Absorption Spectra Of Methemoglobin Research Articles

Noninvasive photoacoustic microscopy of methemoglobin in vivo.

Due to the various causes of methemoglobinemia and its potential to be confused with other diseases, in vivo measurements of methemoglobin have significant applications in the clinic. Using photoacoustic microscopy (PAM), we quantified the average and the distributed percentage of methemoglobin both in vitro and in vivo. Based on the absorption spectra of methemoglobin, oxyhemoglobin, and deoxyhemoglobin, three wavelengths were chosen to differentiate methemoglobin from the others. The methemoglobin concentrations calculated from the photoacoustic signals agreed well with the preset concentrations. Then we imaged the methemoglobin percentage in microtubes that mimicked blood vessels. Average percentages calculated for five samples with different methemoglobin concentrations also agreed well with the preset values. Finally, we demonstrated the ability of PAM to detect methemoglobin in vivo in a mouse ear. Our results show that PAM can quantitatively image methemoglobin distribution in vivo.

Effects of Bacterial Endotoxin on Human Cross-Linked and Native Hemoglobins

Previous investigations have demonstrated that hemoglobin (Hb) is a binding protein for bacterial endotoxin (lipopolysaccharide, LPS) and that the structure and biological activity of LPS are altered in the presence of Hb. In the present study, the influence of LPS on the structure of native human HbA0 and covalently cross-linked Hb (alpha alpha Hb) was studied by analyzing the absorption and circular dichroic spectra of Hb in the wavelength region of 200-650 nm. Incubation of oxyHb with each of several LPSs resulted in a decrease in the intensity of the major Soret band at 414 nm with a shift in the maximum peak to 410 nm, decreases in the intensities of the major visible region peaks at 541 and 577 nm, and the appearance of increased absorbance in the visible region in the range of 630 nm. The resultant spectra are characteristic of methemoglobin formation. These spectral changes were time-dependent and LPS-concentration-dependent. Production of methemoglobin was prominent with chemically modified, partially deacetylated rough LPS, and was observed to a lesser extent both with native, complete rough and with native smooth LPSs. The influence of LPS on the absorption spectrum of methemoglobin also was directly tested. The conversion of methemoglobin to hemichrome in the presence of LPS was demonstrated and was shown to be reversible. Analysis of circular dichroic spectra of Hb demonstrated LPS-induced spectral changes in the visible and Soret regions consistent with the production of a substantial quantity of metHb, but did not demonstrate any alteration in the far-UV region (210-240 nm). Moreover, Hb oxygen affinity was only slightly altered after incubation with any of several LPSs. In conclusion, analyses of absorption and circular dichroic spectra reveal the potential of LPS to produce a facilitated oxidation of both alpha alpha-cross-linked human Hb and native human HbA0, without substantial changes in the secondary structure of the globin.

The determination of cyanide in water and biological tissues by methemoglobin

The authors developed a simple and sensitive method for the determination of cyanide using spectrophotometry to measure changes caused by cyanide in the absorption spectra of methemoglobin. Met-hemes in the hemoglobin molecules react with cyanide rapidly to form a cyanomethemoglobin complex, even at low concentrations of cyanide, in a stoichiometric manner. After adding cyanide, the absorption spectra of methemoglobin changed with a typical decrease in absorbance at 630 nm, and an increase in absorbance at 540 nm. These changes were closely correlated with the concentrations of cyanide ion. From the results, it was shown that 0.2 μg/ml of cyanide ion in water and 0.4 μg/ml in biological tissues can be detected by this method. The method may be useful for the convenient and sensitive detection of cyanide in waste water and biological tissues and fluids.

Enhanced production of ethylene from methional by iron chelates and heme containing proteins in the system consisting of quinone compounds and NADPH-cytochrome P-450 reductase

The addition of iron chelates or heme containing proteins to the systems consisting of NADPH-cytochrome P-450 reductase and quinone compounds, such as vitamin K 3 (menadione), adriamycin, tetrahydropyranyladriamycin, daunomycin, aclacinomycin A, carbazilquinone, and mitomycin C, showed the enhanced production of ethylene from methional. In the vitamin K 3 system, the effective iron chlates were Fe(II)-EDTA, Fe(II)-ADP, Fe(II)-bleomycin A 2, and hemin, and the effective iron containing proteins were methemoglobin, myoglobin, ferredoxin, and partially purified cytochromes P-450, P-420, and b 5, and the reversed effects were observed by horse radish peroxidase and sulfite reductase from yeast. In the system consisting of aclacinomycin A and methemoglobin, the ethylene production was potently inhibited by radical scavengers, such as Tiron, Tirs, thiourea, and KI, and weakly inhibited by some other scavengers. In the system containing vitamin K 3 and methemoglobin, the ethylene production was potently inhibited by catalase, but partially by superoxide dismutase, KCN, and NaN 3. In this system, the absorption spectrum of methemoglobin was immediately changed to oxyform and quenched with time, and catalase protected the decrement of the spectrum. The addition of hydrogen peroxide or cumene hydroperoxide to methemoglobin also produced ethylene from methional.

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.

Effects of p-hydroxymercuribenzoate binding on the visible absorption spectrum of methemoglobin.

The binding of p-hydroxymercuribenzoate to human methemoglobin causes a perturbation of the visible heme abosrption spectrum which is expressed by an increase in absorbance in the high spin band regions, 480 to 510 nm and 590 to 640 nm, concomitant with a decrease in absorbance in the alpha- and beta-band absorption regions. The pH dependence of the p-hydroxymercuribenzoate-induced difference spectrum can be accounted for quantitatively by a 5% shift toward higher spin of the aquo form of methemoglobin, a 15% shift toward higher spin of the hydroxide form, and a shift in the apparent pKa for the water to hydroxide transition from 7.92 to 8.04 when mercurial is bound. The rate of these heme abosrbance changes is consistent with the rapid second order formation of the beta93 cysteine, mercury-mercaptide bond and does not represent a change due to the dissociation of methemoglobin tetramers into dimers, even though the latter, slow process does follow mercurial binding. The observation of an increase in spin produced by the binding of a reagent which also promotes dimer formation argues strongly against any direct correlation between an increase in spin and the appearance of deoxyhemoglobin-like conformations.