Synthesis of protein, nucleosides and other organic compounds from cyanamide and potassium nitrite under possible primitive earth conditions.

The mixing of cyanamide and KNO2 produced changes from white solids to yellow liquid and then to orange solid. The gases cyanogen and ammonia were formed. No external energy was used. The reactions were carried out with a small amount of O2. The presence of proteins in the reaction product formed 13 months after the mixing was indicated by the positive reactions of the cyanamide-KNO2 reaction product with ninhydrin, microbiuret, and Folin reagent; the ultraviolet absorption at about 280 nm; the yield of 24% of 15 amino acids; and molecular weight measurements of more than 160,000. The presence of nucleosides, nucleic acid bases, hydrocarbons, and organic esters in the reaction product formed 2 months after the mixing was indicated by ultraviolet absorption at about 260 nm, and the results of ligand-exchange chromatography, paper chromatography, infrared analysis, mass spectral analysis, and NMR spectroscopy. Possible cyanamide-mediated dehydration reactions and mechanisms are discussed.     

Terephthalic acid: A dosimeter for the detection of hydroxyl radicals in vitro

Hydroxylation reactions of aromatic compounds have been used to detect hydroxyl radicals produced by gamma irradiation and ultrasound. The present study investigated the suitability of terephthalic acid (THA) as a hydroxyl radical dosimeter for general use in biologically relevant reactions. Hydroxyl radicals were generated by: (1) irradiating, THA with a 254 nm ultraviolet; (2) irradiating with gamma rays from a cesium source; and (3) generating hydroxyl radicals with 1 mM H₂O₂ and 10 μM Cu⁺². In each of the three experiments, a fluorescent product was generated which exhibited identical fluorescent excitation and emission spectra. THA is non-fluorescent, eliminating the problem of a high initial background. Because THA has four ring hydrogens, only one mon-hydroxylated isomer was formed. The hydrogen peroxide reaction was dependent on the presence of a metal and cupric ions were effective in enhancing the reaction. With a Cu⁺² concentration of 10 μM, the reation was linear between 0–30 mM H₂O₂. Catalase abolished the reaction at a concentration of 100 μg/ml and the effects could still be observed at 10 ng/ml, consistent with the very high rate at which catalase destroys hydrogen peroxide. Tertbutyl- hydroperoxide did not generate any fluorescence in this system which makes THA a very specific detector of hydroxyl radicals.     

Cyanamide mediated syntheses of peptides containing histidine and hydrophobic amino acids

Summary Using the model of a primitive earth evaporation pond, the synthesis of three histidyl peptides in yields of up to 11% was demonstrated when aqueous solutions of histidine, leucine, ATP, cyanamide, and MgCl₂ were evaporated and heated for 24 h at 80°C. In addition, peptides were formed in yields of up to 56%, 35%, and 21%, respectively for phenylalanine, leucine, and alanine when aqueous solutions of the appropriate amino acid were evaporated and heated with cyanamide and one or more of the following components: ATP, AMP, 4-amino-5-imidazole carboxamide, or MgCl₂. The greatest peptide yield occurred at pH 3. But peptide formation was demonstrated for a system of Leu, cyanamide, and MgCl₂ adjusted to pH 7 with NH₄OH.Peptide synthesis was also studied in the presence of CaCl₂, ZnCl₂, different adenosine nucleotides, and UTP to compare their effects on peptide synthesis. The optimum conditions for cyanamide mediated peptide synthesis were also studied in terms of pH, reaction time, reaction temperature, and cyanamide concentration. The major side product in nearly all reactions studied appears to be an amino acid-cyanamide adduct. Peptides were analyzed and identified by thin layer chromatography, acid hydrolysis, and enzymatic degradation.     

The use of diaminobenzidine for spectrophotometric and acrylamide gel detection of sulfite oxidase and its applicability to hydrogen peroxide-generating enzymes

DAB, in the presence of HRP, sulfite oxidase and FBS, polymerizes to a product with an absorption difference maximum at 352 nm. This reaction has been used as a sensitive and specific spectrophotometric assay for sulfite oxidase. Incubation of acrylamide gels containing sulfite oxidase with assay mixtures produces an insoluble product which precipitates where the enzyme is localized. This stain is at least as sensitive as the amido black protein stain and its specificity is such that no band is seen in the absence of substrate, and that only one band, migrating identically to purified enzyme is seen in rat liver homogenates. This polymerization reaction has been applied to other H₂O₂-generating enzymes and can be used to demonstrate the presence of these enzymes in the midst of other oxidases. DAB polymerization provides a sensitive spectrophotometric assay which can be used with either ultraviolet or visible optics. The application of this procedure to modified enzymes is discussed.     

N-Acetyl-L-Tyrosine (NAT) as a Substrate for Mushroom Tyrosinase

N-acetyl tyrosine (NAT) is hydroxylated by mushroom tyrosinase and the N-acetyl dopa formed is oxidized by the enzyme to N-acetyl dopaquinone (λ_max= 390 ± 10 nm). H₂O₂ and NH₂OH each shortened the lag period of NAT hydroxylation by the enzyme. H₂O₂ had an effect on the changes with time in the spectrum of product(s) formed and on the spectrum of the final product(s) obtained when NAT was hydroxylated by mushroom tyrosinase, in a manner suggesting that H₂O₂ converts N-acetyl dopaquinone to a pink-violet product(s) (λ_max= 490 nm), whereas such a product(s) was not formed in the absence of H₂O₂. A pink-violet product(s) (λ_max 490 ± 20 nm) was also formed when NAT was hydroxylated by mushroom tyrosinase in the presence of NH₂OH or para amino benzoic acid (PABA), probably as a result of an interaction between N-acetyl dopaquinone and NH₂OH or PABA forming mono- or di-oximes. Kojic acid (5-hydroxy-2-hydroxymethyl)-4H-pyran-4-one) inhibited effectively the rate of NAT hydroxylation by mushroom tyrosinase in the absence or presence of H₂O₂. When NAT was oxidized by the enzyme in the absence of kojic acid, N-acetyl dopaquinone was formed at once and a shoulder at 490–530 nm appeared later. Under identical conditions but in the presence of kojic acid, a yellow product(s), characterized by a peak at 320±10 nm, was detected, suggesting that N-acetyl dopaquinone oxidizes kojic acid to the yellow product(s). Maltol(3-hydroxy-2-methyl-4H-pyran-4-one), a γ-pyrone derivative structurally related to kojic acid, also inhibited the rate of NAT hydroxylation by mushroom tyrosinase. The addition of maltol at the plateau phase of the reaction resulted in an immediate decline in absorbance at 400 nm, suggesting that maltol conjugates with N-acetyl dopaquinone, yielding a product(s) characterized by a lower extinction coefficient at 400 nm than that of N-acetyl dopaquinone alone. The final brown-red product(s) formed when NAT was hydroxylated by mushroom tyrosinase was bleached in the presence of ascorbic acid or H₂O₂.     

13C nmr studies of aurothioglucose: Ligand exchange and redox disproportionation reactions

¹³C nmr studies of gold thioglucose, AuSTg, and solutions containing added β-1-D-thioglucose, TgSH, have been conducted at PD 7.4 and interpreted in terms of complexation and ligand exchange reactions that are consistent with the known preference of gold(I) for linear two-coordinate structures. The upper limit of the half-life for ligand exchange between 0.25 M Au(STg)₂^? and TgSH at pD 7.4 is 2.2 msec. The ¹³C nmr spectra of various thioglucose derivatives have been assigned. A novel oxidation reduction reaction was discovered that leads to the formation of metallic gold and a product tentatively identified as the sulfinic acid derivative of thioglucose. The presence of sulfinic acid in AuSTg was indicated by the infrared absorption at 1050 cm^?1. The same product was formed by slow hydrolysis of thioglucose disulfide. A mechanism for the formation of the sulfinic acid derivative from AuSTg is proposed.     

Absorption and circular dichroism spectra of CuCl2 complexed with poly(S-carboxymethyl-L-cysteine) and poly(S-carboxyethyl-L-cysteine)

The interaction of CuCl₂ with poly(S-carboxymethyl-L -cysteine) (poly[Cys(CH₂COOH)]) and poly(S-carboxyethyl-L -cysteine) (poly[Cys(C₂H₄COOH)]) were studied by absorption spectra and circular dichroism (CD). On mixing CuCl₂ with polypeptide solutions, absorption bands appeared at 320–325 nm in both polypeptides, and at 255–260 nm in the case of poly[Cys(CH₂COOH)]. A stable bound species was formed in the case of poly[Cys(CH₂COOH)], since the apparent molar absorption coefficient of the bound species did not depend on the mixing ratio. From the absorption data, it was inferred that Cu²⁺ ions were complexed with the side chains, most probably with sulfur atoms and carboxyl groups. Induced optical activities were observed for the two polypeptides. The CD spectra of poly[Cys(CH₂COOH)] + CuCl₂ gave simpler aspects than those of poly[Cys(C₂H₄COOH)] + CuCl₂.     

Characterization of indoles by thin-layer chromatography and in situ fluorometry

The characterization of microgram quantities of a number of naturally occurring and synthetic indoles through a combination of thin-layer chromatography and in situ fluorescence spectroscopy is reported. Instrumental detection limits of 0.03–0.05 μg of the indoles are possible using the native fluorescence of the indoles in the ultraviolet range, with excitation maxima in the range 285–310 nm and emission maxima in the range 345–360 nm. Spraying with a dilute acid solution (0.1 N H₂SO₄ in methanol) produces an additional pair of maxima, with excitation at about 350 nm and emission at about 450 nm. The presence of a polar compound such as sulfuric acid or dimethyl sulfoxide in the spray produces an enhancement of the indole fluorescence. The procedure should find application in the determination of indoles in biological samples.     

Effects of Halides on Reaction of Nucleosides with Ozone

GRAPHICAL ABSTRACT

Ozone (O₃), a major component of photochemical oxidants, is used recently as a deodorizer in living spaces. It has been reported that O₃ can directly react with DNA, causing mutagenesis in human cells and carcinogenesis in mice. However, little is known about the effects of coexistent ions in the reaction of O₃. In the present study, we analyzed the effects of halides on the reaction of O₃ with nucleosides using reversed-phase high-performance liquid chromatography with ultraviolet detection. When aqueous O₃ solution was added to a nucleoside mixture in potassium phosphate buffer (pH 7.3), the nucleosides were consumed with the following decreasing order of importance: dGuo > Thd > dCyd > dAdo. The effects of addition of fluoride and chloride in the system were slight. Bromide suppressed the reactions of dGuo, Thd, and dAdo but enhanced the reaction of dCyd. The major products were 5-hydroxy-2′-deoxycytidine, 5-bromo-2′-deoxycytidine, and 8-bromo-2′-deoxyguanosine. The time course and pH dependence of the product yield indicated formation of hypobromous acid as the reactive agent. Iodide suppressed all the reactions effectively. The results suggest that bromide may alter the mutation spectrum by O₃ in humans.     

Some novel flavin-nicotinamide biscoenzymes and their reactivities

The present study was undertaken to investigate flavin-nicotinamide reactions and interactions. A series of novel flavin-nicotinamide biscoenzymes have been synthesized by a general three-step procedure. The structures of these compounds were confirmed by nuclear magnetic resonance spectra, absorption spectra and elemental analysis. These compounds consist of short linear hydrocarbon chains interconnecting the N-1 of nicotinamide and the N-10 of the 7,8-dimethyl-isoalloxazine ring. The compounds were reduced with sodium dithionite (Na₂S₂O₄) and the flavin portion was reoxidized with ferricyanide. Re-reduction of the flavin portion by the nicotinamide portion of the molecule was followed anaerobically at 442 nm. When the interconnecting hydrocarbon chain was unsaturated, a second order reaction was observed with a rate equal to that of lumiflavin and 1-propyl-1,4-dihydronicotinamide (NprNicH₂) under the same conditions. When the two halves of the biscoenzymes were connected by saturated three- and four-carbon chains, the expected unimolecular reaction was not observed. Instead, the reduced biscoenzyme, after separation from excess sodium dithionite, was shown to have a strong absorption at 298 nm. This absorption is characteristic of hydration of dihydronicotinamides at the 5,6-double bond.In further studies, the C₃-biscoenzyme exhibited an absorption at 600 nm due to a complex between the reduced flavin and oxidized nicotinamide portions of the molecule. Absorbance at 600 nm increased linearly with the C₃-biscoenzyme concentration, clearly indicating that this is an intramolecular complex. When the C₃-biscoenzyme was at 0°C in 60–75% dimethylformamide buffer solution, no absorption at 600 nm was observed. When excess dithionite was removed, the spectrum under these conditions showed definite peaks at 297 and 357 nm. These respective peaks were attributed to hydrated dihydronicotinamide and dihydronicotinamide species present in the reaction mixture.The reduced flavin was postulated to be a catalyst for the hydration of dihydronicotinamide. This hypothesis was tested by incubating 1-propyl-1,4-dihydronicotinamide alone and with several concentrations of reduced riboflavin under basic anaerobic conditions. The results show that the reduced flavin increases the rate of disappearance of the dihydronicotinamide species and that the product shows an absorption near 298 nm. These results indicate that a reduced form of the flavin nucleus catalyzes the hydration of dihydronicotinamides.     

Spectrophotometric study on the oxidation of rutin by horseradish peroxidase and characteristics of the oxidized products

Rutin (3',4',5,7-tetrahydroxyflavone-3-rutinoside) was oxidized by a horseradish peroxidase-H2O2 system to an ascorbate-reducible product which had an absorption maximum at about 290 nm and a shoulder at about 440 nm at pH 4. At pH 7.8, ascorbate-reducible compounds and sodium hydrosulfite-reducible and -nonreducible compounds were formed by the oxidation. The ascorbate-reducible compounds consisted of at least two components, the absorption bands of which were at 460-480 nm and about 620 nm. The sodium hydrosulfite-reducible compounds also consisted of two components, and one of the components which had an absorption maximum at about 480 nm seems to be formed from the ascorbate-reducible component of an absorption maximum at the blue region by a nonenzymatic reaction. A mixture of oxidized products of rutin formed by tert-butyl hydroperoxide-dependent oxidation was similar to that formed by the enzymatic reaction. It is discussed that the 3'- and 4'-OH groups of rutin were oxidized by the horseradish peroxidase-H2O2 system and that the oxidized product which could be reduced by ascorbate is an o-quinone derivative.     

Preparation of AzoDNA and its use in affinity chromatography

A limited coupling reaction between 4-diazobenzoic acid ([¹⁴C]carboxyl) and sheared single-stranded DNA was employed to prepare a ligand capable of bonding covalently with aminopentane Sepharose C1-4B. The ligand AzoDNA demonstrated small changes in ultraviolet absorbance spectra yet, unlike the parent DNA, had a distinct fluorescence emission peak at 400 nm when excited at 292 nm in neutral or alkaline solutions. On hydroxyapatite thermal chromatography the AzoDNA eluted as single-stranded DNA, while following catalytic reduction, the associated fluorescence and [¹⁴C]azobenzoate radioactivity were removed in large part from the derivatized DNA. In the coupling reaction, prior derivatization of the ligand DNA was required for covalent bonding to aminopentane Sepharose C1-4B and, at optimal polydeoxynucleotide concentrations, about 75 μg was bound/ml of packed gel. DNA:DNA hybridization reactions were accomplished using AzoDNA aminopentane Sepharose C1-4B gels with 50% of the hybridized polynucleotide strands being eluted at temperatures approximating the T_m values measured optically. The use of the AzoDNA gel was extended to the hybridization of adenovirus 2 and vaccinia complementary RNA. The viral complementary RNAs were specifically bound to matrices containing the homologous AzoDNA and eluted under conditions consistent with destabilization of RNA:DNA hybrids. These applications indicate the potential utility of AzoDNA-extensor arm affinity chromatography for the isolation of specific viral RNA molecules.     

Photometabolism of 7-dehydrocholesterol to previtamin D3 in skin.

The photometabolism of [3α-³H]-7-dehydrocholesterol in skin was studied in groups of rats exposed to ultraviolet irradiation. The major photolytic product was identified as previtamin D₃ by its identical migration with authentic previtamin D₃ on high-pressure liquid chromatography. Furthermore, this photometabolite was isolated in pure form from endogenous precursors in skins of rats exposed to ultraviolet irradiation; identification as previtamin D₃ was based on its ultraviolet absorption spectrum, mass spectrum and its thermal conversion to vitamin D₃.     

Formation of 5-carbomethoxyvaleramidine during hydrolysis of the protein cross-linking agent dimethyl adipimidate

Imidoesters have been used in biological studies to measure interresidue distances of proteins and macromolecular complexes, and in hematology as antisickling agents. Treatment of human red blood cells with¹⁴C-labeled dimethyl adipimidate (DMA), a bifunctional imidoester with antisickling properties, was followed by gradual loss of radioactivity from the treated cells. The radioactive compound released was isolated by thin-layer chromatography and identified by high-resolution mass spectrometry and by carbon-13 nuclear magnetic resonance, ultraviolet, and infrared spectroscopy as 5-carbomethyoxyvaleramidine, which was also shown to be the major product of DMA hydrolysis in vitro at physiologic pH in phosphate buffer. High-resolution mass spectrometry studies indicated that this product is formed via cyclization to a reactive intermediate (7-methoxy-2-imino-3,4,5,6-tetrahydro-2H-azepine) followed by hydrolysis. The intermediate exhibited strong UV absorbance, maximal at 232 nm. Such an intermediate would be capable of participating in cross-linking reactions which would have smaller dimensions than those observed with the imidoester in its extended form. The hydrolysis product, an unreactive species, should have no toxic effects on individuals receiving infusions of DMA-treated red cells.     

The participation of a tryptophan residue in the binding of ferric iron to pyrocatechase

The fast reaction technique of pulse radiolysis was used to produce free radicals in aqueous solution from alcohols, deoxyribose, cytosine, uracil, thymine, dihydrothymine and histidine. The electron transfer reactions from these radicals to p-benzoquinone was observed from the formation kinetics of the semiquinone anion ·BQ^? at 430 nm and the efficiency was found to be as high as 90% or more, with k～5×10⁹ M^?1sec^?1. In acid or neutral solutions in the presence of oxygen the peroxy radicals ·O₂RH formed do not essentially transfer an electron to BQ, and the efficiency is <10%. The significance of these results in the fixation of radiation damage in photobiology and radiation biology are indicated. The reactions of the superoxide ·O₂^? radical with BQ are also presented and discussed.     

Liver-microsome-mediated formation of alkylating agents from vinyl bromide and vinyl chloride.

When a mixture of vinyl chloride/oxygen or vinyl bromide/air was passed through a mouse-liver microsomal system, volatile alkylating metabolites were trapped by reaction with excess 4-(4-nitrobenzyl)pyridine. The absorption spectra of the adducts, either from vinyl bromide or vinyl chloride, were identical with that obtained by reaction of chloroethylene oxide with 4-(4-nitrobenzyl) pyridine. Chloroethylene oxide decomposes in aqueous solution with a half-life of 1.6 minutes. After reaction of chloroethylene oxide and 2-chloroacetaldehyde with adenosine and Sephadex chromatography the binding products were compared with those formed in the presence of vinyl chloride, mouse-liver microsomes and adenosine. A common product of these reactions was tentatively characterized as 3-β-ribofuranosyl-imidazo-[2,1-i]purine.     

A solvothermal method to synthesize fluorescent carbon nanoparticles and application to photocatalysis and electrocatalysis

A novel solvothermal approach to synthesize fluorescent carbon nanoparticles (CNPs) was developed using glucose and ammonium oxalate as the carbon source, and glycol as the solvent. The solution of as‐prepared CNPs emitted blue‐green fluorescence under ultraviolet (UV) light at 365 nm, and the carbon nanoparticle formation was investigated through XRD, TEM, DLS, FT‐IR, UV, PL, XPS. The particle was well dispersed with an average diameter of about 10–30 nm. In contrast with previous methods, in this synthesis process neither strong acid treatment nor further surface modification was necessary. The solution of as‐prepared CNPs were applied to photocatalytic degradation of mountain green in the present H₂O₂, and the decolorization rate was over 97% when the reaction time was more than 7 h under visible light. The as‐prepared CNPs were also applied to electrocatalysis and showed excellent electrocatalytic activity. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis and photoluminescence of EuII in barium zinc orthosilicate: a novel green color emitting phosphor for white‐LEDs

A series of Eu²⁺‐activated barium orthosilicates (BaZnSiO₄) were synthesized using a high‐temperature solid‐state reaction. A photoluminescence excitation study of Eu²⁺ shows a broad absorption band in the range of 270–450 nm, with multiple absorption peak maxima (310, 350 and 400 nm) due to 4f–5d electronic transition. The emission spectra of all the compositions show green color emission (in the spectral region 450–550 nm with a peak maximum at 502 nm and a shoulder at ~ 490 nm) with appropriate Comission Internationale de l'Eclairage (CIE) color coordinates. The two emission peaks are due to the presence of Eu²⁺ in two different Ba sites in the BaZnSiO₄ host lattice. The energy transfers between the Eu²⁺ ions in BaZnSiO₄ host are elucidated from the critical concentration quenching data based on the electronic multipolar interaction. All Eu²⁺‐activated BaZnSiO₄ phosphor materials can be efficiently excited in the ultraviolet (UV) to near UV‐region (270–420 nm), making them attractive candidate as a green phosphor for solid state lighting–white light‐emitting diodes.     

Initial Reactions in the Biodegradation of 1-Chloro-4-Nitrobenzene by a Newly Isolated Bacterium, Strain LW1

Bacterial strain LW1, which belongs to the family Comamonadaceae, utilizes 1-chloro-4-nitrobenzene (1C4NB) as a sole source of carbon, nitrogen, and energy. Suspensions of 1C4NB-grown cells removed 1C4NB from culture fluids, and there was a concomitant release of ammonia and chloride. Under anaerobic conditions LW1 transformed 1C4NB into a product which was identified as 2-amino-5-chlorophenol by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. This transformation indicated that there was partial reduction of the nitro group to the hydroxylamino substituent, followed by Bamberger rearrangement. In the presence of oxygen but in the absence of NAD, fast transformation of 2-amino-5-chlorophenol into a transiently stable yellow product was observed with resting cells and cell extracts. This compound exhibited an absorption maximum at 395 nm and was further converted to a dead-end product with maxima at 226 and 272 nm. The compound formed was subsequently identified by ¹H and ¹³C NMR spectroscopy and mass spectrometry as 5-chloropicolinic acid. In contrast, when NAD was added in the presence of oxygen, only minor amounts of 5-chloropicolinic acid were formed, and a new product, which exhibited an absorption maximum at 306 nm, accumulated.     

Isolation and characterization of the transient,luciferase-bound flavin-4a-hydroxide in the bacterial luciferase reaction

Procedures and conditions have been established such that the unstable enzyme-bound flavin intermediate produced in the bacterial luciferase reaction can be isolated as approximately 70% of the flavin product, the remaining being the final product, FMN. The structure of the intermediate is proposed to be that of a luciferase-bound 4a,5-dihydroflavin-4a-hydroxide. The intermediate has a half-life of 33 min at 2°C and decays spontaneously to give H₂O and luciferase-bound FMN with an activation enthalpy of about 120 kJ/mol. It has an absorption spectrum (λ_max = 360 nm) that is consistent with the proposed structure, and a fluorescence emission (λ_max = 485 nm) that matches the bioluminescence emission closely.