Volume 39 Issue 2

April 2011

Extracorporeal Affinity Therapy

Extracorporeal therapies are directed at the removal of potential toxic substances including bilirubin, pathogenic antibodies, various circulating immune-complexes, cholesterol, toxic metal ions, etc. from human plasma. The conventional extracorporeal therapies including plasma exchange, hemodialysis, hemofiltration and hemoperfusion are non-selective techniques. In addition the requirement for plasma substitutes such as albumin is very high. Moreover, the dangers of hepatitis or immune reactions accompany these therapies while using plasma for plasma products. Today, one of the most promising procedures for extracorporeal therapy is specific affinity adsorption. Affinity carriers may be used in hemoperfusion system, where blood is directly perfused through the column filled with these carriers. This type of application is effective, simple and inexpensive. In this article, some interesting applications on extracorporeal affinity therapies for removal of toxic substances from human plasma are briefly reviewed. 

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Bioengineering Functional Copolymers. XIX. Synthesis of Anhydride-Organoboron Functionalized Copolymers and Their Interaction with Cancer Cells

Novel bioengineering functional organoboron homo- and copolymer of maleic anhydride (MA) were synthe- sized by (1) amidolysis of anhydride-containing macromolecules [poly(MA-alt-methyl vinyl ether (MVE) as a bioengineering polymer] with 2-aminoethyldiphenylborinate (2-AEPB), (2) esterification with α-hydroxy-ω-methoxy-poly(ethylene oxide) (PEO) as a compatibilizer and (3) conjugation of organoboron PEO branches with folic acid as a targetting agent. Structure and composition of the synthesized oligomers were characterized by FTIR-ATR and 1H (13C) NMR spectroscopy. Antitumor activity of the organoboron functional (co)polymers were investigated by a combination of various physical and biochemical methods such as cytotoxicity, statistical, apoptotic and necrotic cell indexes, double staining and caspase-3 immunostaining, light and fluorescence inverted microscope analyses. It was found that cytotoxicity and apoptotic/necrotic effects of organoboron macromolecules significantly depend on the structure and composition of studied (co)polymers. Some synthesized (co)polymers at 400 μg.mL-1 concentration as a therapeutic drug exhibits minimal toxicity toward the normal cells, but influential for cancer cells. 

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Affinity Separation and Characterization of IgG Subfragments by Fast Protein Liquid Chromatography with HiTrap_r Protein A Column

It has been previously reported that the antibody molecules can be cleaved into fragments by the proteolytic enzymes and these fragments provide information to determine an antibody’s structure and function. Antibody fragments offer several advantages over intact antibodies for use in experimental applications and immunochemical techniques. These fragments have smaller sizes and higher chemical and physical resistances. In this study, papain was used for the digestion of human IgG and the resulting fragments were separated by Fast Protein Liquid Chromatography (FPLC). For this purpose, GE Healthcare HiTrap_r Protein A FF column was used, the resolution (Rs) and theoretical plate numbers (N) of the column were calculated. The unbound and bound fragments were collected by the Frac 920 fraction unit of the FPLC system and the collected fragments were analysed by enzyme-linked immunosorbent assay (ELISA) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). 

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Inhibition Effects of Some Sulfonamides on α-Carbonic Anhydrase from European seabass (Dicentrachus labrax)

Alpha-carbonic anhydrase (EC:; CA) was purified from European seabass gill and liver. The purification procedure was composed of preparation of homogenate (or hemolysate) and affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. Some sulfonamides exhibited in vitro inhibitory effects on the enzyme activity. Ki constants and IC50 values for these drugs were determined by Lineweaver-Burk graphs and plotting activity % vs. [I], respectively. IC50 values of sulfanilamide, mafenide, acetazolamide, 2-amino-1,3,5-tiyadiazol-5 sulfonamide were 980 μM, 142 μM, 20 μM and 34 μM for gill carbonic anhydrase (GCA), 126 μM, 23 μM, 14 μM and 2.58 μM for liver carbonic anhydrase (LCA), respectively. Some sulfonamides exhibited much stronger inhibitory activity against GCA and LCA at low micromolar concentrations with the Ki values ranging from 0.21 to 76.0 μM as compared with other CAs. 

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Fe3+ Immobilized Cryogel as an Immobilized Metal Affinity Chromatography (IMAC) Support for Dye removal

In this study, the potential use of Fe3+ chelated Cibacron Blue F3GA immobilized poly(acrylamide-allyl glycidyl ether) [p(AAm-AGE)-CB-Fe3+ ] cryogel to remove dyes from aqueous solutions was evaluated. Cryogel was prepared by radical polymerization. Cibacron Blue F3GA was covalently immobilized on p(AAm-AGE) cryogel and then chelated with Fe3+ ions. Maximum adsorption capacities were determined as 14.50 mg/g cryogel for Congo Red and 18.78 mg/g cryogel for Reactive Green. It was also determined that Freundlich isotherm was convenient for both dyes. More than 90% of the adsorbed dyes were desorbed in all cases. This study indicates that p(AAm-AGE)-CB-Fe3+ cryogelic material can be efficiently used as adsorbent for different dyes at high concentrations. 

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Glucose 6-Phosphate Dehydrogenase Activatory Properties of Some Organic Compounds and Amino Acids

The aim of this study was to assess activatory potentials of 4’-(Methylsulfonyl)- acetophenone, 4-(methylsul- fonyl) phenylacetic acid, tyrosine and phenylalanine on human erythrocyte glucose 6-phosphate dehydro- genase. For this purpose, initially erythrocyte glucose 6-phosphate dehydrogenase was managed to be purified 812-fold in a yield of 40.6% by using 2’,5’-ADP Sepharose 4B affinity gel.

4’-(Methylsulfonyl)acetophenone, 4-(methylsulfonyl)phenylacetic acid, tyrosine and phenylalanine exhib- ited activatory effects on the enzyme. % Activity values for these compounds were determined by plotting activity percentage versus [Activator]. 

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Application of Cu2+-attached Magnetite Nanoparticles Embedded Supermacroporous Monolithic Composite Cryogels for DNA Adsorption

Cryogels are known as a novel generation of stationary phases in the separation science, and they have been succesfully used as efficient adsorbents for separation and purification of biomolecules, such as proteins, enzymes, DNA, cell organelles and viruses in downstream processes. In this study, DNA adsorption performance of Cu2+-attached magnetite (Fe3O4) nanoparticles (Cu2+-AMNPs) embedded supermacroporous monolithic composite cryogels were investigated. Firstly, Magnetite nanoparticles (NPs) coated with 3-aminopropyltriethoxysilane (APTES) were prepared by silanization reaction. Then, Cu2+ ions were attached to silanized magnetite NPs through amine groups. After that, Cu2+-AMNPs were embedded into the cryogels for DNA adsorption studies. The prepared cryogel was characterized by scanning electron microscopy (SEM), elemental analysis, FTIR and swelling. SEM analysis indicates that the cryogel have a heteroporous structure with interconnected pores of 10-50 m size, which ascribed to the porogens effect of frozen water crystals. The maximum amount of DNA adsorption was 19.97 mg/g polymer, at pH 8.0. 

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Metallomics as a Junction between Life Sciences

Metallomics is a rapidly growing research area investigating the interaction of metals with biological mol- ecules (e.g. DNA, proteins and metabolites) in living systems. It aims the understanding of all metal de- pendent metabolic processes such as uptake, transport, storage and excretion at molecular level. In order to access the qualitative and quantitative information of metals, which mostly occur at trace amounts and in the form of non-covalent complexes with biological ligands in a highly complex biological matrix, and to elucidate the metal-dependent life processes, metallomics utilizes the state of the art analytical and spectroscopic tech- niques. The mostly used approach for the analysis of metal complexes by preserving native metal species is the hyphenation of a chromatographic or an electrophoretic technique for high resolution separation with an elemental (e.g., ICP-MS) or molecular (e.g., ESI-MS or MALDI-MS) spectrometry technique for detection and/ or identification. X-ray absorption and X-ray fluorescence spectrometry and in-silico approaches with bioinfor- matics are among other main techniques/methodologies contributing the research activities in metallomics. This study highlights the basic terms, primarily used analytical approaches, state-of-the art instrumental tech- niques and very representative recent applications in the field. 

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The Effect of Safranin on Glutathione Reductase and Glucose 6-Phosphate Dehydrogenase Enzymes

The aim of this study was to assess inhibitory effect of safranin on human erythrocyte glutathione reductase (GR) and glucose 6-phosphate dehydrogenase (G6PD). For this purpose, human erythrocyte glutathione reductase was purified 2175-fold (31.35% yield) using 2’,5’-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography and erythrocyte glucose 6-phosphate dehydrogenase was purified 8681.3-fold (24.77% yield) using 2’,5’-ADP Sepharose 4B affinity gel.

Safranin exhibited inhibitory effects on GR and G6PD enzymes in vitro. The IC50 values of safranin were 1.15 mM for GR and 3.22 mM for G6PD, respectively, and the Ki constants were 2.63 ± 0.13 and 4.53 ± 0.17 mM, respectively. Safranin displayed noncompetitive inhibition for GR and G6PD. 

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Affinity Effects of Some Antibiotics on Xanthine Oxidase Enzyme Activities In Vitro

Xanthine oxidase (XO) is the last enzyme of purine catabolism. XO was separately purified by ammonium sulphate precipitation and affinity chromatography. The effect of some antibiotics which is commonly used in clinical on purified xanthine oxidase was determined in vitro. The name of antibiotics was gentamycin sulfate, sodium ampicillin, cefazolin sodium, chlarithromycin, rifamycin SV, clindamycin phosphate and kanamycin sulfate. XO was determined using xanthine as a substrate and IC50 values of these antibiotics exhibiting inhibition effects were found from graphs of activity (%) by plotting concentration of the antibiotics. Macrolid group of antibiotics, the effects of gentamycin sulfate and kanamycin sulfate were determinated on xanthine oxidase. Gentamycin sulfate increased xanthine oxidase enzyme activity but kanamycin sulfate caused an inhibitory effect on xanthine oxidase enzyme activity. In addition, sodium ampicillin and rifamycin SV caused activation on enzyme activity. Cefazolin sodium, chlarithromycin and clindamicin phosphate indicated inhibitory effect on xanthine oxidase enzyme activity. Especially, cefazolin sodium is the most effective inhibitor in studied antibiotics with the value of 5.4 x 10–4 mg/mL. This value is close to the other values found for XO’s classical inhibitors. 

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Activatory Effects of Oxytetracycline on Some α-Carbonic Anhydrases

Three α-carbonic anhydrases, including human carbonic anhydrase I and II (hCA I, II) and sheep kidney carbonic anhydrase (sCA) were purified and kinetic interactions between the enzymes and oxytetracycline were investigated. The purification procedure was composed of preparation of homogenate (or hemolysate) and

affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. Oxytetracycline exhibited in vitro activatory effects on hCA I, II and sCA enzymes activity. Activity % values for this antibiotic were determined by plotting activity % vs. [A]. 

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