The one-electron oxidation of porphyrins to porphyrin pi-cation radicals by peroxidases: an electron spin resonance investigation

For the first time, the enzymatic one-electron oxidation of several naturally occurring and synthetic water-soluble porphyrins by peroxidases was investigated by ESR and optical spectroscopy. The ESR spectra of the free radical metabolites of the porphyrins were singlets (g = 2.0024, delta H = 2-3 G), which we assigned to their respective porphyrin pi-cation free radicals. Several porphyrins were investigated and ranked by the intensity of their ESR spectra (coproporphyrin III greater than coproporphyrin I greater than deuteroporphyrin IX greater than mesoporphyrin IX greater than Photofrin II greater than protoporphyrin IX greater than uroporphyrin I greater than uroporphyrin III greater than hematoporphyrin IX). The porphyrins were oxidized by several peroxidases (horseradish peroxidase, lactoperoxidase, and myeloperoxidase), yielding the same type of ESR spectra. From these results, we conclude that porphyrins are substrates for peroxidases. The changes in the visible absorbance spectra of the porphyrins during enzymatic oxidation were monitored. The two-electron oxidation product, which was assigned to the dihydroxyporphyrin, was detected as an intermediate of the oxidation process. The optical spectrum of the porphyrin pi-cation free radical was not detected, probably due to its low steady-state concentration.     

Near infrared spectral changes of cytochrome aa3 during potentiometric titrations.

Singular value decomposition (SVD) was used to deconvolute the spectral changes occurring in the near infrared region during potentiometric titrations of cytochrome aa3. Overall oxidized minus reduced difference spectra revealed a broad absorbance feature centered near 830 nm with an apparent Em near 250 mV. However, SVD did not isolate any spectral species with an absorbance centered near 830 nm. It was found that the spectral changes occurring in the wavelength region from 650 to 950 nm were associated mainly with cytochromes a and a3. It was concluded that the absorbance at 830 nm should not be used as an independent measure of the concentration of CuA in cytochrome aa3.     

Quantitative analysis of hemoglobin oxygenation state of rat brain in situ by near-infrared spectrophotometry

The light in the near-infrared region (700-900 nm) was illuminated on the rat head, and absorption spectra were measured with the transmitted light under various conditions. The absorbance changes less than 780 nm were attributable to hemoglobin in the brain tissue, whereas those greater than 780 nm were associated with both hemoglobin and cytochrome oxidase. The changes of oxy- and total (oxy- plus deoxy-) hemoglobin content in the rat head could be monitored quantitatively by expressions of delta A700--1.20 delta A730 and delta A700--1.52 delta A730, respectively. The oxyhemoglobin content in the tissue was decreased as the O2 tension in inspired gas was lowered. At 10% O2 approximately 50% of hemoglobin was deoxygenated. The total hemoglobin content was increased under anoxic conditions. Inhalation of 5% CO2 and intravenous injection of a Ca2+ blocker nicardipine increased the O2 saturation of hemoglobin in the brain. These conclusions were confirmed by measuring the difference absorption spectra in the near-infrared region.     

The kinetics of assembly of normal and variant human oxyhemoglobins

The kinetics of assembly have been monitored spectrophotometrically for normal and variant human oxyhemoglobins in 0.1 M Tris, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4, at 21.5 degrees C. Oxyhemoglobin versus oxy chain static difference spectra were performed and revealed subtle but significant absorption changes in both the visible and Soret regions. Kinetic experiments were performed by rapidly mixing equivalent (in heme) concentrations of alpha and beta A chains and following the change in absorbance at 583 nm with time. Over a protein concentration range of 10-100 microM in heme prior to mixing, these time courses were homogeneous and followed first-order kinetics, yielding a value of 0.069 s-1 for the apparent rate constant of dissociation of oxygenated beta A chain tetramers. Under these conditions, the overall assembly of oxyhemoglobins S (beta 6Glu----Val) and N-Baltimore (beta 95Lys----Glu) were also governed by the rates of dissociation of their respective oxygenated beta S and beta N-Baltimore chain tetramers with the apparent first-order rate constants of 0.044 and 0.15 s-1, respectively. In the Soret region, the alpha, beta monomer combination reaction could be observed if the protein concentration (heme basis) was lowered and if protein nonequivalency (beta chain exceeded alpha chain concentration) mixing experiments were performed. A kinetic oxyhemoglobin A, oxy-alpha, oxy-beta A monomer difference spectrum could be generated, and simple second-order kinetics were observed (415 nm) yielding rate constants of 2.3, 3.3, and 4.8 X 10(5) M-1 s-1 for the assembly of oxyhemoglobins S, A, and N-Baltimore, respectively. To our knowledge, this is the first kinetic study to reveal significant differences between the rate of association of alpha and beta monomers of hemoglobin A and those of two distinctly charged hemoglobin variants.     

Spectrophotometric detection of pentachlorophenol (PCP) in water using immobilized and water-soluble porphyrins

The spectrophotometric properties of porphyrins are altered upon interaction with chlorophenols and other organochlorine pollutants. Meso-tetra(4-sulfonatophenyl)porphyrin (TPPS), zinc meso-tetra(4-sulfonato phenyl)porphyrin (Zn-TPPS), monosulfonate-tetraphenylporphyrin (TPPS1), meso-tri(4-sulfonatophenyl)mono(4-carboxyphenyl)porphyrin (C1TPP), meso-tetra(4-carboxyphenyl)porphyrin (C4TPP), and copper meso-tetra(4-carboxyphenyl)porphyrin (Cu-C4TPP) in solution exhibit a broad absorbance in the range 400-450 nm Soret region. The interaction of the above mentioned porphyrins in solution with pentachlorophenol (PCP) induces a red shift in the Soret spectrum with absorbance losses at 413, 418, 403, 405, 407, and 404 nm, respectively, and the appearance of new peaks at 421, 427, 431, 416, 417, and 416 nm, respectively. The intensity of the Soret spectral change is proportional to the pentachlorophenol concentration with a detection limit of 1, 0.5, 1.16, 1, 0.5, and 0.5 ppb, respectively. The interaction of (C4TPP) and (Cu-C4TPP) in solution with PCP shows to concentration dependent for concentrations less than 4 ppb the dependence was log-linear. However, for concentrations greater than 4 ppb the relation was linear. Monosulfonate-tetraphenylporphyrin immobilized as a monolayer on a Kimwipe tissue exhibits an absorbance peak in the Soret region at 422 nm. The interaction of the porphyrin with PCP induces a red shift in the Soret spectrum with absorbance loss at 419 nm and the appearance of new peaks at 446 nm. The intensity of the Soret spectral change is proportional to the log of PCP concentration. The detection limit with immobilized TPPS1 for PCP is 0.5 ppb. These results suggest the potential for development of spectrophotometric chemosensor for PCP residues in water with detection limits less than US EPA maximum contaminate level (MCL) of 1 ppb. The immobilized TPPS1 on the Kimwipe will make it possible to develop a wiping sensors to monitor the PCP or other pesticides residues on the vegetables or wood products.     

Spectrophotometric and spectrofluorimetric determination of indacaterol maleate in pure form and pharmaceutical preparations: application to content uniformity

Two simple, rapid, sensitive and precise spectrophotometric and spectrofluorimetric methods were developed for the determination of indacaterol maleate in bulk powder and capsules. Both methods were based on the direct measurement of the drug in methanol. In the spectrophotometric merthod (Method I) the absorbance was measured at 259 nm. The absorbance‐concentration plot was rectilinear over the range 1.0–10.0 µg mL^?1 with a lower detection limit (LOD) of 0.078 µg mL^?1 and lower quantification limit (LOQ) of 0.238 µg mL^?1. Meanwhile in the spectrofluorimetric method (Method II) the native fluorescence was measured at 358 nm after excitation at 258 nm. The fluorescence‐concentration plot was rectilinear over the range of 1.0–40.0 ng mL^?1 with an LOD of 0.075 ng mL^?1and an LOQ of 0.226 ng mL^?1. The proposed methods were successfully applied to the determination of indacaterol maleate in capsules with average percent recoveries ± RSD% of 99.94 ± 0.96 for Method I and 99.97 ± 0.81 for Method II. In addition, the proposed methods were extended to a content uniformity test according to the United States Pharmacopoeia (USP) guidelines and were accurate, precise for the capsules studied with acceptance value 3.98 for Method I and 2.616 for Method II. Copyright © 2015 John Wiley & Sons, Ltd.     

Photoacoustic spectra of needles as an indicator of the activity of the photosynthetic apparatus of healthy and damaged conifers

Photoacoustic spectra were measured at chopping frequencies of the excitation light at 22 and 515 Hz in order to detect differences between needles from healthy and damaged conifers ( Abies alba Mill, and Picea abies Karst.) with respect to pigment distribution and possibly also to photosynthetic activity. Fully green needles of healthy trees exhibit photoacoustic spectra at 22 Hz with a maximum in the red absorption region of the chlorophyll. This maximum is lost with increasing damage to needles and chlorophyll breakdown. The photoacoustic spectra at 22 Hz of the damaged needles therefore are characterized by a higher signal in the blue-light region as compared to that in the red-light region. This can be quantified by forming the ratio of the photoacoustic signals at 675 and 475 nm (ratio PA 675/475). The needles of the damaged trees possess a lower photosynthetic activity, as seen from the CO₂ fixation rate and the variable fluorescence (Rfd-values). It is assumed that the changes in the PA-spectra of the needles from damaged trees are the result of this decline of the photosynthetic apparatus and its function. In contrast to the PA-spectrum at 22 Hz the PA-spectra at 515 Hz, where the PA-signals primarily emanate from the epidermal layer, exhibit a different shape with a maximum near 550 nm. In the needles from the damaged trees the PA-signals are higher, particularly in this maximum range of the spectrum, than those of fully green needles from healthy trees.     

The detection of cytochrome oxidase heme iron and copper absorption in the blood-perfused and blood-free brain in normoxia and hypoxia.

The resolution of cytochrome and hemoglobin changes in in vivo rat and cat brains has required studies over wide wavelength ranges (580-1100 nm) with a novel spectroscopic technique using blood-free and blood-perfused brains. Tissue oxygen was varied from physiological levels to 0 and hematocrits were varied from normal to less than 1%. The experimental results were subjected to a multicomponent analysis using the Beer-Lambert law. At normal hematocrits, the oxygen saturation of hemoglobin in the brain was found to be 30-50% in rats and cats, indicating that the optical method responded primarily to the saturation of the venous ends of the capillary beds. With low hematocrits, both brains showed the absorption band of reduced cytochrome c, the iron component of cytochrome aa3, plus the absorption band of the oxidized copper component. In cat brains, the background absorption changed at all wavelengths. Thus, no isosbestic points were observed in the spectra. In rat brains, however, they were readily observed. The "overtones" of water absorption in the NIR region were found to be significant in the difference spectra of the cat brain, but not in the rat brain. Parallel absorbance changes in the heme and copper components of cytochrome aa3 were obtained in rat and cat brains during the normoxic-hypoxic transition. The ratio of the iron absorbance at 605 nm to the copper absorbance at 830 nm is much smaller in both brains than the in vitro value due to the shorter path length of photon migration at the shorter wavelengths.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diffuse reflectance spectroscopy of fibrous proteins

UV-visible diffuse reflectance (DR) spectra of the fibrous proteins wool and feather keratin, silk fibroin and bovine skin collagen are presented. Natural wool contains much higher levels of visible chromophores across the whole visible range (700-400?nm) than the other proteins and only those above 450?nm are effectively removed by bleaching. Both oxidative and reductive bleaching are inefficient for removing yellow chromophores (450-400?nm absorbers) from wool. The DR spectra of the four UV-absorbing amino acids tryptophan, tyrosine, cystine and phenylalanine were recorded as finely ground powders. In contrast to their UV-visible spectra in aqueous solution where tryptophan and tyrosine are the major UV absorbing species, surprisingly the disulphide chromophore of solid cystine has the strongest UV absorbance measured using the DR remission function F(R)(∞). The DR spectra of unpigmented feather and wool keratin appear to be dominated by cystine absorption near 290?nm, whereas silk fibroin appears similar to tyrosine. Because cystine has a flat reflectance spectrum in the visible region from 700 to 400?nm and the powder therefore appears white, cystine absorption does not contribute to the cream colour of wool despite the high concentration of cystine residues near the cuticle surface. The disulphide absorption of solid L: -cystine in the DR spectrum at 290?nm is significantly red shifted by ~40?nm relative to its wavelength in solution, whereas homocystine and lipoic acid showed smaller red shifts of 20?nm. The large red shift observed for cystine and the large difference in intensity of absorption in its UV-visible and DR spectra may be due to differences in the dihedral angle between the crystalline solid and the solvated molecules in solution.     

Redox-induced conformational changes in myoglobin and hemoglobin: electrochemistry and ultraviolet-visible and Fourier transform infrared difference spectroscopy at surface-modified gold electrodes in an ultra-thin-layer spectroelectrochemical cell.

Using suitable surface-modified electrodes, we have developed an electrochemical system which allows a reversible heterogeneous electron transfer at high (approximately 5 mM) protein concentrations between the electrode and myoglobin or hemoglobin in an optically transparent thin-layer electrochemical (OTTLE) cell. With this cell, which is transparent from 190 to 10,000 nm, we have been able to obtain electrochemically-induced Fourier-transform infrared (FTIR) difference spectra of both proteins. Clean protein difference spectra between the redox states were obtained because of the absence of redox mediators in the protein solution. The reduced-minus-oxidized difference spectra are characteristic for each protein and arise from redox-sensitive heme modes as well as from polypeptide backbone and amino acid side chain conformational changes concomitant with the redox transition. The amplitudes of the difference bands, however, are small as compared to the total amide I absorbance, and correspond to approximately 1% (4%) of the reduced-minus-oxidized difference absorbance in the Soret region of myoglobin (hemoglobin) and to less than 0.1% of the total amide I absorbance. Some of the bands in the 1560-1490-cm-1 spectral regions could be assigned to side-chain vibrational modes of aromatic amino acids. In the conformationally sensitive spectral region between 1680 and 1630 cm-1, bands could be attributed to peptide C = O modes because of their small (2-5 cm-1) shift in 2H2O. A similar assignment could be achieved for amide II modes because of their strong shift in 2H2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectrophotometry of hemoglobin: a comparison of dog and man

1. The absorptivity at 540 nm of hemiglobincyanide (epsilon 540HiCN) from dog blood was determined on the basis of iron and found to be within the range formerly obtained for human hemoglobin. 2. Consequently, epsilon 540HiCN = 11.0, the established value for human hemoglobin, may be used for dog hemoglobin. 3. On this basis the absorption spectra of oxyhemoglobin, de-oxygenated hemoglobin, carboxyhemoglobin, hemiglobin (methemoglobin) and hemiglobincyanide were determined for dog hemoglobin. 4. No significant differences were found between dog and human hemoglobin, except that dog hemiglobin binds less OH- as reflected in a difference between the absorption spectra of dog and human hemiglobin at the same pH.     

Solution studies on heme proteins. Circular dichroism and optical rotation of Glycera dibranchiata hemoglobins

Circular dichroism (CD) and optical rotatory dispersion (ORD) spectra of several liganded derivatives of the monomer and polymer hemoglobin components of the marine annelid, Glycera dibranchiata were measured over the wavelength range 650--195 nm. The differences observed between the monomer and polymer components for the heme dichroic bands in the visible, Soret and ultraviolet wavelength regions seem to result from changes in the heme environment, geometry and coordination state of the central heme iron in these proteins. Within the Soret region, the liganded derivatives of the monomer hemoglobin exhibit predominantly negative circular dichroic bands. The heme band at 260 nm is also absent for the monomer hemoglobin. The ORD and CD spectra in the far-ultraviolet, peptide absorbing region suggest also differences in the alpha-helix content of the monomer and polymer hemoglobins. The values for the single-chain G. dibranchiata hemoglobin are in the expected range (about 70% alpha-helix) as predicted by the X-ray structure of this protein. The lower estimates of the alpha-helix content for the polymer hemoglobin (approx. 50%), may reflect the differences in amino acid composition, primary structure and polypeptide chain foldings. Changes in oxidation state and ligand binding appears to have no pronounced effect on the helicity of either the monomer or polymer hemoglobins. The removal of the heme moiety from the monomer hemoglobin did result in a major decrease in its helix content similar to the loss of heme from myoglobin.     

Effects of some divalent metal ions on the aging phenomenon of hematoporphyrin and photofrin II

Dilute aqueous solutions of hematoporphyrin (Hp) and its derivative (Hpd or PF II) have been found to undergo a transformation (aging) on keeping at room temperature leading to (i) shift of the Soret band from 395 nm to 405 nm, (ii) disappearance of visible bands I (610 nm) and IV (503 nm) and (iii) shift of the first emission band from 615 nm to 580 nm. The transformation was concentration dependent. The effects of concentration and temperature on the absorption spectra were much more pronounced in Hp than in Photofrin II (PF II). Variation of pH resulted in changes in the relative intensities of the absorption bands, possibly due to formation of different ionic species at different pH. The rate of transformation was accelerated in the presence of Zn ions (0.01 microM) and considerably increased at higher (50 microM) concentration. The effect of Cu ions was different from the effect of aging. It formed the metal-chelate even when present in very small amounts. The results (absorption and fluorescence analysis) suggest that in dilute solutions (conc. less than or equal to 2 microM) of Hp and PF II, Zn ions present in glass and water as impurity, deform the porphyrin nucleus leading to changes in the conjugated ring symmetry and hence changes in the absorption and fluorescence spectra, while in higher concentrations (greater than 2 microM) it forms the metal chelate as evidenced by their absorption and fluorescence spectra.     

Use of photoacoustic imaging for monitoring vascular disrupting cancer treatments

Vascular disrupting agents disrupt tumor vessels, blocking the nutritional and oxygen supply tumors need to thrive. This is achieved by damaging the endothelium lining of blood vessels, resulting in red blood cells (RBCs) entering the tumor parenchyma. RBCs present in the extracellular matrix are exposed to external stressors resulting in biochemical and physiological changes. The detection of these changes can be used to monitor the efficacy of cancer treatments. Spectroscopic photoacoustic (PA) imaging is an ideal candidate for probing RBCs due to their high optical absorption relative to surrounding tissue. The goal of this work is to use PA imaging to monitor the efficacy of the vascular disrupting agent 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) through quantitative analysis. Then, 4T1 breast cancer cells were injected subcutaneously into the left hind leg of eight BALB/c mice. After 10 days, half of the mice were treated with 15 mg/kg of DMXAA and the other half were injected with saline. All mice were imaged using the VevoLAZR X PA system before treatment, 24 and 72 hours after treatment. The imaging was done at six wavelengths and linear spectral unmixing was applied to the PA images to quantify three forms of hemoglobin (oxy, deoxy and met-hemoglobin). After imaging, tumors were histologically processed and H&E and TUNEL staining were used to detect the tissue damage induced by the DMXAA treatment. The total hemoglobin concentration remained unchanged after treatment for the saline treated mice. For DMXAA treated mice, a 10% increase of deoxyhemoglobin concentration was detected 24 hours after treatment and a 22.6% decrease in total hemoglobin concentration was observed by 72 hours. A decrease in the PA spectral slope parameters was measured 24 hours after treatment. This suggests that DMXAA induces vascular damage, causing red blood cells to extravasate. Furthermore, H&E staining of the tumor showed areas of bleeding with erythrocyte deposition. These observations are further supported by the increase in TUNEL staining in DMXAA treated tumors, revealing increased cell death due to vascular disruption. This study demonstrates the capability of PA imaging to monitor tumor vessel disruption by the vascular disrupting agent DMXAA.     

Effect of differences in optical properties of intermediate oxygenated species of hemoglobin A0 on Adair constant determination

Careful evaluation of the so-called isosbestic properties of oxygenated and deoxygenated hemoglobin spectra demonstrates that the spectral changes are not strictly linear with respect to the degree of saturation. In order to quantify the extent of nonlinearity, optical measurements of O2 binding to human hemoglobin were made at different wavelengths in the Soret region approaching the presumed isosbestic point. The results indicate that the extinction coefficient of intermediate oxygenated hemoglobin is 1% less than that of the fully oxygenated hemoglobin, with a resulting 3% (+/- 0.15%) nonlinearity effect on measurements taken at the peak of the oxygenated hemoglobin spectrum (414 nm). The lack of isosbestic conditions allows one to investigate the functional properties of the oxygenated intermediates directly. The small difference in the absorbance of different oxygenated species has practically no influence on the determination of Adair constants at wavelengths removed from the critical isosbestic region.     

Photoinduced changes in adsorption at 800 nm related to photosystem I functioning]

The spectra and kinetics of light-induced absorbance changes in the near-infrared region of subchloroplast fragments enriched by P700 were studied. An increase in absorbancy within the region of 725--900 nm upon illumination was characterized by a maximum around 810 nm and by "shoulders" around 760 and 870 nm. Similar effects of thermal inactivation and low temperatures on the duration of dark recovery of light-induced absorbance changes at 700 nm and within the region of 725--900 nm suggest that the absorbance changes in the near-infrared region are due to photooxidation of P700. The values of P700 differential extinction coefficients at 810 nm are 8,2.10(3) M-1.cm-1 for digitonin fragments and 7,7.10(3) M-1.cm-1 for fragments prepared with the use of diethyl ester. It was shown that the value of midpoint oxidation-reduction potential measured for the absorbance changes at 810 nm (+492 mv) is higher than that measured at 700 nm (+475 mv).     

Polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids oriented in polyvinyl alcohol films

The polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids in unstretched and stretched polyvinyl alcohol films were measured. The intensity ratios of fluorescence bands at 674 nm, 700 nm, 730 nm and 750 nm, and the polarized fluorescence excitation spectra are strongly dependent on light polarization and film stretching. In stretched films, thylakoids exhibit predominantly 674 nm emission. The ratio of photoacoustic signal to absorption is different for light polarized parallel and perpendicular to film stretching. This difference is large in the region of chlorophyll a and carotenoids absorption in which the fluorescence excitation spectra are also strongly dependent on light polarization and film stretching. The observed spectral changes are explained by reorientation of pigment molecules influencing the yield of excitation transfer between different pigments.     

A Method for the Quantitative Assessment of Wound-induced Chitinase Activity in Potato Tubers

A method for the quantitative assessment of chitinase activity was developed. Dilution series of crude potato tuber chitinase extracts were assayed with a colorimetric microtitre plate assay. using CM-chitin-RBV as enzyme substrate. Linearity between absorbance values mea-sured (540 nm) and enzyme concentration was found to be limited to the low concentration range. where depletion of the substrate was no longer limiting. As as absorbance of 0.1 always fell within the concentration range for which absorbance-concentration linearity was valid. one unit of enzyme activity was defined as the amount of enzyme needed to yield and A of 0.1. A more reliable method for the assessment of chitinase activity was established by basing the difinition of enzyme, activity on a concentration rather than on as absorbance value. as was done previously. Using this method, differences in the rate of chitinase induction upon wounding were demonstrated for six commercial potato cultivars.     

A protein and peptide monitor with a hollow-cathode light source.

This communication describes an inexpensive system that will monitor protein and peptide concentration in chromatogram eluates by light absorbance at an adjustable wavelength.Proteins in chromatogram eluate streams are commonly metered for concentration by absorbance measurement at 280 nm. Besides a range of commercially manufactured monitors, there is the apparatus described by Bennett et al. (1) which uses a selectively modulated magnesium lamp. Measurements in the region of 280 nm are of no value, however, when the material does not contain aromatic amino acids. Monitoring then becomes necessary at 230 nm in the region of absorption due to the peptide bond. The common resort in such a case is the standard ultraviolet-visible spectrophotometer, which has the disadvantage of being both unnecessarily elaborate and expensive for the purpose required. The deuterium lamps in these instruments require frequent replacement because of the extended periods of operation, adding to the cost factor.We have investigated the use of a hollow-cathode lamp of the type manufactured for use in an atomic absorption spectrophotometer. These lamps have high stability and a long working life, due to the considerably lower level of power dissipation compared with deuterium lamps. Their emission spectra are discontinuous, but the lamp for the element iron provides adequately strong lines at both 229.5 and 279.2 nm, suitable for a protein monitor.