High-performance affinity chromatography and the analysis of drug interactions with modified proteins: binding of gliclazide with glycated human serum albumin

This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (K _a) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high-affinity sites (average K _a, 7.1–10 × 10⁴ M⁻¹) and a group of lower-affinity sites (average K _a, 5.7–8.9 × 10³ M⁻¹) at pH 7.4 and 37 °C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the K _a values for gliclazide at these sites being 1.9 × 10⁴ and 6.0 × 10⁴ M⁻¹, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification.     

Synthesis of dendrimers with terminal formyl groups

A method for preparation of dendrons and dendrimers with formyl groups at the terminal aromatic rings, ether bonds in the branching blocks, and ester bonds in the core of the macromolecules is proposed. A way for the selective synthesis of p-hydroxymethylbenzaldehyde is described.Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1995–1999, September, 2004.     

Thermal analysis of organically modified siloxane melting gels

Hybrid melting gels were prepared by a sol–gel process, starting with a mono-substituted siloxane and a di-substituted siloxane, methyltrimethoxysilane (MTES) together with dimethyldimethoxysilane (DMDES). Five gel compositions were prepared with concentrations between 50% MTES–50% DMDES and 75% MTES–25% DMDES (in mol.%). The consolidation temperature, the treatment temperature after which the melting gel no longer softens, increased from 135 to 160 °C with a decrease in the amount of the mono-substituted siloxane. The glass transition temperature, recorded with differential scanning calorimetry, decreased from −0.3 to −56.7 °C with a decrease in the amount of the mono-substituted siloxane. When a sample was heat treated isothermally for 2 h at the consolidation temperature, the glass transition temperature increased by about 15°, indicating further crosslinking of the siloxane network.     

Properties of the surface of a porous polymer modified with 5-fluorouracil,according to data of gas chromatography

The effect or modification with 5-fluorouracil on the sorption activity of porous polymeric adsorbent is studied. It is demonstrated that the supramolecular structure formed on the surface is able to addition-ally contribute to the values of the specific retention volumes. It is found that the structure of 5-fluorouracil is capable of size effects corresponding to a molecular window of approximately 7–8 Å. It is concluded that surface polarity diminishes after modification, due to the shielding effect of four fluorine atoms present in the cavity.     

Determination of theophylline binding to human serum proteins by isotachophoresis

Free theophylline was isolated from human serum by ultrafiltration and analysed in a leading electrolyte of 7.5 mM morpholinoethanesulphonic acid with ammediol as a counter ion at pH 8.90 and alpha-alanine as a terminator. The UV (280 nm) absorbance of the theophylline spike between serine and bicine as spacers was integrated. Binding percentages to human pool serum, human albumin and alpha 1-acid glycoprotein (orosomucoid) were determined at physiological concentrations, and found to be 55, 44 and 12%, respectively. The calibration lines were straight from 0 to 30 mg/l, with a standard deviation of 0.2 mg/l. The detection limit was 1 mg/l. The time of analysis was 12 min at 40 microA in a 0.2 mm I.D. capillary.     

Monodisperse porous polymer particles with polyionic ligands for ion exchange separation of proteins

A new “grafting to” strategy was proposed for the preparation of polymer based ion exchange supports carrying polymeric ligands in the form of weak or strong ion exchangers. Monodisperse porous poly(glycidyl methacrylate-co-ethylene dimethacrylate), poly(GMA-co-EDM) particles 5.9 μm in size were synthesized by “modified seeded polymerization”. Poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate), poly(DHPM-co-EDM) particles were then obtained by the acidic hydrolysis of poly(GMA-co-EDM) particles. The hydroxyl functionalized beads were treated with 3-(trimethoxysilyl)propyl methacrylate to have covalently linked methacrylate groups on the particle surface. The selected monomers carrying weak or strong ionizable groups (2-acrylamido-2-methyl-1-propane sulfonic acid, AMPS; 2-dimethylaminoethylmethacrylate, DMAEM and N-[3-(dimethylamino)propyl] methacrylamide, DMAPM) were subsequently grafted onto the particles via immobilized methacrylate groups. The final polymer based materials with polyionic ligands were tried as chromatographic packing in the separation of proteins by ion exchange chromatography. The proteins were successfully separated both in the anion and cation exchange mode with higher column yields with respect to the previously proposed materials. The plate heights obtained for poly(AMPS) and poly(DMAEM) grafted poly(DHPM-co-EDM) particles by using proteins as the analytes were 80 and 200 μm, respectively. Additionally, the plate height exhibited no significant increase with the increasing linear flow rate in the range of 1–20 cm/min. The most important property of the proposed strategy is to be applicable for the synthesis of any type of ion exchanger both in the strong and weak form.     

Time-based analysis of silver-stained proteins in acrylamide gels

Silver staining of proteins after PAGE often remains the method of choice in many laboratories. Nevertheless, it is known that quantification of protein levels is keenly restricted to a small range of protein concentrations leading to an over- or underestimation of protein amounts. To overcome this, a time-based analysis method was developed to avoid the saturation effect of the silver-staining reaction, thus resulting in an improved dynamic range of the gel image produced and therefore better quantification of proteins. Instead of the well-known end-point image analysis, gray intensities of time series images of a developing gel are determined and times until a threshold gray value is reached are calculated. These times are used to calculate a new grayscale image which can be analyzed using commercial image processing software.     

MALDI-MS analysis of peptides modified with photolabile arylazido groups

The ability of MALDI-MS to analyze photolabile arylazido peptide derivatives was investigated. Peptides containing UV-labile p-azidobenzoyl groups were subjected to MALDI-MS analysis in a variety of matrices. As standard MALDI-MS employs a UV laser (337 nm), we investigated conditions that would allow detection of the intact molecule ions for these light-sensitive peptides. When using α-cyano-4-hydroxycinnamic acid (ACHC) or 2,5 dihydroxybenzoic acid (DHB) as the matrix, photoinduced degradation products were prevalent. In contrast, when employing the matrix sinapinic acid, the intact molecule ion corresponding with the azido peptide was the predominant signal. The protection of photolabile azido derivatives correlates with the UV absorbance properties of the matrix employed, i.e., sinapinic acid, which exhibits a strong absorbance near 337 nm, most efficiently protects the azido derivative from photodegradation.     

Silver(I) sorption by silica gels chemically modified with mercaptopropyl or dipropyl disulfide groups

The sorption of silver(I) from nitric and hydrochloric acid solutions with silica gels chemically modified with mercaptopropyl groups (MPSG) and dipropyl disulfide groups (DDSG) has been studied. For nitric acid solutions, the highest silver(I) recovery factor (99.0–99.9%) is reached in the acidity range between 5 M HNO₃ and pH 8 for MPSG and between 2 M HNO₃ and pH 8 for DDSG. In the sorption of silver(I) from hydrochloric acid solutions with MPSG, the recovery factor is 99% at pH 2–7. The recovery of silver(I) from chloride solutions with DDSG depends significantly on the chloride ion concentration. The treatment of silver-containing DDSG with a water-ethanol or water-dimethylformamide solution of Michler thioketone yields an intensely colored, reddish claret, mixed-ligand silver(I) complex on the sorbent surface. The diffuse reflectance spectrum of this complex shows a band peaking at 520 nm.     

Changes of the affinity between fungal proteins and vanillin bonded to thermally modified porous glass support

Summary The affinity of peroxidase and the fungal proteins to vanillin attached to controlled porous glasses depends on the porosity of the glass and additional thermal treatment of the support. The additional thermal treatment of controlled porous glasses leads to an enrichment in boron atoms of their surface.The results presented in this paper show a better resolution of the analyzed substances when glass with a surface enriched in boron was used as the support for vanillin.     

Water-soluble carbosilane dendrimers protect phosphorothioate oligonucleotides from binding to serum proteins

Treatment of dendriplexes formed between water-soluble carbosilane dendrimers and phosphorothioate oligodeoxynucleotides (ODN) with the anionic detergent sodium dodecyl sulfate disrupted the complexes indicating that the nature of the union in such dendriplexes is merely electrostatic. However, dendriplexes were not dissociated by serum proteins like bovine or human serum albumins, as assessed by gel electrophoresis and fluorescence experiments. This would imply a dendrimer-mediated protective effect able to prevent ODN interactions with serum proteins and additionally could translate into a reduction of the ODN doses needed to achieve the biological effects. The employment of carbosilane dendrimers as carriers may solve the problem of ODN kidnapping by plasmatic proteins as a key drawback for therapeutics involving ODNs. As examples, transfection processes on normal primary peripheral blood cells and diagnosis of HIV infection in the presence of serum have been assayed.     

Synthesis of silica gels with a controlled porous structure

Silica gels with a developed porous structure were synthesized by a sol-gel method with the use of templates, drying in supercritical CO₂, and spray drying. A correlation between the synthesis conditions and the textural characteristics of the resulting material was established.     

Cellular binding of nanoparticles in the presence of serum proteins

Cellular binding of cationic nanoparticles in the presence of serum proteins was probed with two-colour fluorescence microscopy. Cationic nanoparticles associate with serum proteins in solution and bind to the cell surface as a single anionic complex. Displacement of serum proteins from the nanoparticles was found to be protein dependent.     

Contributions of various serum proteins to the binding of prasterone sulfate in humans.

The binding of prasterone sulfate (PS) in human plasma was investigated. Binding percentages of PS to human plasma, human serum albumin (HSA), human alpha 1-acid glycoprotein (AGP) and human gamma-globulin (GGL) were independent of the PS concentration between 0.1 and 8.0 micrograms/ml. The mean binding percentages were 99.1% for human plasma, 98.3% for HSA, 12.6% for AGP and 8.1% for GGL. Though PS is an acidic drug, binding of PS to AGP was observed. From the binding index, it was found that PS mainly bound to HSA in human plasma and that the contributions of AGP and GGL to PS in plasma were negligible.     

Micro- and ultramicrodetermination of the formyl and isonitrile groups in the presence of other acyl groups

Gravimetric and colorimetric micromethods for determination of formyl and isonitrile groups are reported. The gravimetric method is based on a quantitative cleavage of the formyl group and decomposition of formed formic acid to carbon monoxide by means of 60% sulfuric acid. Carbon monoxide is oxidized and the formed carbon dioxide is determined gravimetrically.The colorimetric ultramicromethod is based on the basic hydrolysis of the formyl group. After acidifying the hydrolysate, the quantitatively formed formic acid is distilled under special conditions. The acid is then reduced to formaldehyde. Its quantity is determined spectrophotometrically with chromotropic acid.     

Review on recent and advanced applications of monoliths and related porous polymer gels in micro-fluidic devices

This review critically summarises recent novel and advanced achievements in the application of monolithic materials and related porous polymer gels in micro-fluidic devices appearing within the literature over the period of the last 5 years (2005-2010). The range of monolithic materials has developed rapidly over the past decade, with a diverse and highly versatile class of materials now available, with each exhibiting distinct porosities, pore sizes, and a wide variety of surface functionalities. A major advantage of these materials is their ease of preparation in micro-fluidic channels by in situ polymerisation, leading to monolithic materials being increasingly utilised for a larger variety of purposes in micro-fluidic platforms. Applications of porous polymer monoliths, silica-based monoliths and related homogeneous porous polymer gels in the preparation of separation columns, ion-permeable membranes, preconcentrators, extractors, electrospray emitters, micro-valves, electrokinetic pumps, micro-reactors and micro-mixers in micro-fluidic devices are discussed herein. Procedures used in the preparation of monolithic materials in micro-channels, as well as some practical aspects of the micro-fluidic chip fabrication are addressed. Recent analytical/bioanalytical and catalytic applications of the final micro-fluidic devices incorporating monolithic materials are also reviewed.