Collisional Spectroscopy in Structural Characterization of Melanins: I. A First Study on |C8H7ON|+· Ions Originating From Pyrolysis of Biosynthetic and Synthetic Tryptophan Melanins

The structural investigation of four bio- and synthetic melanins, obtained by the action of polyphenol oxidase from potatoes and Psalliota campestris mushrooms and of tyrosinase from Sepia officinalis on tryptophan, or by means of performic oxidation, has been carried out by thermal decomposition performed in the ion source of a mass spectrometer. The structural characterization of the ionic species |C₈H₇ON|⁺· common and abundant for all the examined compounds, has been obtained by accurate mass measurements and collisional spectroscopy. These mass spectrometric techniques have shown unequivocally for ionic species | C₈H₇ON| ⁺· the structure of 2-indolinone.     

NMR studies of melanins: characterization of a soluble melanin free acid from Sepia ink.

This paper deals with the nuclear magnetic resonance characterization of a soluble derivative (melanin free acid) of Sepia melanin obtained by a peroxidative treatment of the parent (insoluble) species. High resolution 13C and 15N solid state NMR spectroscopies allow the assessment of the chemical changes occurring in the macromolecule upon solubilization. 1H and 13C NMR solution spectra are discussed in light of the results obtained from the solid state spectra. Furthermore, the coordination properties of melanin have been investigated through 27Al NMR spectroscopy and proton relaxation enhancement studies of the paramagnetic gadolinium complex of melanin free acid. Through these experiments it has been possible to evaluate the molecular reorientational time tau R (and from it an estimated molecular weight close to 20 KDa) and the strength of the metal-macromolecule interaction.     

Characterisation of non-activated and activated estrogen- and antiestrogen-receptor complexes by high performance ion exchange chromatography

The ionic species of cytosol estrogen receptors from mature rat uteri have been compared by HPIEC on a SynChrom AX-1000 column when complexed with either [125I]iodoestradiol, [3H]estradiol or [3H]4-hydroxy tamoxifen. Three species of receptors (isoforms) each suppressible by excess competitor were fractionated at identical salt concentrations regardless of ligand employed. One species eluted in the column void volume (10 mM) and the others at congruent to 90 mM and congruent to 155 mM phosphate. Activation of receptor complexes by increasing time of incubation with ligand from 1 to 24 h at 4 degrees C or addition of 10 mM GTP increased the proportion of the congruent to 90 mM species for all 3 ligands. The addition of 10 mM molybdate to homogenization and HPIEC buffers resulted in only two species being resolved at 10 and 110 mM phosphate. These species were again identical regardless of ligand employed. Increasing concentrations of estradiol (1-40 nM) tamoxifen (20 nM-4 microM) and 4-hydroxy tamoxifen (2-400 nM) were able to compete for binding of [125I]iodoestradiol to each of the three ionic species. Binding to each species was inhibited equally by each concentration of competitor. There was no preferential or unique association of estrogen or antiestrogen with any of the ionic species and all ligands gave identical ionic species of non-activated and activated receptor complexes.     

Effects of 1-octyl-3-methylimidazolium bromide on the antioxidant system of Lemna minor

Ionic liquids have gained more attention due to their excellent properties in many different scientific fields. However, previous researches indicated that ionic liquids have adverse effects on organisms. The objective of this study was to evaluate the effects of 1-octyl-3-methylimidazolium bromide ([C(8)mim]Br) on the aquatic plant duckweed (Lemna minor) by exposure of the plant to 0.25 to 2 mg L(-1) of [C(8)mim]Br for 28 days. Exposure to [C(8)mim]Br significantly decreased the photosynthetic pigment contents at 21 and 28 days. The activities of superoxide dismutase and catalase and the total antioxidant capacity level increased at 7 days of exposure and decreased at the termination of exposure. In contrast, the H(2)O(2) content and peroxidase activity in all treatments increased during the period of exposure. Furthermore, marked increase of malondialdehyde content occurred in duckweed after 21 to 28 days of exposure. In addition, reactive oxygen species (ROS) scavenger dimethyl thiourea prevents duckweed from oxidative damage caused by [C(8)mim]Br. These results suggest that ROS might be involved in the mechanism of ionic liquid-induced toxicity in L. minor.     

Melting of short DNA fragments. Relation between the nucleation constant of a spiral region and the ionic strength

Melting of two DNA duplexes of known nucleotide sequences containing 14 and 36 base pairs has been investigated within the range of ionic strength from 0.2 to 0.02 M [Na+]. The values of melting enthalpy of base pair delta H were measured for the duplex of 14 base pairs in the solutions of varying ionic strength. The values of delta H were obtained from slopes of linear plots of reciprocal melting temperature versus logarithm of oligonucleotide chains concentration. In the aforementioned range the decrease of the ionic strength causes a 5% decrease of delta H. By fitting the theoretical profiles to the experimental ones the ionic strength dependence of the nucleation constant beta was measured for DNA fragments of various lengths. With the decrease of the ionic strength the value of beta drops 2 times for the short duplex and 8 times for the long one.     

Neuraminic acid-specific modification and tritium labelling of gangliosides.

1. A crude ganglioside mixture and pure GM1 and GD1a from bovine brain grey matter were prepared on a large scale. 2. The C7- and G8-analogues of NeuNAc were prepared from Collocalia mucoid and their structures established by gas-liquid chromatography and mass spectrometry. 3. Using model compounds in addition to various gangliosides, the conditions for the periodate oxidation and subsequent borohydride reduction of gangliosides were investigated with regard to the yield of C7- and C8-analogues of NeuNAc and the integrity of other monosaccharides in the oligosaccharide chain. These conditions were optimised to yield maximum C8-NeuNAc production and low C7-NeuNAc formation. Thus products were obtained which closely resemble the native gangliosides. 4. Using boro [3H] hydride, ganglioside derivatives with high specific radioactivity were prepared for the first time, containing either NeuNAc and labelled C8-NeuNAc or mainly labelled C7-NeuNAc depending on the prevailing conditions.     

A stable isotope dilution assay for disopyramide and its [13C, 15N]labelled analogue in biological fluids

The mass spectra of disopyramide phosphate and two stable isotopically labelled analogues have been obtained using electron impact and chemical ionization. The low isotopic purity of [13C, 15N)disopyramide phosphate was shown to be due to the low isotopic purity of the 15N label. A stable isotope dilution assay for disopyramide and [13C, 15N]disopyramide in biological fluids has been developed using [2H14]disopyramide phosphate as the internal standard. This assay will be used to analyse samples obtained after the co-administration of disopyramide phosphate intravenously and [13C, 15N]disopyramide phosphate orally to several animal species.     

8-aza-7-deazaadenine and 7-deazaguanine: synthesis and properties of nucleosides and oligonucleotides with nucleobases linked at position-8

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N8-(2'-deoxyribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-amin-(3H)-4-one) C8-(2'-deoxyribonucleoside) (4) were synthesized and incorporated in oligonucleotides employing phosphoramidite chemistry. Oligonucleotides carrying compound 2 are able to form base pairs with the four canonical DNA constituents without significant structural discrimination. The nucleoside 4 was obtained from the corresponding ribonucleoside by deoxygenation. Oligonucleotides containing compound 4 showed similar base pairing properties as those with 2'-deoxyisoguanosine.     

Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates

The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies.     

The effects of selective amino acid substitution upon neuropeptide Y antisecretory potency in rat jejunum mucosa

The antisecretory potency of NPY and a series of truncated and structural analogues of NPY have been tested upon mucosal preparations of rat small intestine. Single amino acid substitutions, i.e., [Ile34]NPY, [Pro34]NPY, resulted in severe attenuation and loss of biological activity, respectively, and neither peptide affected NPY responses. An agonist order of potency: NPY greater than or equal to [Glu16,Ser18,Ala22,Leu28,31]NPY (ESALL-NPY) greater than [Cys2,Aoc5-24,DCys27]NPY (C2-NPY) greater than [Aoc5-24]NPY greater than [Des-Ser3,Des- Lys4]C2-NPY much greater than [Cys5,Aoc7-20,DCys24]NPY (C5-NPY) greater than equal to [DCys7,Aoc8-17, Cys20]NPY (C7-NPY) greater than [Aoc8-17]NPY greater than or equal to [Ile34]C7-NPY much greater than [Aoc2-27]NPY much greater than [Pro34]C2-NPY was obtained. The use of analogues based upon the tertiary structural model of NPY with varying amounts of N- and C-terminal helical regions removed and replaced with a single 8-aminooctanoic acid residue (Aoc) has allowed us to assess the structural requirements for activation of the regions in close apposition to each other. The polyproline helix, beta-turn and majority of the amphipathic alpha-helix serve a structural role bringing N- and C-terminal residues together for optimal receptor recognition and activation.     

Amino acid sequence of a 12-kDa inhibitor of protein kinase C

The complete primary structure of a bovine-brain-derived inhibitor of protein kinase C has been established. Fragments of the purified protein were obtained by cleavage with cyanogen bromide, Staphylococcus aureus V8 protease, trypsin and chymotrypsin. Subsequent analysis of the resulting fragments by fast-atom-bombardment mass spectrometry and Edman degradation revealed a calculated molecular mass of 11,779 Da with the following 107-amino-acid sequence: [sequence: see text] This inhibitor does not share significant primary structural identity with any other known protein.     

Ferricytochrome c oxidation of cobaltocytochrome c. Comparison of experiments with electron-transfer theories.

Electron transfer from cobaltocytochrome c to ferricytochrome c has been studied by stopped-flow kinetics. The second-order rate constant at pH 7.0, 0.1 ionic strenght, 0.2 M phosphate, and 25 degrees C is 8.3 x 103 M-1 s-1. The activation parameters obtained from measurements made between 20 and 50 degrees C are deltaHnot equal to = 2.3 kcal mol-1 and deltaSnot equal to = -33 eu. The rate constant is not significantly dependent on ionic strength; it is also relatively independent of pH between the pK values for conformation transitions. The rate diminishes at pH greater than 12. The self-exchange reaction of cobalt cytochrome c was investigated with pulsed Fourier transform 1H NMR. The rate is too slow on the 1H NMR scale; it is estimated to be less than 133 M-1 s-1. These results together with the self-exchange rates of iron cytochrome c [Gupta, R.K., Koenig, S. H., and Redfield, A. G. (1972), J. Magn. Reson. 7, 66] were analyzed by theories of Jortner and Hopfield. The theories predict the self-exchange of Cocyt c to be too slow for 1H NMR determination. The rate constant calculated by the nonadiabatic multiphonon electron-tunneling theory for the Fecyt c-Fecyt c+ and Cocyt c-Fecyt c+ electron transfers are in good agreement with experiments.     

Energetics of the equilibrium between two nucleotide-free myosin subfragment 1 states using fluorine-19 nuclear magnetic resonance

A new fluorine-containing reagent has been synthesized and used to specifically label the reactive sulfhydryl [sulfhydryl-1 (SH1)] of myosin subfragment 1 (S-1). The labeled S-1 (S-1-CF3) demonstrates activated calcium and magnesium adenosinetriphosphatase (ATPase) activities relative to S-1 and a lower potassium ethylenediaminetetraacetate (EDTA) ATPase activity. Maximal effect is obtained with the modification of one thiol per S-1. The 19F NMR spectrum of S-1 CF3 contains only one resonance with a line width of 110 Hz, which implies a rotational correlation time of 2.3 X 10(-7) s. The chemical shift of this resonance is sensitive to temperature, PH, ionic strength, and nucleotides bound in the active site. The temperature dependence of the chemical shift clearly indicates two limiting states for the S-1-CF3 with a highly temperature-dependent equilibrium between 5 and 40 degrees C. The low-temperature state appears to be identical with the state resulting from the binding of Mg.ADP or Mg.AMPPNP at 25 degree C. The energetics of the conformational change have been studied under various conditions. At pH 7 in 25 mM cacodylate, 0.1 M KCl, and 1 mM EDTA, delta H degree = 30 kcal/mol and delta S degree = 105 cal deg-1 mol-1. A decrease in pH to 6.5 results in an increased population of the low-temperature state with delta H degree = 31 kcal/mol and delta S degree = 107 cal deg-1 mol-1. Similarly, the low-temperature state is favored by low ionic strength. In 5.8 mM piperazine-N,N'bis(2-ethanesulfonic acid) and 1 mM EDTA (pH 7), delta H degree = 8 kcal/mol and delta S degree = 27 cal deg-1 mol-1. We have also obtained 19F NMR spectra of S-1-CF3 in D2O solution with 30% ethylene glycol at pH 7.1. Increasing concentrations of ethylene glycol progressively stabilize the high-temperature states.     

A naturally occurring deaminated neuraminic acid, 3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN). Its unique occurrence at the nonreducing ends of oligosialyl chains in polysialoglycoprotein of rainbow trout eggs

An unknown deaminated sialic acid has been isolated from Salmo gairdneri (rainbow trout) egg polysialoglycoprotein. A combination of structural methods including gas-liquid chromatography, chemical and enzymatic analyses, mass spectrometry, and 400-MHz 1H NMR spectroscopy was used to determine the structure as 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (= 3-deoxy-D-glycero-D-galacto-nonulosonic acid; KDN). This structure has been confirmed by comparison with a chemically synthesized authentic sample of KDN. The natural occurrence of deaminated sialic acid in biological material has not been previously reported. A series of KDN-containing oligosialic acids were isolated from the polysialoglycoprotein after pH 4.7-catalyzed hydrolysis. Structural studies including methylation analysis, mass spectrometry, 1H NMR spectroscopy, and chemical reactivity were also used to confirm the structures of the sialyloligosaccharides as KDN alpha 2[8NeuGc alpha 2-]n (n = 1-7). The exclusive location of KDN at the nonreducing termini in polysialoglycoproteins protects oligo(poly)sialyl chains from exosialidases. Terminal capping of these chains may be important in egg activation in salmonid fishes.     

Physicochemical properties of salt-soluble,unsheared chromatin

The mass per unit length of and the corresponding DNA packing ratio of about 14 for the chromatin soluble at moderate ionic strengths has been determined by light scattering. With the increase in ionic strength and corresponding release of histone H1 the DNA packing ratio has been found to decrease down to 4.4. The data obtained are consistent with the idea suggested previously that the salt-soluble chromatin is organized in double nucleosome chains arranged side-by-side and stabilized by H1. With salt-induced H1 release the double chain dissociates and the nucleosomal DNA partially unravels.     

Fast Atom Bombardment and Tandem Mass Spectrometry for the Identification of Nucleoside Adducts with Phenyl Glycidyl Ether

Abstract

The present study deals with the use of fast atom bombardment (FAB) in combination with constant neutral loss (CNL) scanning, high resolution mass spectrometry and tandem mass spectrometry (MS-MS) with collisionally activated decomposition (CAD), as complementary methods for the identification and structural analysis of phenyl glycidyl ether-nucleoside adducts. Selective detection of the parent ions of the modified nucleosides at the 1–10 ng level has been achieved by suitably designed CNL scans. The elemental composition of the adducts has been determined by accurate mass measurements. CAD-MS has been carried out on the [M + H]⁺ and [M - H]^? ions to derive structural data on the size and nature of the base, sugar and alkyl substituent. In some cases, information on the alkylation site has been obtained, which is very useful for distinguishing isomeric adducts.     

Structure and function of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Species difference in molecular properties of the receptors from mouse and rat hepatic cytosols

Molecular properties of cytosolic Ah receptors from livers of Sprague-Dawley rats and C57BL/6N mice were assessed by velocity sedimentation on sucrose gradients and by gel permeation chromatography on Sephacryl S-300. Analyses were done under conditions of both moderate ionic strength (presence of 0.1 M KCl) and high ionic strength (0.4 M KCl). [3H] 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was used as the radioligand. In conditions of moderate ionic strength the receptor from Sprague-Dawley rat liver sedimented at 8.8 +/- 0.05 S, had a Stokes radius of 7.0 +/- 0.21 nm, and an apparent relative molecular mass (Mr) of 257,000 +/- 7,700. In conditions of high ionic strength the Ah receptor from rat hepatic cytosol dissociated to a [3H]TCDD-binding subunit which sedimented at 5.6 +/- 0.58 S, had a Stokes radius of 5.2 +/- 0.24 nm, and an apparent Mr of 121,000 +/- 5,600. The Ah receptor from liver of C57BL/6N mice, in moderate ionic strength conditions, sedimented at 9.4 +/- 0.54 S, had a Stokes radius of 7.1 +/- 0.12 nm, and an apparent Mr of 277,000 +/- 4,800. Whereas the Ah receptor from rat liver readily dissociated into a [3H]TCDD-binding subunit during brief exposure to 0.4 M KCl, the mouse Ah receptor resisted dissociation. When exposed to 0.4 M KCl for 2 h, the mouse Ah receptor remained at the same molecular size that it had exhibited in moderate ionic strength conditions. Prolonged exposure (16 h) to 0.4 M KCl prior to analysis partially converted the mouse Ah receptor into a smaller [3H]TCDD-binding subunit which sedimented at 4.9 +/- 0.07 S, had a Stokes radius of 5.2 +/- 0.19 nm, and an apparent Mr of 105,000 +/- 3,800. The potency of seven different Ah receptor agonists in competing with [3H]TCDD for specific receptor sites was slightly different in mouse cytosol than in rat cytosol. By criteria of size, response to high ionic strength environments, and ligand binding preferences the mouse and rat Ah receptors appear to be similar but not identical molecular species.     

Structure of iturine A, a peptidolipid antibiotic from Bacillus subtilis.

A mixture of iturines extracted from Bacillus subtilis gave, on column chromatography, iturine A, iturine B, and iturine C. Iturine A has the entire antifungal activity. It is a mixture of two homologous peptidolipids C48,H74N12O14 and C49H76N12O14 (mp 177 degrees C, [alpha]D-1.7 degrees in methanol (c 0.05 g/mL); mol wt 1042 and 1056). The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid and 3-amino-12-methyltetradecanoic acid. The peptide moiety contains 7 mol of amino acids: D-Asn2, L-Asn, L-Gln, L-Pro, L-Ser, and D-Tyr. A cyclic structure for iturine A with the serine residue linked to the fatty amino acids through a peptide bond has been domonstrated. By mild HCl hydrolysis, lipid-soluble and water-soluble peptides were obtained. They were analyzed by chemical methods and by mass spectrometry. Permethylated and perdeuteriomethylated derivatives of iturine A were also subjected to mass spectrometric analysis. Both chemical analysis and mass spectrometry led to the cyclic structure I for iturine A.     

Biosynthesis of [7-3H]16 alpha-hydroxy-dehydroepiandrosterone having high specific activity.

Biosynthesis of [7-3H]16alpha-hydroxy-dehydroepiandrosterone in high specific activity has been studied. [7-3H] dehydroepiandrosterone (13.9 C/mM) in trace quantity was oxidized by Streptomyces roseochromogenes (NRRLB-1233) for 5 min at 27 degrees C. The radioactive products were chromatographically separated, identified and their radiochemical purity established by isotopic dilution analysis. [7-3H]16alpha-hydroxy-dehydroepiandrosterone (2.5 x 10(7) dpm) was obtained by microbial hydroxylation of substrate (1.9 X 10(9) dpm). In some cases [7-3H])5-androstene-3beta, 16alpha, 17beta-triol in a small amount of radioactivity could be found at the prolonged reaction for 30 hr.     

Synthesis and structure determination of [8(-13)C]guanosine 5'-diphosphate

A combined chemical and enzymatic synthesis of [8(-13)C]guanosine 5'-diphosphate (GDP) from H13COOH is described. About 35 mg nucleotide was obtained from 500 mg H13COOH. Analysis of the [8(-13)C]GDP by negative ion fast atom bombardment mass spectrometry and by 13C NMR confirmed that one atom of 13C was incorporated at the 8-position of the guanine ring at 90 +/- 10% enrichment. The chemical shift of the C(8) was 140.2 ppm downfield from internal trimethylsilylpropionate at neutral pH and room temperature, with a spin-spin coupling 1J(13C(8)-H(8] of 217 Hz and a 3J(13C(8)-H(1'] of 3.9 Hz.