Study of prostaglandins and thromboxanes B2 using mass-spectrometry of secondary ions

Prostaglandins E, F, I2 and thromboxane B2 have been studied by secondary ion mass spectrometry. It is shown that the method is suitable for direct identification of these compounds either as free acids or as their sodium salts. The spectra of the former reveal their structural features, while with the latter information on the molecular weight can be obtained. The limit of detection (about 1 microgram) allows the analysis of prostaglandin solutions of 1 microgram/microliter concentrations used in pharmacological tests.     

Computer-assisted analysis of photoacoustic spectra of solid and solution state cyanide methemoglobin

A computer-assisted method for analyzing photoacoustic spectra has been developed. Using this analysis, the relative absorption spectrum and either the chromophore concentration or thermal diffusivity characteristic of a sample can be derived from its photoacoustic spectrum. We have demonstrated the accuracy of the method by analyzing photoacoustic spectra of solution and crystalline-phase bovine cyanide methemoglobin. BASIC and FORTRAN routines used to collect and to analyze photoacoustic spectra are described. Photoacoustic spectroscopy can be used in conjunction with the analytical method presented here to obtain accurate absorption spectra from a variety of solid, opaque, and/or turbid samples.     

The uptake of lead,zinc, cadmium,and copper by the pulmonate mollusc,Helix aspersa muller,and its relevance to the monitoring of heavy metal contamination of the environment

Summary The occurrence of lead, zinc, cadmium, and copper in individuals of Helix aspersa from two sites of varying degrees of contamination was studied. Zinc, cadmium, and copper were shown to increase in a linear fashion with animal weight. The rate of uptake for zinc and cadmium in particular was significantly greater at the more contaminated site. Statistical analysis of the data, using correlation and regression techniques provided information on apparent intermetallic effects.It is concluded that because metal uptake and body weight show a positive linear relationship only the use of animals of similar weight and/or size can be used for monitoring purposes. Even then, different patterns of uptake into different organs and interactions between metal uptakes are such as to seriously question the use of Helix, and other molluscs, for monitoring purposes unless specific organs from comparably sized and/or aged animals are used.     

Measurement of opioid peptides with combinations of reversed phase high performance liquid chromatography, radioimmunoassay, radioreceptorassay, and mass spectrometry

Novel state-of-the-art mass spectrometric methods have been developed and are now used to identify and to quantify enkephalins and other neuropeptides in biological tissue extracts. As the first step, RP-HPLC gradient elution is performed of a Sep-Pak treated peptide-rich fraction from a tissue extract, and the eluent is monitored by a variety of post-HPLC detectors. In an effort to maximize the structural information that can be obtained from the analysis, UV (200 nm) provides the analog absorption trace; receptorassay analysis (RRA) data of all (90) fractions that are collected are used to construct the profile of opioid-receptoractive peptides; radioimmunoassay (RIA) of selected HPLC fractions at retention times corresponding to the retention time of standards, or in some special cases of all 90-fractions, provides immunoreactivity information; and fast atom bombardment mass spectrometry (FAB-MS) in two modes - corroboration of the (M+H)+ of the expected peptide, or MS/MS to monitor an amino acid sequence-determining fragment ion unique to that peptide in the selected ion monitoring (SIM) mode - provides structural information. As a demonstration of the level of quantification sensitivity that can be attained by these novel MS methods, FAB-MS-MS-SIM of solutions of synthetic leucine enkephalin was sensitive to the 70 femtomole level. This paper discusses RIA versus RRA data, and recent MS measurements of peptides in human tissues.     

Size-Exclusion Chromatography with On-Line Light-Scattering, Absorbance, and Refractive Index Detectors for Studying Proteins and Their Interactions

Techniques of using size-exclusion chromatography (SEC) with on-line light-scattering, uv absorbance, and refractive index detectors to characterize the polypeptide molecular weights of simple proteins or glycoproteins or to determine the stoichiometry of protein complexes are described. Two unique advantages of this approach over conventional SEC are that the molecular weight measurement is independent of elution position and can exclude the contributions from carbohydrates. When a protein or complex contains no carbohydrates, a two-detector method, i.e., light scattering combined with refractive index, can be used to calculate the molecular weight. When a protein contains carbohydrates, a three-detector method is used to calculate the molecular weight of polypeptide alone. Finally, a self-consistent three-detector method is used to determine the stoichiometry of a protein complex containing carbohydrates. Example applications for all these methodologies are described.     

Management of dietary essential metals (iron,copper, zinc,chromium and manganese) by Wistar and Zucker obese rats fed a self-selected high-energy diet

The balances and content of essential elements (iron, copper, zinc, chromium and manganese) in the body of Wistar, Zucker lean and Zucker obese rats fed a reference or cafeteria diet from day 30 to 60 after birth have been studied. Intestinal iron absorption compensated for low iron content of the cafeteria diet and the extra needs of growth and fat deposition. It can be assumed that the altered energy regulation processes that afflict the genetically obese rat are not directly related to altered iron metabolism. Obese Zucker rats had lower copper tissue concentrations than lean rats, but when fed a cafeteria diet the differences between Zucker rats strains disappear. This cannot be traced to large differences in diet copper concentration. A low diet availability of zinc—such as that of cafeteria-fed fa/fa rats—is easily compensated for by increasing absorption. So, as a consequence, we can conclude that genetic obesity did not impair zinc absorption. There was no deficit of zinc in any of the groups studied; the rats have enough capacity to extract zinc within a wide range of dietary concentrations. The absorption of dietary chromium was inversely proportional to its concentration. The ability to extract chromium from the diet and the very low urinary losses are a consequence of its scarcity in most dietary items. Despite wide variations in the manganese of the diets, the absorption rates were practically unchanged except for obese rats fed the cafeteria diet. It seems that this low absorptive capacity is enough to supply the rat with the manganese it needs, since a sizeable—but subjected to 8-fold-span variations-proportion is lost in the urine. This alone points towards a considerable excess of manganese in both diets studied. Obesity does not have a significant effect on the abilities to absorb and retain minerals, since these processes were more related to dietary availability. Management of essential metals by obese rats depends whether this condition is genetic or induced by diet. Most of the differences observed can be related to differences in diet concentration, to the excess fat content or different metabolic attitude to use substrates of obese animals. The data presented show that the cafeteria diet used adequately serves the mineral needs of the rat, since the rat adapts its absorbing and retaining strategies to match the dietary availability of these minerals.     

Intrinsic Thermodynamics and Structure Correlation of Benzenesulfonamides with a Pyrimidine Moiety Binding to Carbonic Anhydrases I,II, VII,XII, and XIII

The early stage of drug discovery is often based on selecting the highest affinity lead compound. To this end the structural and energetic characterization of the binding reaction is important. The binding energetics can be resolved into enthalpic and entropic contributions to the binding Gibbs free energy. Most compound binding reactions are coupled to the absorption or release of protons by the protein or the compound. A distinction between the observed and intrinsic parameters of the binding energetics requires the dissection of the protonation/deprotonation processes. Since only the intrinsic parameters can be correlated with molecular structural perturbations associated with complex formation, it is these parameters that are required for rational drug design. Carbonic anhydrase (CA) isoforms are important therapeutic targets to treat a range of disorders including glaucoma, obesity, epilepsy, and cancer. For effective treatment isoform-specific inhibitors are needed. In this work we investigated the binding and protonation energetics of sixteen [(2-pyrimidinylthio)acetyl]benzenesulfonamide CA inhibitors using isothermal titration calorimetry and fluorescent thermal shift assay. The compounds were built by combining four sulfonamide headgroups with four tailgroups yielding 16 compounds. Their intrinsic binding thermodynamics showed the limitations of the functional group energetic additivity approach used in fragment-based drug design, especially at the level of enthalpies and entropies of binding. Combined with high resolution crystal structural data correlations were drawn between the chemical functional groups on selected inhibitors and intrinsic thermodynamic parameters of CA-inhibitor complex formation.     

Interaction between fatty acid salts and elastin: Kinetics,absorption equilibrium,and consequences for elasticity

Elastin from bovine ligamentum nuchae is incubated in aqueous solutions of sodium salts of fatty acids (FAS). The FAS are laurate, myristate, and palmitate. Absorption of FAS in the elastin network is studied as a function of time, FAS concentration, and ionic strength. The consequences of this uptake for the elasticity of the elastin are studied by static and dynamic stress–strain measurements. Generally, distinction must be made between the initial time‐dependent stage (I) and the final equilibrium stage (II). In I the initial rate of absorption follows a second‐order binding mechanism, with the rate constant increasing with decreasing length of the FAS. In this regime, the elasticity modulus remains more or less unaffected. Especially in regime II the absorption of FAS is enhanced by a reduction in the cross‐link density in the elastin network. This is ascribed to an osmotic pressure primarily caused by the concomitant uptake of low molecular weight ions in the elastin. The absorption equilibrium can be described by Langmuir theory. The absorption affinity increases with increasing hydrocarbon chain length of the FAS, indicating the contribution of hydrophobic interaction. Although the elasticity is not lost, the modulus is now reduced and a concomitant viscous component is developed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 472–485, 1999     

Strategies for gathering structural information on unknown peaks in the GC/MS analysis of Corynebacterium glutamicum cell extracts

A comprehensive analysis of low molecular weight compounds in biological samples by the hyphenated method of GC/MS in general detects a large number of peaks which can not be identified by searching of commercially available mass spectral libraries. Therefore, more information is required for a successful identification of these compounds. Some structural features like molecular weight and number of derivatization groups present in the molecule can be determined by variation of the derivatization prior to GC/MS. The use of deuterated derivatizing reagents and the newly developed N-methyl-N-ethyldimethylsilyl-trifluoracetamide for this purpose is described. The knowledge of these structural properties alone undoubtedly will not lead to the structure elucidation of novel metabolites. However, it may be helpful in identifying derivatives of known metabolites or artifacts. Thus, by use of the molecular weight as search criterion it is possible to find plausible candidates in metabolic pathway databases. It is shown that the application of this method to a hydrophilic extract of C. glutamicum lead to the identification of 31 in previous analyses unidentified peaks, in their majority representing minor derivatives of common metabolites.     

On the action of ozone on gelatin

Gelatin, the lower molecular weight derivative of collagen, was treated with ozone and the structural changes were studied with polarimetry, electronic absorption and FT-IR spectroscopy. The resulting ozonized gelatins were studied by thermal analysis (DSC and TGA) in comparison to a reference gelatin. It has been found that at relatively low ozone dose, for instance at ozone/gelatin molar ratios between 0.1 and 0.35 the structural damage introduced by ozone oxidation of gelatin is minimal to negligible and this may facilitate the industrial utilization of ozone in the cold sterilization of gelatin as well as in its bleaching processes required for certain applications. At higher O(3)/gelatin molar ratios the gelatine damage is evident both polarimetrically as suggested by the drop of its specific optical rotation and by spectrophotometrical analyses.     

当归及其混淆品独活、欧当归的紫外鉴别

为寻找当归及其混淆品独活、欧当归的紫外吸收光谱鉴别特征，采用紫外谱线组法对三者在不同极性溶剂中的紫外吸收光谱图进行比较。结果表明，当归和独活在四种极性溶剂中均存在明显差异，而当归和欧当归仅在无水乙醇和蒸馏水溶剂中鉴别差异显著。紫外谱线组法可以鉴别当归及其混淆品独活、欧当归。     

Studies on safety aspects of contraceptive Magainin-A in rabbits.

We have previously reported the contraceptive potential of Magainin-A in rats and rabbits under in vitro and in vivo condition. In this report we evaluated the effect of Magainin-A on the structural organisation of vaginal epithelium in rabbits. The effect of this compound on the erythrocytes and its rate of absorption and clearance from systemic circulation was also studied. The effective contraceptive dose of Magainin-A (1 mg) when administered intra-vaginally for five consecutive days did not induce any structural or morphological abnormalities in vaginal epithelial cells. No adverse effect was observed on the erythrocytes. The rate of Magainin-A absorption and clearance from the circulation was found to be rapid. These results suggest that Magainin-A may be used as a safe intra-vaginal contraceptive compound in future.     

High-throughput determination of identity, purity, and quantity of combinatorial library members using LC/MS/UV/ELSD

We have used a high-throughput LC/MS/UV/ELSD method to rapidly determine the absolute quantity and purity of 42 organic compounds from seven lead discovery libraries. A general calibration curve generated from a different set of 42 compounds with seven different scaffolds was used in this analysis. We have also studied 33 organic compounds with different molecular weight (MW) by LC/MS/UV/ELSD to investigate the effect of MW on ELSD response and the accuracy for purity and quantity measurement using UV(214) and ELSD. A general ELSD calibration curve from these compounds was also generated to quantify 42 library compounds. Purity measurement by ELSD underestimates the amounts of impurities due to a reduced ELSD response from smaller molecular weight impurities often produced in library synthesis. Absolute quantity determination by ELSD is more accurate (RSD 28%) than that by UV(214) (48%) using a calibration curve generated from the same set of compounds with diverse MWs. Error assessment for the measurement of absolute quantity of a class of commercial compounds and a class of representing reference compounds from seven diverse lead discovery libraries shows that structurally related compounds should be used to generate calibration curves to sustain smaller deviation.     

Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy

An important part of characterizing any protein molecule is to determine its size and shape. Sedimentation and gel filtration are hydrodynamic techniques that can be used for this medium resolution structural analysis. This review collects a number of simple calculations that are useful for thinking about protein structure at the nanometer level. Readers are reminded that the Perrin equation is generally not a valid approach to determine the shape of proteins. Instead, a simple guideline is presented, based on the measured sedimentation coefficient and a calculated maximum S, to estimate if a protein is globular or elongated. It is recalled that a gel filtration column fractionates proteins on the basis of their Stokes radius, not molecular weight. The molecular weight can be determined by combining gradient sedimentation and gel filtration, techniques available in most biochemistry laboratories, as originally proposed by Siegel and Monte. Finally, rotary shadowing and negative stain electron microscopy are powerful techniques for resolving the size and shape of single protein molecules and complexes at the nanometer level. A combination of hydrodynamics and electron microscopy is especially powerful.     

Lack of evidence in vivo for nitrergic inhibition by Escherichia coli (STa) enterotoxin of fluid absorption from rat proximal jejunum

Fluid absorption from the proximal jejunum of the anaesthetised rat was measured in vivo by fluid recovery. As expected, heat stable (STa) enterotoxin from E. coli reduced fluid absorption. Neither intraperitoneal L-NAME, thought to inhibit a putative neurally mediated action of STa, nor similar doses of D-NAME, ameliorated the inhibitory effect on jejunal fluid absorption of STa. Luminally perfused 10 mM sodium nitroprusside (SNP) had no effect on fluid absorption when expressed per gram dry weight per hour but reduced fluid absorption when expressed per cm length per hour. Similarly, 80 but not 40 mg/Kg of L-NAME reduced fluid absorption when expressed per cm length per hour, while the same dose of D-NAME did not. L-NAME and SNP significantly increased the wet weight to dry weight and the length to dry weight ratio of perfused loops. We conjecture that smooth muscle relaxation caused by these compounds increases interstitial fluid volumes that can be misconstrued as changes in absorption when this is expressed per cm length or per tissue wet weight. When fluid absorption is expressed per gram dry weight of tissue, there is no evidence for a role of nitric oxide in normal or STa inhibited fluid absorption.     

Identification of peptides containing tryptophan, tyrosine, and phenylalanine using photodiode-array spectrophotometry

The characteristic absorption spectra of aromatic amino acids between 240 and 310 nm were used to identify tryptophan, tyrosine, and phenylalanine-containing peptides. In acidic solution, the absorption spectra of these amino acids exhibit minima or maxima at 255, 270, and 286 nm. Based on these characteristics, the content of the aromatic amino acid in peptide can be estimated. For this study, 2 nmol of tryptic peptides from human apolipoprotein A-1 was separated by high-performance liquid chromatography using a reverse-phase column. The peptide fragments were monitored by a photodiode-array spectrophotometer. This new approach offers a rapid, simple, sensitive, and direct identification of peptides containing aromatic amino acids. Those containing Trp, which may be of interest for DNA sequencing and important in sequence analysis of proteins, can be selectively purified using this technique.     

A structural study of bovine lens alpha-crystallin and its subunits by absorption and linear dichroism spectroscopy

The structure of the bovine alpha-crystallin aggregate and its reaggregated isolated subunits has been studied by measurement of their absorption and linear dichroism spectra over the range 250-350 nm. Also, changes in structure with respect to time have been monitored in this way. From the absorption spectra it appears that the aromatic residues in subunit aggregates are in the same chemical environment as those in native protein. The light scattering due to the size of the protein molecules increases when the proteins are kept in solution, this effect being much stronger for the subunits. The linear dichroism spectra point to strong structural ordering in alpha-crystallin, the absorption transition dipoles of the aromatic residues being preferentially aligned along the long axis of the molecules. Moreover, a considerable deviation from a spherical or tetrahedrally symmetric structure of alpha-crystallin is inferred. The subunit aggregates show less ordering and might be more spherical. When kept in solution, their structural order seems to be decreased. The linear dichroism spectra show absorption at 325 nm, which is not detectable in the normal absorption spectra.     

Chromatin models. Thermal denaturation studies of (Lysx, Leuy)n-and (Lys)n(Leu)m-DNA complexes.

The structural transitions of (Lysx, Leuy)n-DNA and (Lysx)n(Leuy)m-DNA complexes have been studied by thermal denaturation utilizing simultaneous absorption and circular dichroism (CD) measurements [R. Mandel and G.D. Fasman (1974), Biochem. Biophys. Res. Commun. 59, 672]. These complexes are used as models for nucleohistones. At amino acid/nucleotide ratios r less than 1, the copolymers bind to DNA in a ratio of one amino acid residue per nucleotide, and such binding stabilizes the DNA double helix against thermal denaturation relative to the unbound regions. The leucine residues in the copolymers stabilize the bound portion of the complex against thermal denaturation but to a lesser degree than does poly(L-lysine). This study confirms the hypothesis that absorption melting profiles reflect only the change in secondary structure (helix-coil transition) of DNA. It was found that, in the absence of a higher ordered structure (condensed), the CD melting profile also reflects this same conformational transition, and the melting temperatures, Tm, in CD are equal to those in absorption. However, when a higher ordered structure (tertiary) exists in the complex, then the CD melting profile will be dominated by the structural transitions related to the melting of the higher ordered asymmetric structure in the condensed state, followed by the melting of the secondary structure. Under such circumstances, the Tm obtained from absorption may be slightly different from that of the CD, since only the secondary structural changes are being reflected in absorption. The relevance of these studies to the structure of chromatin is discussed.     

Mass spectrometric approaches using electrospray ionization charge states and hydrogen-deuterium exchange for determining protein structures and their conformational changes

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.     

Development and evaluation of a liquid chromatography tandem mass spectrometry method for simultaneous determination of salivary melatonin, cortisol and testosterone

Circadian disruption can have several possible health consequences, but is not well studied. In order to measure circadian disruption, in relation to shift or night work, we developed a simple and sensitive method for the simultaneous determination of melatonin, cortisol and testosterone in human saliva. We used liquid-liquid extraction (LLE) followed by liquid chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS) recorded in positive ion mode. Saliva samples were collected by spitting directly into tubes and 250 μL were used for analysis. The limits of detection were 4.1 pmol/L, 0.27 nmol/L and 10.8 pmol/L for melatonin, cortisol, and testosterone, respectively. The developed method was sensitive enough to measure circadian rhythms of all 3 hormones in a pilot study among four healthy volunteers. It can therefor be used to study the impact of night work and working in artificial light on the workers circadian rhythms. To our knowledge this is the first LC-ESI-MS/MS method for simultaneous determination of salivary melatonin, cortisol and testosterone.