Synthesis of a New Series of Bone Affinity Compounds

As known that tetracycline possesses bone affinity1, which can be used as carrier of bone-targeting drugs. Chrysophanol is one of the anthraquinone components isolated from Rheum palmatum L., its structure is similar to tetracycline. The bone affinity of …     

Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.     

Principles of multienzyme purifications by affinity chromatography

Recently in our laboratory, up to 20 different enzymes and their genetic variants have been purified from mouse andDrosophila by affinity chromatography. By virtue of the specific coenzyme requirements, up to ten different enzymes could be copurified from a single tissue extract either by biospecific elutions with different coenzymes or inhibitors, or by sequential passages of the extract through several cofactor-related affinity columns. Important principles were developed to purify enzymes exhibiting low affinity to the affinity columns. By “affinity filtration” of the extract through the affinity column, enzymes of low affinity can be retarded and separated effectively from strongly bound and nonadsorbed proteins. By the “saturation readsorption” procedure, enzymes of low affinity could be effectively separated from those of high affinity by overloading of the extracts on the affinity columns. Readsorption of the leaked low affinity enzymes to a second affinity column often results in better enzyme purification because of the elimination of competitive high affinity enzymes. With the application of these principles, the following enzymes and their genetic variants were highly purified via a single- or two-step affinity column procedure: lactate dehydrogenase-A, lactate dehydrogenase-B, lactate dehydrogenase-X, phosphoglycerate kinase-A, phosphoglycerate kinase-B, cytoplasmic and mitochondrial isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase, glutathione reductase, phosphoglucose isomerase and pyruvate kinase from mouse tissues; alcohol dehydrogenase, malate dehydrogenase, α-glycerol-phosphate dehydrogenase, malic enzyme, and glucose-6-phosphate dehydrogenase fromDrosophila.     

A General Strategy for Targeting Drugs to Bone

Targeting drugs to their desired site of action can increase their safety and efficacy. Bisphosphonates are prototypical examples of drugs targeted to bone. However, bisphosphonate bone affinity is often considered too strong and cannot be significantly modulated without losing activity on the enzymatic target, farnesyl pyrophosphate synthase (FPPS). Furthermore, bisphosphonate bone affinity comes at the expense of very low and variable oral bioavailability. FPPS inhibitors were developed with a monophosphonate as a bone‐affinity tag that confers moderate affinity to bone, which can furthermore be tuned to the desired level, and the relationship between structure and bone affinity was evaluated by using an NMR‐based bone‐binding assay. The concept of targeting drugs to bone with moderate affinity, while retaining oral bioavailability, has broad application to a variety of other bone‐targeted drugs.     

膜亲和色谱的现状、发展和应用

文章对膜亲和色谱的原理、特点、设备、过程和发展情况做了介绍，并对亲和膜在整个膜分离技术中的地位、所占的比重及市场预测做了述评，对膜亲和色谱与其它色谱分离技术的优缺点进行了比较。重点介绍了制备亲和膜的材料，活化方法，间隔臂和配基的种类、选择和共价键合方法，配基和配合物产生亲和作用的机理及解离过程和方法。并对膜亲和色谱在酶、蛋白质、核糖核酸等生物大分子纯化分离方面的应用情况做了述评。     

Affinity Biosensors Based on Electropolymerized Films

This review gives an overview on different types of affinity biosensors based on electropolymerized polymer films that are becoming an important class of analytical tools. These affinity biosensors may be classified according to the strategy used for their fabrication, namely entrapment within polymers during their electrochemical growth, simple adsorption onto electropolymerized films, chemical coupling or affinity interactions between bioreceptors and electropolymerized films or direct electrochemical polymerization of the bioreceptor itself. Recently opened perspectives and potential research directions are also discussed.     

High-performance affinity chromatography

High-performance affinity chromatography is a new technique for the fast and efficient purification of biologically active molecules. It combines the biospecificity of affinity chromatography with the high speed and resolution obtained in high-performance liquid chromatography. In particular, the immobilization of ligands to different silica derivatives and their suitability for high-performance affinity chromatography are discussed.     

Boronate affinity saccharide electrophoresis: a novel carbohydrate analysis tool

The incorporation of specialised carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for fluorophore-assisted carbohydrate electrophoresis greatly improved the effective separation of saccharides that show similar mobilities in standard electrophoresis. Polyacrylamide gel electrophoresis using methacrylamido phenylboronic acid in low loading (typically 0.5-1% dry weight) was unequivocally shown to alter retention of labelled saccharides depending on their boronate affinity. While conventional fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone labelled glucose oligomers showed an inverted parabolic migration, an undesired trait of small oligosaccharides labelled with this neutral fluorophore, boron affinity saccharide electrophoresis separation of these carbohydrates completely restored their predicted running order, based on their charge/mass ratio, and resulted in improved separation of the analyte saccharides. These results exemplify boron affinity saccharide electrophoresis as an important new technique for analysing carbohydrates and sugar-containing molecules.     

Studies on the heterogeneity of anti-hapten antibodies by means of two-dimensional affinity electrophoresis

The molecular heterogeneity of rabbit anti-hapten antibodies has been investigated by two-dimensional affinity electrophoresis (2D-AEP). Anti-dansyl and anti-arsanilic diazo-antibodies were separated into several hundred IgG spots as in the case of anti-DNP antibodies. They were grouped into a number of monoclonal IgG families. At the beginning of immunization, IgG spots having low pI and low affinity were predominant but one or two weeks after immunization the IgG spots with high affinity and high pI increased. After the second or third immunization, the 2D-AEP patterns became stable and constant. Anti-arsanilic diazo and anti-DNP antibodies exhibited only weak cross-reactivity with other aromatic haptens. In contrast, anti-dansyl antibodies cross-reacted to a considerable degree with DNP-hapten. A few anti-dansyl IgG families which have cross-reactivity with DNP-hapten were separated. Their apparent dissociation constants to the haptens and their affinity ratios were calculated from their 2D-AEP patterns, according to the affinity theory.     

Boronic acid–lectin affinity chromatography. 1. Simultaneous glycoprotein binding with selective or combined elution

We introduce a novel combination of boronic acid affinity chromatography with lectin affinity chromatography, dubbed as boronic acid–lectin affinity chromatography (BLAC). Concanavalin A and wheat germ agglutinin lectins were mixed with the pesudo-lectin boronic acid to form the BLAC affinity column and their performance was evaluated with standard glycoproteins. Optimization of the binding and elution buffers for the BLAC system is described. The BLAC columns were employed to isolate glycoproteins of interest using both selective and/or combined elution.     

Sialoside analogue arrays for rapid identification of high affinity siglec ligands

The siglec family of sialic acid binding proteins participates in diverse cell surface biology that includes regulation of immune cell signaling and the interaction of neuronal cells with glial cells. The weak intrinsic affinity of the natural sialoside ligands has hampered the development of synthetic ligand based probes needed to elucidate their roles in siglec function. In this report, we describe a glycan microarray comprising a library of 9-acyl-substituted sialic acids incorporated into sialosides containing the Neu5Acalpha2-3Gal and Neu5Acalpha-6Gal linkages commonly recognized by the siglecs. The array is demonstrated to exhibit utility for detecting 9-acyl substituents that increase the affinity of siglecs for their ligands. Substituents that increase affinity are anticipated to be useful for the design of high affinity ligand based probes of siglec function.     

Synthesis, docking studies and biological evaluation of benzo[b]thiophen-2-yl-3-(4-arylpiperazin-1-yl)-propan-1-one derivatives on 5-HT1A serotonin receptors

A series of novel benzo[b]thiophen-2-yl-3-(4-arylpiperazin-1-yl)-propan-1-one derivatives 6a-f, 7a-f and their corresponding alcohols 8a-f were synthesized and evaluated for their affinity towards 5-HT(1A) receptors. The influence of arylpiperazine moiety and benzo[b]thiophene ring substitutions on binding affinity was studied. The most promising analogue, 1-(benzo[b]thiophen-2-yl)-3-(4-(pyridin-2-yl)piperazin-1-yl)propan-1-one (7e) displayed micromolar affinity (K(i) = 2.30 μM) toward 5-HT(1A) sites. Docking studies shed light on the relevant electrostatic interactions which could explain the observed affinity for this compound.     

Chromatographic separation simulation of metal‐chelating peptides from surface plasmon resonance binding parameters

Some metal‐chelating peptides have antioxidant properties, with potential nutrition, health, and cosmetics applications. This study aimed to simulate their separation on immobilized metal ion affinity chromatography from their affinity constant for immobilized metal ion determined in surface plasmon resonance, both technics are based on peptide‐metal ion interactions. In our approach, first, the affinity constant of synthetic peptides was determined by surface plasmon resonance and used as input data to numerically simulate the chromatographic separation with a transport‐dispersive model based on Langmuir adsorption isotherm. Then, chromatographic separation was applied on the same peptides to determine their retention time and compare this experimental t_R with the simulated t_R obtained from simulation from surface plasmon resonance data. For the investigated peptides, the relative values of t_R were comparable. Hence, our study demonstrated the pertinence of such numerical simulation correlating immobilized metal ion affinity chromatography and surface plasmon resonance.     

亲和层析研究进展

亲和层析具有高选择性,高活性回收率和高纯度等特点,已成为纯化蛋白质等生物大分子最有效的技术之一,本文综述了亲和层析的类型,配基的种类,选择方法以及亲和层技术的最新研究进展,重点介绍了多肽作为亲和配基的制备及筛选方法及其在生物大分了职的应用,并比较了不同亲和层析方法的优缺点及其发展趋势。     

Quantitative structure-affinity relationship of 5-HT1A receptor ligands by the classification tree method

The influence of molecular structure of 346 ligands on their affinity for 5-HT1A receptors was investigated. It was shown that the effectiveness of the proposed novel approach for interpretation of decision tree models compared favourably with the PLS method. In the context of the proposed approach, molecular fragments and their values of the relative influence on the affinity for 5-HT1A receptors were defined.     

A new series of emodin derivatives with bone affinity

A new series of bone affinity compounds were synthesized by linking emodin with 5-fluorouracil derivatives. Their bone affinities were established by hydroxyapative (HA) affinity experiment in vitro, and their cytostatic effects were shown by the MTT assay.     

Affinity Chromatography with Triazine Dyes Immobilized onto Poly(styrene-divinylbenzene)(PSDVB) Microbeads

Recently high-performance liquid affinity chromatography (HPLAC) has been developed very rapidly. There are many materials used as affinity sorbents. The rigid PSDVB microbeads are widely used because of their chemical stability in the entire pH range.     

Arginine-modified DNA aptamers that show enantioselective recognition of the dicarboxylic acid moiety of glutamic acid

We have screened glutamic acid-binding aptamers from a modified DNA pool containing arginine residues using the method of systematic evolution of ligands by exponential enrichment (SELEX). Thirty-one modified DNA molecules were obtained from the enriched pool after the 17th round of selection, and their binding affinities for the target were evaluated by binding assays using affinity gels. Three modified DNA molecules having higher affinity were sequenced and we determined their affinity and specificity for the target by surface plasmon resonance (SPR) measurements. The SPR studies indicated that two of these three aptamers distinguished the dicarboxylic acid moiety of the D-isomer from that of the L-isomer; however, the third aptamer did not show enantioselectivity.     

The tumour-localizing properties of porphyrin derivatives

The tumour-localizing abilities of various kinds of porphyrin derivatives in tumour-bearing hamsters were assessed by nitrogen-pulsed laser spectrofluorometry (N2-PLS). On examination of porphine derivatives (from haemoglobin), it was found that the dimer and acetylated and amidated compounds had a high affinity for tumour tissue; the dimer and hydroxylated compound of phorbine derivatives (from chlorophyll) also showed a high affinity. Furthermore, of the metalloporphines (gallium, zinc and indium complexes), those which contained hydrophilic groups showed a high affinity for tumour tissue; of the metallophorbines (gallium, zinc and indium complexes), those which contained hydrophobic groups showed a high affinity. A correlation was found between the side-chain structure of the porphyrins and metalloporphyrins and their affinity for tumour tissue.     

Affinity separation of enzymes from mixtures containing suspended solids

Agarose beads containing immobilized enzymes or affinity ligands have been made magnetically responsive by adsorbing freshly precipitated magnetite on their surface. These beads are used for affinity adsorption of proteins from complex mixtures containing suspended solids. The magnetically responsive beads and the unwanted (diamagnetic) solids are then separated by magnetic filtration. This magnetic adsorption scheme for direct affinity separation of enzymes from mixtures containing suspended solids is compared with a similar, but nonmagnetic, scheme in which the affinity matrix is supported on fiberglass cloth. The enzyme is allowed to adsorb in this matrix, and the matrix is simply removed physically from the suspension to achieve separation from the unwanted solids. The two methods seem comparable in their ability to separate a desired enzymatic activity. The magnetic methods are technically the more complex of the two, but are significantly the more rapid. The efficiency of separation of diamagnetic and ferrimagnetic solids in these biological systems by high gradient magnetic filtration is good.