1:1 dioxygen-Co complex was reversibly formed when the solution of CoP-PSP was exposed to oxygen atmosphere at low temperature. While the visible spectra and esr parameters for the dioxygen complexes of CoP-PSP were the same as those of the CoP-pyridine complex, the equilibrium constant for the oxygen binding increased with the vinylpyridine-unit content of the PSP-ligand. The larger entropy change was observed for the oxygenation in the CoP-PSP system especially, of which the vinylpyridine-unit content was large.     

Involvement of lipids in ferriprotoporphyrin IX polymerization in malaria.

Approximately 70% of the initial ferriprotoporphyrin IX polymerizing activity in cell-free preparations of erythrocytes infected with Plasmodium berghei was recovered in a chloroform extract. No polymerizing activity remained in the residue. In studies to identify substances that promote FP polymerization, arachidonic, linoleic, oleic, and palmitoleic acids, 1-mono- and di-oleoylglycerol, and the detergents, SDS, Tween 80, and n-octyl-glucopyranoside, were active. Tri-oleoylglycerol, cholesterol, di-oleoylphosphatidylethanolamine, and stearic and palmitic acids were inactive. The model lipid, mono-oleoylglycerol (250 nmol), co-precipitated with FP from a 0.09 M acetate medium at pH 5 and promoted the polymerization of 215 nmol (61%) of the ferriprotoporphyrin IX in the precipitate during a 24-h incubation at 37 degrees C. Polymerization was maximal at pH 5, it was approximately linear for 2 h, and it continued at a decreasing rate for 24 h. The polymer contained exclusively ferriprotoporphyrin IX (97+/-1.3%, mean+/-S.E., n=4) and exhibited the solubility and the electronic absorption and infrared spectral characteristics of the sequestered ferriprotoporphyrin IX of hemozoin. Detergents presumably promote polymerization in an acid medium by helping to dissolve monomeric FP. We suggest that unsaturated lipids co-precipitate with FP in the parasite's acidic food vacuole and also dissolve sufficient monomeric FP to allow polymerization.     

The state of ferriprotoporphyrin IX in malaria pigment

To evaluate the state of ferriprotoporphyrin IX (FP) in malaria pigment, mouse erythrocytes infected with Plasmodium berghei NYU-2 parasites were lysed by hypotonic shock, and hemoglobin and other soluble material were removed by extensive washing. The amount of FP recovered in the insoluble pellet was 2.1 mumol/ml of packed infected erythrocytes, of which approximately 1% was attributable to hemoglobin contamination. This crude preparation then was digested with a nonspecific protease from Streptomyces griseus and extracted with chloroform/methanol. The residue of insoluble dark brown material had the spectral and solubility properties characteristic of the FP of malaria pigment, and various different preparations contained from 82 to 99% of FP by weight. By elemental analysis, highly purified preparations contained no chlorine and had an oxygen content consistent with 1 mol of hydroxyl/mol of FP (oxygen content: calculated, 12.6%; found, 12.5%). In comparison to hematin purchased from Sigma, which had a measured oxygen content of 14.7%, the low oxygen form of hematin purified from malaria pigment was remarkably less soluble in ethanol, 3% sodium bicarbonate, and chloroform.     

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.     

Oxygen binding to cobalt(II) proto-, deutero- and meso-porphyrin IX dimethyl ester complexes in organic solvents

The binding of oxygen to cobalt(II) meso, deutero- and proto-porphyrin IX dimethyl esters complexed with pyridine or 2-methylimidazole was investigated at −10°–−60°C in toluene or DMF solution, and the thermodynamic data related with the binding were presented. The oxygen affinity of cobalt meso-porphyrin complex was larger by the factor of 2.0–1.4 than those of the other complexes where oxygen affinities were not explained by a simple electron-withdrawing capability of 2,4-substituents of the porphyrin ring. The oxygen binding property was, generally, dependent on the solvent, suggesting that the solvation affects appreciably the oxygen binding to the complexes.The oxygen affinities of cobalt porphyrin complexes in various organic solvents were compared with those of their apomyoglobin complexes. The differences of oxygen affinities between both systems decreased with increasing the size of 2,4-substituents, and it was in the following order on 2,4-substituted porphyrins: Deutero ⪢ Proto ⪢ Meso. It was suggested that the 2,4-substitutent effect on the oxygen affinity of cobalt myoglobin complexes was not only caused by the direct electronic effect on the central cobalt atom, but also controlled by the stereochemical interaction between apomyoglobin and the porphyrin.     

Proton NMR study of the interaction of tin(IV) protoporphyrin IX monomers and dimers with apomyoglobin.

Events during the reconstitution of apomyoglobin to form the holoprotein were probed by porphyrin-metal substitution. Thus interactions between tin(IV) protoporphyrin IX (SnPP) and equine apomyoglobin (apoEqMb), and between tin(IV) protoporphyrin IX dimers [(SnPP)2] and apoEqMb, were observed by 1H NMR and optical absorbance spectroscopic techniques. The chief advantages of using SnPP are that products and intermediates can easily be related to SnPP.EqMb which has been studied [Deeb, R.S., & Peyton, D.H. (1991) J. Biol. Chem. 266, 3728-3733] and that at least one step during reconstitution is slowed considerably as compared to heme. Reactions of apoEqMb with SnPP and (SnPP)2 produce different intermediates, although the final product, SnPP.EqMb, is the same for each. An intermediate observed for reaction of SnPP with apoEqMb at pH 10 is in exchange with free SnPP, with the observed rate constant koff approximately 1 s-1. meso-Proton resonances were assigned for this intermediate by correlation to SnPP resonances via chemical exchange. The intermediate observed for reaction of (SnPP)2 with apoEqMb at pH 7.5 is heterogeneous. The reaction of either SnPP or (SnPP)2 with apoEqMb at neutral pH produces another species which may be the alternate porphyrin-insertion isomer arising from a 180 degree rotation about the alpha, gamma-meso axis of the porphyrin. Although optical absorbance spectroscopy of the Soret region shows evidence for each reaction, only in combination with 1H NMR are the various processes assigned.     

Interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX, a solid state spectroscopy study

To investigate the nature of binding of quinoline antimalarial drugs to heme and to extract experimental evidence for this binding, the interaction of ferriprotoporphyrin IX (FP) with chloroquine and quinacrine (both of which have a similar side chain) and quinoline methanol antimalarials quinine and mefloquine has been studied using IR and NIR-Raman spectroscopy in the solid state. Attenuated total reflectance infrared spectroscopic data clearly show that heme in chloroquine-FP complex is not μ-oxo dimeric indicating that the hypothesis that chloroquine binds to FP μ-oxo dimer with a stoichiometry of 1 chloroquine:2 μ-oxo dimers is not valid in the solid state. Moreover, the first vibrational spectroscopy evidence is presented for the formation of hydrogen bonding between a propionate group of heme and the tertiary amino nitrogen of chloroquine and quinacrine. Raman spectroscopy data does not provide any evidence to support the formation of a similar salt bridge in the complexes of FP with quinine and mefloquine; however, it suggests that the interaction of these drugs with FP happens through coordination of the Fe(III) center of the porphyrin to the 9-hydroxy group of the drug.     

Electronic spectra for nitrosyl(protoporphyrin IX dimethyl ester)iron(II) and its complexes with nitrogenous bases as model systems for nitrosylhemoproteins

Soret and visible absorption spectra for nitrosyl(protoporphyrin IX dimethyl ester)iron(II) (Fe(PPIXDME)(NO] and its complexes with nitrogenous bases (imidazoles, pyridines, aliphatic amines, and cyclic secondary amines) as model systems for nitrosylhemoproteins have been measured in various solvents. As the solvent polarity increases, the Soret and visible absorption bands for the five-coordinate Fe(PPIXDME) (NO) were shifted to shorter wavelengths. Accompanying the coordination of a nitrogenous base to the vacant axial position of Fe(PPIXDME)(NO), the Soret band becomes sharp and the band maximum is shifted to longer wavelengths. The band positions for the six-coordinate Fe(PPIXDME)(NO)(Base) complex are not sensitive to the pi-bonding ability of the axial ligand trans to NO group. The electronic spectra of five-coordinate Fe(PPIXDME)(NO) and six-coordinate Fe(PPIXDME)(NO)(Base) complexes are interpreted in relation to the structural information. The comparison of the spectra for model systems with those for nitrosylhemoproteins is discussed.     

The reaction of the superoxide ion with iron (3) protoporphyrin IX dimethyl ester perchlorate: evidence for a stable dioxygen complex

The reaction of superoxide ion with one equivalent of iron(III)protoporphyrin IX dimethyl ester perchlorate in NN-dimethylformamide at ?50°C yields a complex with an absorption spectrum comparable to that of oxymyoglobin. The complex decomposes at ?10° to iron(II)protoporphyrin IX dimethyl ester which does not react with oxygen.     

Regulation of human erythrocyte glyceraldehyde-3-phosphate dehydrogenase by ferriprotoporphyrin IX

Erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) is a glycolytic enzyme containing critical thiol groups and whose activity is reversibly inhibited by binding to the cell membrane. Here, we demonstrate that the insertion of ferriprotoporphyrin IX (FP) into the red cell membranes exerts two opposite effects on membrane bound G3PD. First, the enzyme is partially inactivated through oxidation of critical thiols. Dithiothreitol restores part of the activity, but some critical thiols are irreversibly oxidized or crosslinked to products of FP-induced lipid peroxidation. Second, G3PD binding to the membrane is modified and the enzyme is activated through displacement into the cytosol and/or release from its binding site.