Isolation of immunosimilar proteins from cotton seeds by immunoaffinity chromatograph

A sorbent highly specific forVerticillium proteins has been obtained from BrCN-Sepharose and rabbit immunoglobulins. By affinity chromatography using this sorbent a protein immunologically similar to the proteins of the mycelium of the fungusV. dahliae has been isolated from cotton seeds of the Tashkent-1 variety. The molecular mass of the protein has been determined, and its proteolytic activity has been established. Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 746–749, September–October, 1993.     

Immobilization of snake venom on epoxy-activated Silokhroms

The production of immobilized phospholipase A₂ preparations by the covalent addition of the proteins of viper venom to Silokhroms containing epoxide groups is described. The dependence of the amount of protein bound to the support on its concentration and on the amount of epoxide groups in the sorbent has been investigated. The phospholipase activity of the preparations obtained did not depend on the amount of immobilized enzyme but was determined by diffusional factors.M. V. Lomonosov Institute of Fine Chemical Technology, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 126–129, January–February, 1989.     

Equilibrium of Br− and Cl− exchange on strong-basic anion-exchanger

The equilibrium of Br^– and Cl^– exchange is studied on the strong-basic anionexchangers. Quantitative characteristics which can be used for elaboration of dual-temperature separation processes have been obtained. An empirical equation for the dehydration extent of an anion in a phase of a sorbent has been suggested, allowing one to calculate the Br^– and Cl^– exchange equilibrium constants on the anion-exchanger in a wide temperature range.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2083–2089, December, 1994.     

Application of solid-phase extraction for determination of phenolic compounds in barrique wines

A fast, selective and sensitive chromatographic method has been developed for determination of gallic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, benzoic, ferulic, sinapic, cinnamic, and ellagic acids and p-hydroxybenzaldehyde, vanillin, syringaldehyde, 2-furfural, 5-methylfurfural, and 5-methoxyfurfural. The compounds from untreated wine samples were pre-concentrated and cleaned using solid-phase extraction on RP-105 polymeric sorbent. The cartridge was conditioned with methanol and water. Co-extracted ballast substances were rinsed from the sorbent with 0.1 mol L^–1 hydrochloric acid–methanol, 1:4 (v/v). Retained phenolic compounds were selectively eluted with diethyl ether. A linear mobile phase gradient containing 0.3% acetic acid and methanol was used for final baseline chromatographic separation on a Hypersil BDS C18 column. Limits of detection (LOD=3s_bl) in the range 5.2 to 181.2 g L^–1, resolution (R) better than 1.7, and repeatability of 2.7–5.1% (RSD for real samples) were achieved. The method was applied for quantification of individual phenolic compounds in barrique wines.     

Investigation of bound diffusion of ultrafine particles of FeIII,hydroxide in a polymeric sorbent by Mössbauer spectroscopy

Bound diffusion of ultrafine particles of Fe^III hydroxide in the pores of a solvated polymeric sorbent has been discovered and investigated by Mössbauer spectroscopy. Particles with diameters of 30 Å <-d <- 90 Å were precipitated from an aqueous solution in the pores of the poly(divinylbenzene-ethylstyrene) Porolas A sorbent. The Mössbauer spectra obtained atyT > 250 K (i.e., above the crystallization and glass transition points of the liquid in the pores,viz., water or glycerol) were found to assume the shape of a superposition of broadened and nonbroadened components, which is characteristic of bound diffusion. Experimental data were treated in terms of two alternative models of bound diffusion,viz., in harmonic (Brownian overdamped oscillator) and rectangular potentials. The following values for the parameters of bound diffusion (diffusion coefficientD and diffusion displacementr) at room temperature were found: for glycerol,D = 0.3 · 10^–9; 0.5 · 10^–9 cm² s^–1 andr = 0.14; 0.38 Å for water,D > 2 × 10^–8 cm² s^–1 andr = 0.22; 0.45 Å. Unlimited diffusion of particles in the solvated sorbent was not observed. No diffusion of the particles was observed in the dry sorbent.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 886–895, April, 1996.     

Study of the disulfide reduction of denatured proteins by liquid chromatography coupled with on-line cold-vapor-generation atomic-fluorescence spectrometry (LC–CVGAFS)

Hydrophobic-interaction chromatography coupled on-line with chemical-vapor-generation atomic-fluorescence spectrometry (HIC–CVGAFS), optimized recently for the analysis of thiol-containing proteins under denaturing conditions, has been used to study the chemical reduction of denatured proteins. Four proteins chosen as models (human serum albumin (HSA), bovine serum albumin (BSA), -lactalbumin (-Lac) from bovine milk, and lysozyme from chicken egg (Lys)) were denatured with urea and reduced with dithiothreitol (DTT), with selenol as catalyst. The method is based on derivatization of the –SH groups of proteins with p-hydroxymercurybenzoate (PHMB), followed by HIC separation and post-column on-line reaction of the derivatized reduced, denatured proteins with bromine generated in situ. Hg^II, derived from rapid conversion of uncomplexed and protein-complexed PHMB, is selectively detected by AFS in an Ar/H₂ miniaturized flame after sodium borohydride (NaBH₄) reduction to Hg°. The yield of the reduction was studied as a function of reductant concentration, reduction time (t_red), and urea concentration. Results showed that the optimum values for DTT and selenol concentrations and for t_red were between 1 and 100 mmol L^–1 and between 1 and 20 min, respectively, depending on the protein studied. The percentage disulfide bond reduction increases as the urea concentration used for protein denaturation increases, giving a single-step sigmoid increment for single-domain, low-MW proteins (-Lac and Lys), and a two-step sigmoid increment for multi-domain, high MW proteins (HSA and BSA). The shapes of plots of percentage reduced disulfide against urea concentration are characteristic of each protein and are correlated with the location of S–S in the protein. Under the adopted conditions complete protein denaturation is the conditio sine qua non for obtaining 100% S–S reduction. The detection limit for denatured, reduced proteins examined under the optimized conditions was found to be in the range 1–5×10^–12 mol L^–1 (10–30 pg), depending on the protein considered.     

Organomineral sorbents based on clinoptilolite-containing tuffs

The anion-exchange properties of a new organomineral sorbent obtained by modification of clinoptilolite-containing tuffs by polyhexamethyleneguanidine have been studied after different periods of storage (time after synthesis) and numbers of sorption-regeneration cycles. The sorbent can be used as a cation- and an anion-exchanger simultaneously. Selectivity coefficients (exchange constants) for F^–, SO₄ ^2–, and HPO₄ ^2– ionsvs. Cl^– ions on modified clinoptilolite-containing tuffs have been determined. The modification improves the mechanical properties of clinoptilolite tuffs.For part 1, see Ref. 1.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1554–1556, September, 1994.     

Fractional and amino acid compositions of micromycete proteins

A comparative study has been made of the fractional and amino acid compositions of the proteins of the biomasses ofTrichoderma harzianum, Fusarium moniliforme, and the hybrid culture FT-2. It has been established that the micromycete strains investigated are high-protein cultures containing from 36.0to 49.7% of protein. A study of the amino acid compositions of the micromycetes showed that the hybrid culture FT-2 possessed the largest amount of amino acids among the water-soluble proteins of the biomass (61.3 g/100g of biomass protein).Institute of Microbiology, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 41 71 29. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 428–431, May–June, 1997.     

A natural undecaprenol and its influence on the biochemical reactions of cotton seedling nuclei

A method has been developed for isolating polyprenols from the leaves of cotton plants of lines L-463, L-501, and L-4. The influence of an undecaprenol on the level of biosynthesis of the proteins of the nuclei of cotton seedlings has been studiedin vivo andin vitro. It has been found that they double the level of biosynthesis nuclear proteins when the seeds are first wetted in a 0.1% solution.Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 65–69, January–February, 1998.     

Sorption of radioactive strontium on a silica-titania mixed hydrous oxide gel

A silica-titania (Si–Ti) mixed hydroxide gel with Ti to Si mole ratio of 11 was prepared. Studies on the sorption of radiostrontium from alkaline solutions having various concentrations of sodium were carried out with Si–Ti. The distribution coefficient (K _d) of strontium decreased with increasing concentrations of sodium from 0.1 to 4.0 mol·dm^–3. The plot of logK _d versus log [Na⁺] gave a slope of –0.55 as against the theoretical value of –2.0. This suggests that some other paths in addition to the simple stoichiometric ion exchange is also existing for sorption. From pH titrations the pH_pzc of Si–Ti was found to be 4.22 coinciding nearly with the pH sorption edge. An attempt was made to correlate the sorption results with the surface characteristics of the sorbent. It was found that sorption is favored when the surface of the sorbent transforms into an anionic form. Sharp increase in the sorption of strontium was observed when the equilibrium pH was between 7 and 10. This behavior could be attributed to the abrupt increase in net negative charge on the surface of the sorbent.     

Adsorption of low level51Cr(VI) from aqueous solution by bismuth trioxide: Kinetic and IR study

Radiotracer technique has been used for the investigation of adsorption of chromium (VI) traces on bismuth trioxide from aqueous solution. The effect of pH (2–10), concentration of chromate solution (10^–6–10^–2M) and temperature (303–323 K) has been thoroughly investigated. The influence of certain foreign ions has also been studied. The calculated kinetic and thermodynamic parameters indicate the first order rate law, spontaneity and exothermic nature of the adsorption process. Further, IR studies have established the chemical interaction between the sorbate and sorbent and a possible mechanism of the sorption process based on ligand exchange has been proposed.     

Modeling of iron adsorption on HTTA-loaded polyurethane foam using Freundlich,Langmuir and D-R isotherm expressions

The sorption of Fe(III) at low pH range from 1 to 4.5 on open cell polyether type HTTA-loaded polyurethane foam has been carried out using batch technique. The optimum shaking time for 2.5· 10^–4M solution of Fe(III) was found to be 30 minutes. The concept of macropore and micropore nature of polyurethane foam sorbent offers unique advantages of adsorption. Freundlich and Langmuir adsorption isotherms are followed at low concentration range from 1·10^–4 to 3·10^–4M solution of Fe(III). The Freundlich constant (1/n=0.46±0.013 andK=9.16±1.39 mg·g^–1) and Langmuir isotherm constants(M=21.78 mg·g^–1 andb=88.41±9.731·g^–1) were established. The sorption mean free energyE=12.22±0.09 kJ·mol^–1 and loading capacityC _m =145.21±6.1 mg·g^–1 were evaluated using Dubinin-Radushkevich isotherm, which suggested that the adsorption mechanism was chemisorption.     

Simplified Solid-Phase Extraction Procedure and Liquid Chromatographic Determination of Celecoxib in Rat Serum

A simplified solid phase extraction method, eliminating a preliminary protein precipitation has been developed for the determination of celecoxib in rat plasma. The technique included a solid phase extraction of the serum samples on a [poly (divinylbenzene-co-N-vinylpyrrolidone)] sorbent. After conditioning, the cartridge was loaded with 0.5 mL of acidified serum containing internal standard. Elution was made with 1 mL of a mixture of acetonitrile and methanol (1/1, v/v). After evaporation of the eluate to dryness and reconstitution with methanol, the samples were analyzed on an octadecyl bonded phase with several mobile phases containing acetonitrile and a phosphate buffer. Detection was carried out using a Photodiode Array Detector. Full validation of the proposed method was provided (linearity range: 0.01–2 mg. L^–1, average extraction efficiency: 92.4%; average intra-day variability: 4.6% with an accuracy of 94.8%; average interday variability: 5% with an accuracy of 95.3%, limit of detection: 0.005 mg. L^–1, limit of quantification: 0.002 mg. L^–1). The proposed method was successfully utilised to quantify celecoxib in rat plasma for a pharmacokinetic study.Revised: 26 January and 23 April 2004     

Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems: Effect of fusion localization on endoglucanase I of Trichoderma reesei

Genetic engineering has been used for fusion of the peptide tag, Trp–Pro–Trp–Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-β- -glucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)–propylene oxide (PO) random copolymer EO–PO (50:50) (EO₅₀PO₅₀) and dextran. The Trp–Pro–Trp–Pro tag was found to direct the fusion protein to the top EO₅₀PO₅₀ phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro–Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.     

Comparative investigation of the molecular structures of the species-specific proteins of the seeds ofGossypium hirsutum andG. barbadense

The N-terminal amino acids and the amino acid compositions of four species-specific proteins of two species of the cotton plant —Gossypium hirsutum andG. barbadense — have been determined. Peptide maps of tryptic hydrolysates of these proteins have been obtained and compared. Individual tryptic peptides have been isolated and purified. The N-ends, amino acid compositions, and amino acid sequences of individual peptides have been determined. The N-terminal sections of the proteins ofG. hirsutum andG. barbadense as far as the 16th amino acid residue have been sequenced. A comparative analysis has been made of the peptides and proteins of these cottonplant species.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 40 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 858–864, November–December, 1997.     

Affinity chromatography of carboxylic proteinases on a polymeric sorbent containing gramicidin-C as ligand

A new polymeric sorbent for proteinases has been synthesized by the radical copolymerization of N-vinylpyrrolidone, bis-N-acryloylgramicidin C, and N,N-methylenebisacrylamide. Biospecific chromatography on the new sorbent has enabled an industrial preparation of procine pepsin to be purified by a factor of 2.5. With the aid of the new sorbent, a carboxylic proteinase has been isolated from the industrial preparation Tsellolignorin with a 15-fold purification factor.M. V. Lomonosov State University. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 215–218, March–April, 1986.     

Sepharose 4B-DNP-hexamethylenediamine as a sorbent for the chromatography of carboxylic proteinases

Summary 1. The chromatography of the carboxylic proteinases porcine pepsin, aspergillopepsin A, and chymosin on the hydrophobic sorbent Sepharose 4B-DNP-hexamethylenediamine has been studied. It has been shown that the nature of the binding of the proteinases with the sorbent depends on the pH.2. A shortening of the length of the carbohydrate chain of the ligand by four methylene units substantially weakens the interaction of pepsin with the sorbent.3. With chymotrypsin and pepsin as examples, the possibility has been shown of using ionic effects for separating these enzymes.M. V. Lomonosov Moscow State University. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 191–199, March–April, 1979.     

Electrophoretic investigation of the seed proteins of the cotton plantGossypium hirsutum

The proteins of the buffer-soluble fraction of the seeds of cotton plants of the speciesGossypium hirsutum, wild and ruderal varieties, have been investigated by electrophoresis in polyacrylamide gel and have been subdivided into two classes. Characteristic components have been detected in the protein spectra of these samples. In diploid species of the cotton plant, in addition to characteristic protein components, individual components are also observed.Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 736–738, September–October, 1995. Original article submitted November 21, 1994.     

Ab initio and density functional theory based studies on collagen triplets

An understanding of the amino acid sequence dependent stability of polypeptides is of renowned interest to biophysicists and biochemists, in order to identify the nature of forces that stabilize the three-dimensional structure of proteins. In this study, the role of various collagen triplets influencing the stability of collagen has been addressed. It is found from this study that proline can stabilize the collagen triplet only when other residues are also in the polyproline II conformation. Solvation studies of various triplets indicate that the presence of polar residues increases the free energy of solvation. Especially the triplets containing arginine residues displays a higher solvation free energy. The chemical hardness of all the triplets in collagen-like conformation has been found to be higher than that in the extended conformation. Studies on Gly–X–Y, Gly–X–Hyp, and Gly–Pro–Y triplets confirm that there will be local variations in the stability of collagen along the entire sequence.     

Analytical Method Development for the Simultaneous Determination of Five Human Pharmaceuticals in Water and Wastewater Samples by Gas Chromatography-Mass Spectrometry

Summary Effective analytical methods for the simultaneous determination of five pharmaceuticals from various therapeutic classes in a variety of aqueous samples have been developed and method performance data are presented. The method involves the simultaneous extraction of the selected pharmaceuticals from the aqueous phase by solid phase extraction using a hyper cross linked, polystyrene-divinylbenzene polymer based sorbent. Analytes were eluted with methanol, derivatised with N-methyl-N-trimethylsilyltrifloroacetamide and analysed by gas chromatography – electron ionisation mass spectrometry (GC-EI-MS). Recoveries of 50 to 98% were established for waters spiked with the studied compounds at the low ng L^–1 level with the highest detection sensitivities being achieved in the selected ion monitoring (SIM) mode and the quantification limit of the procedure for sample sizes of 1000 ml was approximately 5 ng L^–1 for all matrices except sewage which was only tested to 20 ng L^–1. Analysis of domestic sewage from a large treatment works demonstrate the presence of all five compounds in both influents and effluents.