A near-infrared analysis of water-macromolecule interactions:hydration and the spectra of aqueous solutions of intact proteins.
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Crystallization↗
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Results for “Spectroscopy, Near-Infrared”
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[Laser spectroscopy--a new procedure in fetal monitoring].
Monitors for laser spectroscopy are available as an additional tool for foetal surveillance. Using this technique, continuous and non-invasive registration of four biochemical parameters (HbO2, HbR, blood volume and cytochrome aa3) can be performed. The calculation is based on the optical density after irradiation of the biological sample with pulsed laser light in the near-infrared range (755, 805, 845, 904 nm). We evaluated the new technique in a clinical trial of 20 patients. The application of the laser sensor was performed, using a modified amnioscope. During prenatal tracings in cases with suspect CTG patterns, hypoxia was not verified. In cases without late decelerations a decrease of the cytochrome aa3 signal was observed in less than 1%. During intrapartual measurements, near-infrared signals were compared with the result of the PO2 measurement in foetal blood analysis. We found a significant correlation for HbO2 and cytochrome aa3 with a correlation coefficient of r = 0.96 respectively r = 0.94. We conclude, that laser spectroscopy will potentially be of benefit for foetal monitoring as it might provide a basis for differentiation between foetuses at risk and non-hazardous cases.
Cardiotocography↗
Cytochrome c'' isolated from Methylophilus methylotrophus. An example of bis-histidine-co-ordinated Fe3+ haem, with near-perpendicular orientation of the ligands.
Cytochrome c'' (Methylophilus methylotrophus) is a soluble protein, Mr 15,000, possessing one haem which is high-spin in the reduced state but switches to a low-spin form on oxidation. Low-temperature electron-paramagnetic-resonance spectroscopy of the oxidized state shows a low-spin signal at gz = 3.65 with a folded line-shape typical of a haem of low rhombicity, and the near-infrared magnetic-circular-dichroism (m.c.d.) spectra reveal an unusually intense (delta epsilon = 400 M-1.cm-1 at 5 T, 4.2 K) charge-transfer band at 1560 nm, establishing that the oxidized haem is co-ordinated by two His residues in a near-perpendicular orientation. This conformation is well established for transmembrane b cytochromes, but this appears to be the first example in a water-soluble cytochrome. The low-temperature m.c.d. spectra of the reduced form of the protein confirms that the haem contains a high-spin Fe2+ ligated by one His residue. The redox-linked spin-state change releases a His group. Since this residue is likely to bind a proton at pH values less than 6.5, this cytochrome may provide a useful model of a molecular mechanism of a redox-linked proton uptake and release process.
Bacteria↗
Fluorescence polarization and low-temperature absorption spectroscopy of a subunit form of light-harvesting complex I from purple photosynthetic bacteria.
Measurements of polarized fluorescence and CD were made on light-harvesting complex 1 and a subunit form of this complex from Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodobacter capsulatus. The subunit form of LH1, characterized by a near-infrared absorbance band at approximately 820 nm, was obtained by titration of carotenoid-depleted LH1 complexes with the detergent n-octyl beta-D-glucopyranoside as reported by Miller et al. (1987) [Miller J. F., Hinchigeri, S. B., Parkes-Loach, P. S., Callahan, P. M., Sprinkle, J. R., & Loach, P. A. (1987) Biochemistry 26, 5055-5062]. Fluorescence polarization and CD measurements at 77 K suggest that this subunit form must consist of an interacting bacteriochlorophyll a dimer in all three bacterial species. A small, local decrease in the polarization of the fluorescence is observed upon excitation at the blue side of the absorption band of the B820 subunit. This decrease is ascribed to the presence of a high-energy exciton component, perpendicular to the main low-energy exciton component. From the extent of the depolarization, we estimate the oscillator strength of the high-energy component to be at most 3% of the main absorption band. The optical properties of B820 are best explained by a Bchl a dimer that has a parallel or antiparallel configuration with an angle between the Qy transition dipoles not larger than 33 degrees. The importance of this structure is emphasized by the results showing that core antennas from three different purple bacteria have a similar structure.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacteriochlorophylls↗
Origin of optical activity in the purple bacterial photoreaction center.
The photoreaction center (RC) of purple bacteria contains four bacteriochlorophyll (Bch) and two bacteriopheophytin (Bph) molecules as prosthetic groups. Their optical activity, as measured by circular dichroism (CD) spectroscopy, is largely increased in situ as compared to organic solutions. The all-exciton hypothesis posits that this enhanced optical activity is entirely due to excitonic interactions between the electronic transitions of all six bacteriochlorin molecules. Using the simple exciton theory, this model predicts that the near-infrared CD spectra should be conservative. The fact that they are not, whether the special pair of Bch (SP) that constitutes the primary electron donor is reduced or oxidized, has been explained by hyperchromic effects. The present work tests this hypothesis by successively eliminating the absorption and, therefore, the optical activity of the Bphs and of the non-special-pair (non-SP) Bchs. This was accomplished by trapping these pigments in their reduced state. RC preparations with the four non-SP bacteriochlorins trapped in their reduced state and, therefore, with an intact SP displayed conservative CD spectra. RC preparations with only the electronic transitions of SP and of one non-SP Bch also showed conservative CD spectra. These conservative CD spectra and their corresponding absorption spectra were simulated using simple exciton theory without assuming hyperchromic effects. Bleaching half of the 755-nm absorption band by phototrapping one of the two Bph molecules led to the complete disappearnce of the corresponding CD band. This cannot be explained by the all-exciton hypothesis. These results suggest that the optical activity of the SP alone, or with one non-SP Bch, is due to excitonic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)
Ascorbic Acid↗
Oxido-reduction of B800-850 and B880 holochromes isolated from three species of photosynthetic bacteria as studied by electron-paramagnetic resonance and optical spectroscopy.
Certain redox properties of bacteriochlorophyll alpha were used to probe the structure of several light-harvesting pigment-protein complexes or holochromes. To attribute redox properties unequivocally to a given holochrome, we worked with purified holochromes. We developed purification procedures for the B880 holochromes from Rhodospirillum rubrum, Rhodopseudomonas sphaeroides and Ectothiorhodospira sp. and for the B800-850 holochromes from the latter two species. In all these holochromes, bacteriochlorophyll alpha could be oxidized by ferricyanide as witnessed by the bleaching of their near-infrared absorption bands. However, only in B880 holochromes was this oxidation reversible. Another important difference between the B800-850 and the B880 holochromes is that oxidation of the latter gives rise to a g = 2.0025 electron paramagnetic resonance (EPR) signal with linewidth varying, according to species, from 0.37 mT to 0.48 mT. Both the reversible EPR signal and absorption changes titrate with a midpoint redox potential (pH 8.0) of approximately 570 mV. Linewidth narrowing can be interpreted by delocalization of the free electron spin over approximately 12 bacteriochlorophyll molecules. While the B880 holochromes from the three species considered had indistinguishable redox properties, the B800-850 holochromes differed from one another by their circular dichroic spectra and by the relative ease of oxidation of their 800-nm and 850-nm bands. This indicates that, contrary to the B880 holochromes, the B800-850 holochromes may not form a homogeneous class.
Bacterial Proteins↗
Low temperature excitation and emission spectroscopy of the photosynthetic bacteria Rhodopseudomonas sphaeroides 'wild-type' strain ATCC 17023.
Prompt and delayed emission of Rhodopseudomonas sphaeroides 'wild-type' strain ATCC 17023 was measured in the spectral range 550-950 nm at 1.7 K. The broad emission spectra could be resolved into separate bands by excitation spectroscopy. Through a comparison of the separated excitation and emission spectra of the present study with absorption and fluorescence spectra of photosynthetic pigments given in the literature, the emitting species could be identified. Beside the well-known pigments bacteriochlorophyll (BChl), bacteriopheophytin (BPh) and carotenoids, additional pigments could be detected. In the visible and near-infrared range, the fluorescence of pigments could be observed which were produced by the biosynthesis of BChl in the cells. Further, fluorescence bands at 753 and 813 nm are interpreted as originating from BPh (F-753) and BChl (F-813). Delayed emission signals between 700 and 800 nm were attributed to a metalloporphyrin phosphorescence. Surprisingly, the excitation spectra of the delayed BChl emissions measured at 872 and 912 nm showed the same excitation spectra as the short-wavelength emission, typical of metalloporphyrins. This is strong indication of an energy transfer between metalloporphyrins and BChl.
Bacteriochlorophylls↗
O2 and CO reactions with heme proteins: quantum yields and geminate recombination on picosecond time scales.
Picosecond time-resolved absorption spectroscopy and low-temperature studies have been undertaken in order to understand the nature of the intrinsic quantum yields and geminate recombination of carbon monoxide and oxygen to hemoglobin and myoglobin. We find that the photoproduct yields at 40 ps and long times (minutes) after photolysis at 8 K are similar; however, the yield of oxygen photoproducts is 0.4 +/- 0.1 while the yield of carbon monoxide photoproducts is 1.0 +/- 0.1 for both myoglobin and hemoglobin. Measurements in the Soret, near-infrared, and far-IR are used to quantitate the photoproduct yields. These results call into question previous cryogenic kinetic studies of O2 recombination. Significant subnanosecond geminate recombination is observed in oxyhemoglobin down to 150 K, while below 100 K this geminate recombination disappears. The lower photoproduct yields for oxyheme protein complexes can be attributed to both subnanosecond and subpicosecond recombination events which are ligand and protein dynamics dependent.
Animals↗
The site of the redox-linked proton pump in eukaryotic cytochrome c oxidases.
The electronic spectra of fully oxidized derivatives of some cytochrome c oxidase preparations are distinctly pH dependent. In general, the observed spectral shifts are greater in the case of pulsed derivatives compared to resting preparations and also, greater for preparations of the enzyme from shark skeletal muscle compared to beef heart. The low temperature near-infrared magnetic circular dichroism spectrum of the fully oxidized shark enzyme is not pH dependent in the experimental range, indicating the sensitivity of the visible region electronic spectrum to variation in pH to be due principally to changes at the heme a3-CuB chromophore. The results are discussed in relation to proposed mechanisms of proton translocation in cytochrome c oxidase.
Animals↗
The effect of hyperbaric oxygen on cerebral hemoglobin oxygenation and dissociation rate of carboxyhemoglobin in anesthetized rats: spectroscopic approach.
By measuring near-infrared transmittance spectra, we examined the effect of HBO on cerebral Hb oxygenation in normal and ischemic brain in the anesthetized rat. The oxygenation state of Hb was around 80% in the rat brain under 1 ATA air breathing. HBO did not induce further cerebral Hb oxygenation above 2 ATA in control animals but improved tissue oxygenation in the ischemic brains. The oxidation-reduction state of cyt. aa3 in the normal brain was not affected by HBO. In the ischemic brain, however, HBO prevented ischemia-induced reduction of cyt. aa3. Non-invasive optical monitoring of COHb with visible reflectance spectrophotometry was also examined. HBO markedly accelerated dissociation of COHb. Tight correlation was found between the optical signal and COHb content determined from blood samples. These results demonstrated the usefulness of optical monitoring in vivo under hyperbaric conditions.
Anaerobiosis↗
Thiobacillus ferrooxidans cytochrome c oxidase: purification, and molecular and enzymatic features.
Cytochrome c oxidase from Thiobacillus ferrooxidans was purified to homogeneity and some of its properties were studied. The oxidase was solubilized with n-octyl-beta-D-thioglucoside (OTG) under acidic conditions (pH 4.0) and purified by one step of ion-exchange chromatography with a CM-Toyopearl column. The absorption spectrum of the oxidase showed peaks at 420 and 595 nm in the oxidized form and at 440 and 595 nm in the reduced form. Its CO compound showed a novel absorption spectrum; a double-peaked gamma band appeared at 429 and 438 nm. The oxidase seemed to have CuA-like copper atom from its ESR and near-infrared spectra. The oxidase molecule consisted of three polypeptides with molecular weights of 53,000, 22,000, and 17,000, respectively, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme in a solution containing detergents was estimated to be 169,000 on the basis of the results obtained by gel filtration, while the molecular weight per heme alpha was estimated to be 83,700. The copper content of the oxidase was 1.01 g atom per mol of heme alpha. Therefore, the cytochrome seemed to contain one molecule of heme alpha and one atom of copper in the minimal structural unit consisting of one molecule each of the three subunits, and to occur as a dimer of the unit in the solution. The oxidase oxidized ferrocytochrome c-552 of the bacterium, and the optimal pH of the reaction was 3.5.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Fractionation↗
Spectroscopic studies of cobalt and nickel substituted rubredoxin and desulforedoxin.
The single iron site of rubredoxin was replaced by nickel and cobalt. The near-infrared/visible/UV spectra of these metal derivatives show ligand-field transitions and charge-transfer bands which closely resemble those of simple tetrathiolate complexes, indicating a tetrahedral arrangement of the sulfur cysteinyl ligands around the metal core. The 1H NMR spectra of the nickel and cobalt derivatives reveal extremely low-field contact shifted resonances of one proton intensity assigned to beta-CH2 and alpha-CH cysteinyl protons. Other well resolved resonances shifted out of the main protein spectral envelope are also observed and probably arise from contact plus pseudocontact shift mechanisms. Rubredoxins from different sulfate reducers were metal substituted and assignments of aliphatic protons are tentatively proposed, taking advantage of the amino acid sequence homologies. The present data is promising in terms of structural analysis of the coordination sphere of the metal core. It was also shown that replacement of the iron atom of desulforedoxin, a close analogue of rubredoxin, by cobalt and nickel was possible.
Amino Acid Sequence↗
The axial ligands of heme in cytochromes: a near-infrared magnetic circular dichroism study of yeast cytochromes c, c1, and b and spinach cytochrome f.
Room temperature near-infrared magnetic circular dichroism and low-temperature electron paramagnetic resonance measurements have been used to characterize the ligands of the heme iron in mitochondrial cytochromes c, c1, and b and in cytochrome f of the photosynthetic electron transport chain. The MCD data show that methionine is the sixth ligand of the heme of oxidized yeast cytochrome c1; the identify of this residue is inferred to be the single conserved methionine identified from a partial alignment of the available cytochrome c1 amino acid sequences. A different residue, which is most likely lysine, is the sixth heme ligand in oxidized spinach cytochrome f. The data for oxidized yeast cytochrome b are consistent with bis-histidine coordination of both hemes although the possibility that one of the hemes is ligated by histidine and lysine cannot be rigorously excluded. The neutral and alkaline forms of oxidized yeast cytochrome c have spectroscopic properties very similar to those of the horse heart proteins, and thus, by analogy, the sixth ligands are methionine and lysine, respectively.
Amino Acid Sequence↗
Chemical modification of the CuA center in cytochrome c oxidase by sodium p-(hydroxymercuri)benzoate.
Cytochrome c oxidase contains a copper ion electron-transfer site, CuA, which has previously been found to be unreactive with externally added reagents under conditions in which the protein remains structurally intact. We have studied the reaction of cytochrome oxidase with sodium p-(hydroxymercuri) benzoate (pHMB) and found that the reaction proceeds, under appropriate conditions, to give an excellent yield of a particular derivative of the CuA center that has electron paramagnetic resonance and near-infrared absorption spectroscopic properties which are distinctly different from those of the unmodified center. Spectroscopic and chemical characterization of the other metal ion sites of the enzyme reveals little or no effect of the pHMB modification on the structures of and reactions at those sites. Of particular interest is the observation that the modified enzyme still displays a substantial fraction of the native steady-state activity of electron transfer from ferrocytochrome c to O2. Although the modified copper center retains the ability to receive electrons from the powerful reductant Na2S2O4 and to transfer electrons to O2, it is not significantly reduced when the enzyme is treated with milder (higher potential) reductants such as NADH/phenazine methosulfate or the physiological substrate ferrocytochrome c. CuA exhibits many spectroscopic and chemical properties which make it highly atypical of cuproprotein active sites; the singular nature of this site has prompted speculation about the importance of the structural peculiarities of this metal ion center in the catalytic cycle of the enzyme. In this work, we demonstrate that the unusual features of this site are not prerequisites for competent catalysis of electron transfer and O2 reduction by the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Rapid verification of identity and content of drug formulations using mid-infrared spectroscopy.
A general method for the rapid verification of both identity and content of complete solid drug formulations has been devised. Infrared spectra for the samples were recorded using the diffuse reflectance technique, and specially written software was employed to identify the type of formulation and level of active ingredient. This software was devised to ensure reliable use when applied by those with minimal operator skills. Three differing drug tablet formulations containing simvastatin, enalapril maleate and lovastatin, as well as a capsule formulation containing finastride were studied. Adequate precision was obtained to reliably verify drug dosage levels. Near-infrared (NIR) and mid-infrared (MIR) spectrometers were evaluated for use with the method. The MIR instrument allowed sufficient resolution and spectral/structural selectivity to reliably verify correctness of either of two near derivative drugs necessarily present in the same clinical study. Drug tablet and capsule dosage levels tested ranged from 0.2 to 40 mg of drug. Approximately 1% (w/w) of the drug in the formulation was the minimum amount determined. Parameters affecting method ruggedness in routine use were optimized. Experimental addition of an extraneous material to a simvastatin formulation was easily detected and flagged by the routine test procedure. Subsequent data retrieval and searching against spectral libraries was used to demonstrate identification of the additive.
Anticholesteremic Agents↗
Near-infrared measurement of relative and absolute humidity through detection of water adsorbed on a silica gel layer.
Near-IR spectroscopy is especially well suited to moisture determination because of the relatively high absorptivity of water compared to most other substances. In the present work, near-IR diffuse-transmittance spectroscopy is applied to the measurement of humidity via observation of adsorbed water on a high-performance thin-layer chromatography silica gel plate. The adsorbed water is detected through both ordinary absorption of radiation by water molecules and the increased scattering of near-IR radiation by the silica gel as it adsorbs more water. This technique shows promise as a highly accurate and sensitive humidity sensor with a time constant of less than 1 min. The addition of inorganic salts to the silica gel layer is shown to increase the response to water vapor. However, it also increases the time constant of the sensor. A miniature humidity sensor using a commercially available near-IR transmissive switch is also demonstrated. Possible interferences and hysteresis effects are investigated.
Adsorption↗
Infrared spectroscopy of photodissociated carboxymyoglobin at low temperatures.
We have studied the infrared spectra of the bound and photodissociated states of Mb-12CO and Mb-13CO from 5.2 to 300 K. The absorbance peaks seen between 1800 and 2200 cm-1 correspond to CO stretching vibrations. In the bound state of Mb-12CO, the known lines A0 at 1969, A1 at 1945, and A2 at 1927 cm-1, have center frequencies, widths, and absorbances that are independent of temperature between 5.2 and 160 K. Above 160 K, A2 gradually shifts to 1933 cm-1. The low-temperature photodissociated state (Mb) shows three lines (B0, B1, B2) at 2144, 2131, and 2119 cm-1 for 12CO. The absorbances of the three lines depend on temperature. B0 is tentatively assigned to free CO in the heme pocket and B1 and B2, to CO weakly bound to the heme or heme pocket wall. The data are consistent with a model in which photodissociation of MbCO leads to B1 and B2. B2 decays thermally to B1 above 13 K; rebinding to A occurs from B1. The barriers between B2 and B1 and between B1 and A are described by activation enthalpy spectra. Heme and the central metal atom in state Mb have near-infrared, EPR, and Mössbauer spectra that differ slightly from those of deoxyMb. The observation of essentially free CO in state B implies that the difference between Mb and deoxyMb is not due to an interaction of the flashed-off ligand with the protein but is caused by an incomplete relaxation of the protein structure at low temperatures.
Animals↗
X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. II. Comparison of diffraction patterns of photosynthetic units from various purple bacteria.
Comparative X-ray diffraction studies, in conjunction with infrared absorption spectroscopy, were performed on chromatophores isolated from various purple photosynthetic bacteria in order to achieve a better understanding of the molecular structure of the photosynthetic unit. Purple non-sulfur bacteria used were Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas palustris. Chromatophores of Chromatium vinosum, as a typical example of purple sulfur bacteria, were also investigated. The results were as follows. Distinct equatorial X-ray diffraction patterns were obtained from chromatophores of all the bacteria examined. They showed diffuse, continuous diffraction patterns having several maxima, and the patterns are evidently distinguished from those of either crystalline or amorphous material. The pattern indicates that the photosynthetic unit in the chromatophore has a highly organized molecular structure in the plane of the membrane. Bacteria whose major photosynthetic pigment is bacteriochlorophyll alpha can be categorized in three groups from the viewpoint of near infrared absorption spectra. X-ray diffraction patterns are also grouped accordingly, although the differences are minimal and the patterns display common features. In other words, the bacteriochlorophyll forms, which are bacteriochlorophyll-protein complexes exhibiting different near-infrared absorption spectra, show different X-ray patterns: the molecular structure of photosynthetic units is closely related to the state of pigment in each complex, although the "X-ray" molecular structure is mainly concerned with the arrangement of constituent protein molecules at the present resolution, whereas the "spectroscopic" structure reflects the local environment of pigment.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacterial Chromatophores↗