Clear Isosbestic Points Research Articles

The formation and structure of aggregates in hemicyanine monolayers

Absorption spectra of a hemicyanine monolayer on a sodium sulfate subphase were recorded as a function of surface pressure. Upon compression, the intensity of the monomer band is reduced and two new bands are observed. The results from principal factor analysis of the spectra and the clear isosbestic points indicate that the two bands are due to exciton splitting from an aggregate structure with two molecules per unit cell. By comparison with spectra obtained previously, the influence of counterions and the dilution of the dye monolayer on the formation and structure of the aggregates is investigated. The addition of sulfate counterions to the subphase raises the threshold surface concentration for the formation of aggregates, and produces an aggregate structure in which one long molecular axis is oriented closer to the surface normal. Dilution of the dye monolayer results in a small change to the aggregate structure. Furthermore, if it is assumed that the dye-diluent monolayer is inhomogeneous, dilution has no significant effect on the surface concentration dependence of the formation of aggregates.

The Mechanism of Electron Donation to Molecular Oxygen by Phagocytic Cytochrome b558

Phagocytic cytochrome b558 is a unique heme-containing enzyme, which catalyzes one electron reduction of molecular oxygen to produce a superoxide anion with a six-coordinated heme iron. To clarify the mechanism of the superoxide production, we have analyzed oxidation-reduction kinetics of cytochrome b558 purified from porcine neutrophils by stopped-flow and rapid-scanning spectroscopy. Reduced cytochrome b558 was rapidly reoxidized by O2 showing spectral changes with clear isosbestic points, which were also observed during the reduction of ferric cytochrome b558 with Na2S2O4 under anaerobic conditions. The single turnover rate for the reaction with O2 linearly depended on the O2 concentration but was not affected by addition of CO. The rate of the reaction decreased with an increase of pH giving a pKa of 9.7. Under complete anaerobic conditions, ferrous cytochrome b558 was oxidized by ferricyanide at a rate faster than by O2. The thermodynamic analysis shows that the enthalpic energy barriers for the reactions of cytochrome b558 are significantly lower when compared to the autoxidation of native and modified myoglobins through the formation of the iron-O2 complex. These findings are most consistent with the electron transfer from the heme to O2 by an outer-sphere mechanism.

Proton interactions in the resting form of cytochrome oxidase

The effect of pH on the near-UV absorption spectrum of cytochrome oxidase has been examined. Several lines of evidence implicate a proton binding site that can modulate the optical properties of cytochrome alpha 3 in the resting enzyme. Changing the pH within the range 6.5-10.5 was found to reversibly shift the position of the Soret band over an 11-nm range. The lower pH values caused a progressive blue shift in the Soret band, whereas the high-pH range promoted a gradual red shift. Limiting band positions were approximately 416 and 427 nm. The incubation time required to reach a stable band position varied somewhat as did the actual extent of the shift. In most cases, the shift was associated with an isosbestic point. A pH titration profile for the apparent equilibrium position of the Soret band was obtained. Nonlinear least-squares fitting to a scatter plot, assuming a single acid/base group, showed an apparent pKa of 7.8. Magnetic circular dichroism (MCD) spectra of the low-pH form at 416 nm, the high-pH form at 427 nm, and the cyanide derivative at 428 nm were compared. No evidence of a high-pH-dependent low-spin transition or a change in the redox state of cytochrome a3 was found, confirming earlier work [Baker, G. M., Noguchi, M., & Palmer, G. (1987) J. Biol. Chem. 262, 595-604]. Subtraction of ferricytochrome a [spectrum taken from Vanneste, W. H. (1966) Biochemistry 5, 838-848] from a series of blue-shifting spectra showed a band at 414 nm that progressively gained amplitude and a band at 430 nm that correspondingly lost amplitude. A series of red-shifting spectra showed the opposite behavior with a clear isosbestic point being evident in both cases. The difference extinction change at 414 and 430 nm depended linearly on the position of the Soret band, both showing a reversible dependence on pH. The 430-nm band is noted to be unusually red-shifted for high-spin ferric heme a. An additional, pH-insensitive band was observed at 408-410 nm which was eliminated by treatment with cyanide. The kinetics of the pH-induced blue shift and red shift were obtained at 416 nm by using dual-wavelength method and found to be biphasic, despite the occurrence of an isosbestic point.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of chloride on the redox and EPR properties of myeloperoxidase.

Myeloperoxidase was purified from human polymorphonuclear leukocytes and the effect of chloride upon the EPR and potentiometric properties was studied. The redox titration between the ferrous and ferric states of the enzyme yielded n = 1 Nernst plots between pH 9 and 4, with clear isosbestic points in the optical spectra during the redox change. The midpoint potential (Em) between the ferric and ferrous forms of the enzyme exhibited a pH-dependent change between pH 4 and 9, and the effect of added chloride ion indicated that Cl- competed with OH- for a binding site on the enzyme. Interestingly, the pH dependence of the Em indicated that the overall redox reactions of the enzyme was: ferric myeloperoxidase + 2e- + 1H+ = ferrous myeloperoxidase. Myeloperoxidase exhibited a rhombic high spin EPR signal which exhibited reduced rhombicity upon the binding of chloride. Our results strongly suggest that chloride binds to the sixth coordination position of the chlorin iron in myeloperoxidase by replacing the water which is the sixth ligand in the resting state. It is also concluded that the two iron centers are identical and that there is no interaction between them.

Interaction of the antileukemic alkaloid, 2-hydroxy-3,8,9-trimethoxy-5-methylbenzo[ c]phenanthridine (fagaronine), with nucleic acids

The novel alkaloid 2-hydroxy-3,8,9-trimethoxy-5-methylbenzo[ c]phenanthridine was isolated from the roots of Fagara zanthoxyloides, an indigenous plant of Ghana, structurally characterized and assigned the trivial name fagaronine. Fagaronine is a potent antileukemic agent. We presently report a detailed isolation procedure and the results of studies designed to examine its mechanism of action. Admixture of calf thymus DNA produced a hypsochromic shift in the absorption spectra of fagaronine, with a remarkable increase in intensity in the range of 400 nm. The extent of the spectral alterations were dependent on the concentration of DNA which was added, and clear isosbestic points, indicative of a single mode of interaction, were generated. As determined by gel filtration column chromatography, the DNA-fagaronine complex formed with an association constant of 4.7 × 10 4 M −1 and the maximum extent of binding was 1.0 fagaronine molecule per 4.5 base pairs. Contrary to previous literature reports, the binding of fagaronine was not limited to regions of DNA containing adenine and thymine. Isosbestic points were observed following admixture with poly-(dA-dT), as well as poly(dG) · poly(dC). The most likely mode of interaction is intercalation. As judged by spectral titrations, fagaronine could also interact with ribonucleic acids. As compared with single-stranded homopolymers [e.g., poly(A), poly(U)], the interaction with double-stranded species [e.g., poly(A) · poly(U), poly(I) · poly(C)] was more extensive. Although the spectra differed from those obtained with deoxyribonucleic acids, single isosbestic points could be generated. Additionally, utilizing Salmonella typhimurium strain TM677, fagaronine was found to be bactericidal but not mutagenic. The bactericidal activity was enhanced about 1000-fold in the presence of a 9000 × g supernatant fraction derived from rat liver and the activating component was further characterized as being heat labile and localized in the microsomal subfraction. Preliminary investigations did not support the notion that fagaronine was metabolized to an active species responsible for this effect. When KB cells were treated with fagaronine, it was also found to be cytotoxic. In contrast, N-demethylfagaronine was not bactericidal or cytotoxic, nor could it effectively interact with DNA. Thus, the cationic (quaternary) nitrogen atom of fagaronine is of requisite importance, and a key mechanism of cytotoxic activity may be interaction with cellular nucleic acids.