Analysis of cyanobacterial pigments and proteins by electrophoretic and chromatographic methods

Cyanobacteria are a diverse and ubiquitous group of prokaryotes with several unifying features. Amongst these is the macromolecular structure known as the phycobilisome, which is composed of water-soluble phycobiliproteins covalently bound by linker peptides or proteins in a configuration designed to optimize energy transfer to the photosynthetic reaction center of the organism. Phycobiliproteins are highly fluorescent by virtue of their covalently bound, linear tetrapyrrole chromophores known as bilins. Analysis of these prosthetic pigments, along with other non-water soluble pigments, such as the chlorophylls and carotenoids, can provide insight into microbial diversity. The effects of environmental growth conditions and stresses can also be probed by measuring pigment and protein concentrations. This review will focus, therefore, on applications of various chromatographic and electrophoretic methods for the analysis of cyanobacterial pigment and protein constituents. Although the greatest emphasis will be placed on the measurement of bilins and phycobiliproteins, this review will also consider other pigments and proteins important to cyanobacterial growth and survival, such as chlorophyll a, carotenoids, ectoenzymes, linker and membrane proteins, and extracellular proteins.     

LACK OF CORRELATION BETWEEN SUPPRESSION OF CONTACT HYPERSENSITIVITY BY UV RADIATION AND PHOTOISOMERIZATION OF EPIDERMAL UROCANIC ACID IN THE HAIRLESS MOUSE

Abstract The immunological consequences of exposure to UVA (320–400 nm) radiation are unclear. This study describes the relationship between the generation of epidermal cis -urocanic acid and the ability to respond to a contact-sensitizing agent, in hairless mice exposed to different UV radiation sources, which incorporate successively greater short-wavelength cutoff by filtration of the radiation from fluorescent UV tubes. Mice were exposed to these radiation sources at doses systematically varying in UVB radiation content but supplying increasing proportions of UVA radiation. All radiation sources were found to generate approximately 35% cis -urocanic acid in the epidermis, thus normalizing the sources for cis -urocanic acid production. However, only those sources richest in short-wavelength UVB resulted in suppression of the systemic contact hypersensitivity response. These sources also induced the greatest erythema reaction, measured as its edema component, in the exposed skin. A strong correlation was thus demonstrated between the induction of edema and the suppression of contact hypersensitivity, but there appeared to be no correlation between the generation of epidermal cis-urocanic acid and suppression of contact hypersensitivity. The sources richest in UVA content did not result in suppression of contact hypersensitivity: furthermore mice previously irradiated with such UVA-rich sources were refractory to the immunosuppressive action of exogenous cis-urocanic acid. A protective effect of the increased UVA content thus appeared to be inhibiting immunosuppression by the available endogenously generated or exogenously applied cⁱs-urocanic acid.     

Pragmatic Studies on Protein-Resistant Self-Assembled Monolayers

Summary. The present study describes new synthetic routes to oligo(ethylene glycol)-terminated alkanethiols (OEG-ATs), starting from α,ω-dibromoalkanes, which are reacted either with OEG or with trityl mercaptan in the first step. In addition to these ether conjugates of OEG and AT, analogous ester and amide conjugates were prepared by established procedures. All thiols were used to form self-assembled monolayers (SAMs) on cleaned gold surfaces and these were stored for 1–2 weeks under water at 4°C before the extent of nonspecific protein adsorption was tested with IgG, BSA, and lysozyme at 1 mg cm⁻³ protein concentration in phosphate-buffered saline. Under these practice-oriented testing conditions, SAMs with tri(ethylene glycol) chains (EG ₃) exhibited nonsatisfactory protein resistance, in sharp contrast to EG ₄ or longer OEG chains. The effectiveness of EG ₃ was partially restored when they were linked to a long acyl chain (16-mercaptohexadecanoic acid) instead of 12-mercaptododecane or 11-mercaptoundecane. Furthermore it was found that (i) SAM formation at 20 μM thiol versus 500 μM OEG-AT gave identical results, (ii) gel-filtered proteins were much less adsorbed than the unpurified commercial products, and (iii) the method for gold-precleaning was very critical. In conclusion, this study offers convenient synthetic routes to OEG-AT and helps to choose molecules and procedures for reliable preparation of protein-resitant SAMs with prolonged stability during storage.     

Spectral Study of the Reaction of Functionally Substituted Enehydrazides with Proton Donors

Reactions of new enehydrazides, N',N'-dimethyl-N-vinylpropenohydrazide and N',N'-dimethyl-N-vinylbenzohydrazide with chloroform, phenol, and hydrogen chloride in carbon tetrachloride were studied by IR spectroscopy. In the first two cases, molecular complexes are formed between the hydrazide and proton donor. The reaction of N',N'-dimethyl-N-vinylpropenohydrazide with HCl results in formation of dihydropyrazole derivative which exists as a tautomeric mixture of the major lactam and minor lactim forms. N',N'-dimethyl-N-vinylbenzohydrazide reacts with hydrogen chloride to give protonated form in which proton is localized on the amino nitrogen atom. The structure of the initial compounds and the products was analyzed in terms of AM1 quantum-chemical calculations.     

Extraction and determination of sulfonamides, macrolides, and trimethoprim in sewage sludge

Pressurized liquid extraction (PLE) was optimized and validated for the determination of sulfonamide and macrolide antimicrobials and trimethoprim in sewage sludge samples. A mixture of water/methanol (50:50, v/v) was found as the most efficient extraction solvent. A temperature of 100 degrees C and a pressure of 100 bar were chosen for extraction. Two cycles of 5 min each efficiently extracted at least 97% of the total extractable amount of all studied analytes from activated sludge. The limits of quantification (S/N= 10) varied between 3 and 41 microg/kg dry weight (dw) and the relative recoveries ranged between 78 and 142%. Additionally, the influence of pH and different LC/MS/MS systems on the absolute recoveries was assessed. Of the investigated antimicrobials sulfapyridin, sulfamethoxazole, trimethoprim, azithromycin, clarithromycin and roxithromycin were detected in municipal sewage sludge samples. Concentrations in activated sludge ranged up to 197 microg/kgdw. In comparison, results obtained by ultrasonic solvent extraction were significantly lower for sulfonamides and in tendency lower for macrolides.     

Etching of metals by means of organic radicals

A new method for the etching of In, Sn, Pb, Sb, Bi, and Zn films in methane and acetone discharges was examined with respect to various parameters such as electrode temperature, power density, bias, and reaction period. The etching species are assumed to be CH₃ radicals forming a volatile metallorganic compound, since etch rates in hydrogen plasmas or argon sputter rates are orders of magnitude lower. Etch rates up to 1600 Å/min could be obtained.Presented in part at the I.S.P.C.-7, Eindhoven, 1985.     

Proteinase assay by capillary electrophoresis employing fluorescence-quenched protein-dye conjugates

Determination of proteinases--enzymes that catalyze the hydrolysis of peptide bonds--is often difficult due to the presence of interferences in complex biological media and limited sample size. Capillary electrophoresis (CE), with laser-induced fluorescence (LIF) can serve as a useful tool for such determinations. LIF detection offers the advantages of increased sensitivity and increased selectivity. However, direct LIF detection requires the proteinase analyte to be fluorescently derivatized prior to analysis. A viable alternative is offered by the present work, in which protein substrates are first labeled with BODIPY dye, a relatively pH-insensitive, high-fluorescence quantum yield dye. Upon binding of some 4-10 molecules of dye to a single protein, the dye is effectively fluorescence-quenched. Digestion of the BODIPY--labeled and quenched protein by an unlabeled enzyme yields smaller peptide fragments in which the fluorescence of associated BODIPY tags is restored. We will present how the fragmentation pattern of BODIPY-labeled casein changes as a function of incubation time with trypsin, as well as the effect of varying concentrations of trypsin on the BODIPY-casein digest.     

Stereoselective Synthesis of Isomers of the Naturally Occurring 13-Hydroxy-2,4,9-tetradecatrienoic Acid. Part II [1]

Summary. The stereoselective syntheses of four unsaturated hydroxy fatty acids 13S,2E,4E,9E)- 13-hydroxy-2,4,9-tetradecatrienoic acid, (13S,9Z,11E)-13-hydroxy-9,11-tetradecadienoic acid, (13S,9E, 11E)-13-hydroxy-9,11-tetradecadienoic acid, and (13S,2E,4E,9E)-13-hydroxy-2,4,9,11-tetradecatrienoic acid, are described. Wittig reactions, regioselective oxidation of dialcohol 3, and diastereomerization were used.     

DETECTION OF OXYGEN RADICALS IN BIOLOGICAL REACTIONS

Abstract— Recent data are presented on the mechanisms or selected assay procedures for superoxide anions (O₂^-) and hydroxyl radicals (OH). The systems discussed include the autoxidation of adrenalin to adrenochrome and other indole compounds, the oxidation of hydroxylamine to nitrite, the generation of ethylene from methional, and the scavenging of OH radicals by p -nitroso-dimethylaniline. The results are compared with other assay procedures to aid in the search for absolute and specific tests for these oxygen radicals. Particular emphasis is placed on the interrelation of 0₂^- OH and hydrogen peroxide.     

Fluorimetric studies and noncovalent labeling of protein with the near-infrared dye HITCI for analysis by CE-LIF

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.