Topic:Spectroscopy
Spectroscopy is an analytical technique used to measure the interaction between matter and electromagnetic radiation. In equine research, spectroscopy is applied to assess various biological and chemical properties of horses. Techniques such as infrared spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry are utilized to analyze tissues, fluids, and other biological samples. These methods provide insights into metabolic processes, nutritional status, and disease states in horses. Spectroscopy aids in the identification and quantification of biomolecules, contributing to the understanding of equine physiology and pathology. This page compiles peer-reviewed research studies and scholarly articles that explore the application, methodology, and findings of spectroscopic techniques in equine science.
1H NMR resonance assignments in a paramagnetic heme protein by two-dimensional spectroscopy: heme resonances in equine met-azido myoglobin. Specific heme protons for the majority of resonances in the downfield resolved region of equine met-azido myoglobin have been assigned using solely the two-dimensional 1H NMR experiments NOESY and COSY. Metazido myoglobin provides a useful test case for the applicability of these techniques to paramagnetic proteins for the following reasons. First met-azido myoglobin is a mixed spin-state protein, with significantly shorter relaxation times and broadened lines relative to pure low-spin systems (eg., met-cyano myoglobin). Second, met-azido hemoglobin and met-azido myoglobin are important as mod...
1H and 119Sn magnetic resonance study of the SnIV protoporphyrin IX complex of equine myoglobin. Structure of the porphyrin-binding pocket. Tin protoporphyrin IX (SnPP) is being used in the treatment of hyperbilirubinemia. We have studied the SnPP complex with equine myoglobin (EqMb) by 1H and 119Sn nuclear magnetic resonance spectroscopy (NMR) as a general model for SnPP interaction with hemoproteins. The complex formed from SnPP and EqMb, SnPP.EqMb, was found to have essentially the same porphyrin-binding pocket as EqMbCO, including the same porphyrin orientation in the major form of EqMbCO. 119Sn NMR spectroscopy has been used to demonstrate that the proximal His93F8-metal coordination is likely to be intact in SnPP.EqMb. Minor...
[Local structure of cytochrome c from horse heart in solution. Conformational analysis using data of two-dimensional nuclear Overhauser effect spectroscopy]. Using the earlier suggested method the calculation of the backbone conformations of horse heart cytochrome c in oxidized (ferricytochrome c) and reduced (ferrocytochrome c) states has been performed by the two-dimensional nuclear Overhauser effect spectroscopy data. For both protein forms the secondary structure elements have been revealed and the conformations of the irregular polypeptide chain segments have been analysed. The similarity of the secondary structures of ferri- and ferrocytochrome c in solution was established from the comparison of their conformations. Small differences between...
Chemical structures of three neutral oligosaccharides obtained from horse (thoroughbred) colostrum. 1. Three neutral oligosaccharides were obtained from horse colostrum by ion-exchange, activated charcoal column and preparative paper chromatographies. 2. The following structures were elucidated by methanolysis, methylation analysis and 75 MHz 13C-NMR spectroscopy; Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (HM-3a), Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4Glc (HM-3b) and Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc (HM-5). 3. HM-3a and HM-5 have been found in human milk, named as lacto-N-neotetraose and lacto-N-neohexaose, respectively. HM-3b has been isolated from g...
Characterization of hydrophobic cores in apomyoglobin: a proton NMR spectroscopy study. A proton nuclear magnetic resonance spectroscopic study of horse apomyoglobin was undertaken in order to define the regions of myoglobin that are and that are not structurally affected by the binding of the prosthetic group. It was found that, in spite of the poor spectral resolution, a number of spin systems could be identified by using standard correlated methods. Four clusters consisting mostly of hydrophobic residues were detected by nuclear Overhauser spectroscopy, two of which involved the tryptophan side chains. Extensive similarities to nuclear Overhauser spectroscopy data collected on...
Biochemical analysis of normal articular cartilage in horses. Articular cartilage specimens from the distal articular surface of 32 radiocarpal bones from 24 2- to 5-year-old horses were analyzed. The total collagen content was determined on the basis of the 4-hydroxyproline content, using a colorimetric method. A method for estimating the proportions of types-I and -II collagen by measuring spectrophotometric densities of specific cyanogen bromide peptide bands from mixtures of types-I and -II collagen on sodium dodecyl sulfate-polyacrylamide gels was used. The cyanogen bromide peptides representative of each collagen types-I and -II were identified. Th...
Characterisation of compounds isolated from the sera of horses with acute grass sickness. Isolates were prepared from the sera of 12 horses with acute grass sickness, using methods reported to yield serum fractions associated with neurotoxicity, and their components identified by liquid chromatography and spectroscopy. All isolates were found to contain cortisol and six isolates also contained a degradation product of an analgesic drug, dipyrone. However, no recognised neurotoxin was detected.
Identification of a major iodolipid from the horse thyroid gland as 2-iodohexadecanal. The incorporation of iodide into proteins (PBI) and lipids (LBI) of horse thyroid slices was measured in various conditions. Their dependency on the concentration of extracellular iodide was strikingly different. For PBI the relationship was biphasic with a decrease above 10 microM, likely to correspond to the Wolff-Chaikoff effect. On the contrary, LBI increased as a function of iodide concentration up to 100 microM. Methimazole (MMI) inhibited the incorporation of iodide into both LBI and PBI, but higher concentrations of MMI were required to depress LBI as compared to PBI. The inhibition of...
Effect of inositol hexakisphosphate on the spectroscopic properties of the nitric oxide derivative of ferrous horse and bovine hemoglobin. The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous horse and bovine hemoglobin (Hb) has been investigated. In the absence of IHP, the nitric oxide derivative of ferrous horse Hb shows spectroscopic properties similar to those of the corresponding derivative of ferrous human Hb that are generally taken as typical of the high affinity state of tetrametric hemoproteins. Similar to human Hb, the addition of IHP to the nitric oxide derivative of ferrous horse Hb induces a transition toward a species characteri...
Primary structure of horse serotransferrin glycans. Demonstration that heterogeneity is related to the number of glycans and to the presence of N-acetylneuraminic acid and N-acetyl-4-O-acetylneuraminic acid. Three serotransferrin variants Tf 2a, Tf 4b and Tf 5b were isolated in an homogeneous form from a preparation of homozygous horse serotransferrin Tf 0. On the basis of the results concerning molecular mass determination and the carbohydrate analysis, it is concluded that the serotransferrin variant Tf 2a contains only one glycan while variants Tf 4b and Tf 5b contain two glycans. The structure of all of the glycans has been established by combining methylation analysis, mass spectrometry and 400-MHz 1H-NMR spectroscopy. From the obtained results, it appears that the two glycans of Tf 5b varian...
Mössbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated Fe(III) atoms and oxo-bridged Fe(III) dimers as early intermediates. Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room ...
The sequence-specific assignment of the 1H-NMR spectrum of an enzyme, horse-muscle acylphosphatase. A complete range of two-dimensional NMR experiments was used for the assignment of the 1H-NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long-range patterns characteristics for secondary structure elements. This result had also provided the ...
Identification and description of beta-structure in horse muscle acylphosphatase by nuclear magnetic resonance spectroscopy. Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed s...
A spectrophotometric procedure for the determination of objective measurements of equine spermatozoan motility. A spectrophotometric procedure was developed and evaluated for the objective measurement of equine spermatozoan motility. A 100 mul sample of a sperm suspension, prepared by the removal of seminal plasma, was layered under a column of optically clear medium in a specially designed spectrophotometric cuvette maintained at 37 degrees C. Changes in light transmittance above the interface of the sperm suspension and medium were recorded on chart paper. As sperm cells swam into the medium, a decrease in light transmittance was recorded as a deflection on the chart paper. Chart recordings were analy...
Analysis of N-acetyl-4-O-acetylneuraminic-acid-containing N-linked carbohydrate chains released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Application to the structure determination of the carbohydrate chains of equine fibrinogen. The carbohydrate chains of equine fibrinogen were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500-MHz 1H-NMR spectroscopy. Four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains occur: (formula; see text)
Identification and description of alpha-helical regions in horse muscle acylphosphatase by 1H nuclear magnetic resonance spectroscopy. It has been proposed that combination of intraresidue, sequential and longer range nuclear Overhauser enhancements occurring in 1H nuclear magnetic resonance spectra of protein chains folded in a helix show a regular characteristic pattern. As a test case the spectra of horse muscle acylphosphatase were searched for this pattern together with other typical signs of a helical conformation (i.e. chemical shift, coupling constants and slow 2H-H exchange). Two amino acid sequences complying with these requirements were found. Just a few amino acid spin system assignments were then sufficient to lo...
The structure and properties of horse muscle acylphosphatase in solution. Mobility of antigenic and active site regions. The solution structure of acylphosphatase determined by proton nuclear magnetic resonance spectroscopy is described. The results allow us to discuss the fold of the protein (101 amino acids), to correlate the exposure and the mobility of the backbone with the antigenicity, and to locate the active site.
Monoclonal antibody recognizes a conformational epitope in a random coil protein. The antigenic determinants for two monoclonal antibodies directed against horse apo-cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme-linked immunoassay, and differential acetylation of free and antibody-bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen-antibody complex is compared to the accessibility in the free antigen. The two antibodies against the heme-free protein do not re...
Assessment of copper and zinc status of farm horses and training thoroughbreds in south-east Queensland. The copper and zinc concentrations in the blood of stabled thoroughbred horses and in Australian Stock Horses mares at pasture, either late pregnant or lactating were determined by an atomic absorption spectroscopic method. The plasma concentration of the trace elements in these apparently normal horses were generally below the "normal" range. The plasma copper, caeruloplasmin copper, whole blood copper and plasma zinc concentrations in the stabled thoroughbreds were 0.76 +/- 0.19 micrograms/ml (n = 82), 0.56 +/- 0.14 micrograms/ml (n = 83), 0.75 +/- 0.18 micrograms/ml (n = 82) and 0.47 +/- 0....
Metabolism of naturally occurring [13C]glucose given orally to horses. In 5 horses, 13CO2/12CO2 ratios in expired air were determined using isotope mass spectroscopy to investigate metabolism of naturally occurring [13C]glucose. Oral glucose tolerance tests (OGTT) were performed using maize or beet glucose. Maize has a higher 13C concentration than that of most plants. The 13CO2/12CO2 ratios after OGTT was performed using maize glucose were compared with 13CO2/12CO2 ratios in expired air after OGTT was performed using beet glucose. The ratio also was determined during the period horses were not fed. Using OGTT, all horses were glucose tolerant. The OGTT performed...
Evidence for eumelanin and pheomelanin producing genotypes in the Arabian horse. The ultrastructural imaging of melanocytes coupled with analyses to detect sulfur-containing melanosomes by energy-dispersive X-ray spectroscopy were used to test the hypothesis that the yellowish-red and black pigments found in Arabian horses result from pheomelanogenesis and eumelanogenesis, respectively. These procedures detected pheomelanosomes in follicles at the base of hairs in chestnut horses and eumelanosomes in follicles at the base of hairs in black horses. By analyzing tissue obtained by skin biopsy, these procedures also demonstrated that skin melanocytes in a chestnut horse produ...
Identification of a flunixin metabolite in the horse by gas chromatography-mass spectrometry. The main metabolite of flunixin, a hydroxylated product, has been identified by gas chromatography-mass spectrometry and 1H NMR spectroscopy in equine urine and plasma. The method also permits the qualitative monitoring of the urinary elimination of the drug and its metabolite. The two products are detected up to 175 and 54 h, respectively, after a single intravenous administration at the dose of 1 mg/kg. Simultaneous detection of the two compounds increases the reliability of anti-doping control analysis.
Reconstituted and native iron-cores of bacterioferritin and ferritin. The structural and magnetic properties of the iron-cores of reconstituted horse spleen ferritin and Azotobacter vinelandii bacterioferritin have been investigated by high-resolution transmission electron microscopy, electron diffraction and Mossbauer spectroscopy. The structural properties of native horse spleen ferritin, native Az. vinelandii, and native and reconstituted Pseudomonas aeruginosa bacterioferritins have also been determined. Reconstitution in the absence of inorganic phosphate at pH 7.0 showed sigmoidal behaviour in each protein but was approximately 30% faster in initial rate f...
Identification of the ligand-exchange process in the alkaline transition of horse heart cytochrome c. Magnetic-circular-dichroism (m.c.d.) spectra over the wavelength range 300-2000 nm at room temperature and at 4.2K of horse heart cytochrome c are reported at a series of pH values between 7.8 and 11.0, encompassing the alkaline transition. The effect of glassing agents on the e.p.r. spectrum at various pH values is also reported. Comparison of these results with spectra obtained for the n-butylamine adduct of soybean leghaemoglobin support the hypothesis that lysine is the sixth ligand in the alkaline form of horse heart cytochrome c. The m.c.d. and e.p.r. spectra of horse heart cytochrome c ...
The role of aromatic side chain residues in micelle binding by pancreatic colipase. Fluorescence studies of the porcine and equine proteins. Fluorescence techniques have been employed to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles. Nitrotyrosine-55 of porcine colipase is obtained by modification with tetranitromethane (low excess, in the presence of taurodeoxycholate) of the protein followed by gel filtration and ion-exchange chromatography. Verification of the residue modified was obtained by h.p.l.c. peptide purification and sequence analysis. Reduction and quantitative reaction with dansyl chloride yields a fluorescent derivative that is twice as active in conjunction with ...
Proton hyperfine resonance assignments using the nuclear Overhauser effect for ferric forms of horse and tuna cytochrome c. Proton hyperfine resonance assignments for cytochromes c from several species are currently being successfully pursued by several laboratories. These efforts focus mostly on the ferrous forms. In contrast to that work, we have pursued assignments of the proton hyperfine shifted resonances for horse and tuna ferricytochromes c. Our results indicate that assignments are nearly identical in those two proteins. Using the pre-steady state nuclear Overhauser effect, several additional assignments have been made for the tuna protein, whereas for the horse protein, the following protons have been assi...
A proton NMR study of the non-covalent complex of horse cytochrome c and yeast cytochrome-c peroxidase and its comparison with other interacting protein complexes. Cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) forms a noncovalent 1:1 complex with horse cytochrome c in low ionic strength solution that is detectable by proton NMR spectroscopy. When the entire proton hyperfine-shifted spectrum is considered only five hyperfine resonances exhibit unambiguously detectable shifts: the heme 8-CH3 and 3-CH3 resonances, single proton resonances near 19 ppm and -4 ppm and the methionine-80 methyl group. These shifts are very similar to those observed for the covalently crosslinked complex of cytochrome-c peroxidase and h...