Preparation and characterization of a macroporous agarose monolith as a stationary phase in IMAC chromatography

A macroporous monolith used as stationary phase for the separation of biomolecules by immobilized metal ion affinity chromatography (IMAC), based on D5 agarose (D5) chemically modified was proposed. The characterization of physical properties was studied. Pressure drop was <0.4?MPa, being a very low value compared to other similar chromatographic supports. The adsorption/desorption process was carried out using bovine serum albumin (BSA) at pH 7.4 as a target protein. The monolith was re-used for 20 adsorption/desorption cycles and it was possible to verify that the average percentage of adsorption in all cycles was 89.65%. It was also possible to apply a model in order to obtain the kinetic adsorption constant (k_a), desorption constant (k_d) and equilibrium constant (K_e) by the proposed system. These results indicate that this system is governed by the adsorption process.     

A DNA‐Encoded Library of Chemical Compounds Based on Common Scaffolding Structures Reveals the Impact of Ligand Geometry on Protein Recognition

A DNA‐encoded chemical library (DECL) with 1.2 million compounds was synthesized by combinatorial reaction of seven central scaffolds with two sets of 343×492 building blocks. Library screening by affinity capture revealed that for some target proteins, the chemical nature of building blocks dominated the selection results, whereas for other proteins, the central scaffold also crucially contributed to ligand affinity. Molecules based on a 3,5‐bis(aminomethyl)benzoic acid core structure were found to bind human serum albumin with a K_d value of 6 nm , while compounds with the same substituents on an equidistant but flexible l ‐lysine scaffold showed 140‐fold lower affinity. A 18 nm tankyrase‐1 binder featured l ‐lysine as linking moiety, while molecules based on d ‐Lysine or (2S,4S)‐amino‐l ‐proline showed no detectable binding to the target. This work suggests that central scaffolds which predispose the orientation of chemical building blocks toward the protein target may enhance the screening productivity of encoded libraries.     

Photoprocesses of chlorin e6 bound to lysozyme or bovin serum albumin

The ground and excited state processes of chlorin e6 in aqueous solution were studied in the presence of lysozyme and also bovine serum albumin. Non-covalent binding to proteins was analyzed using fluorescence, UV–visible and circular dichroism absorption spectroscopy. The number of binding sites, n, was 1.5–2.4 and the apparent macroscopic dissociation constant, K_d was 0.2–2.5 μM. The binding of chlorin e6 to lysozyme, in contrast to that of bovine serum albumin, imparted fluorescence quenching; circular dichroism spectra revealed a chiral environment upon binding to β-sheets of bovine serum albumin. Time-resolved photolysis showed a longer triplet lifetime upon interaction with the proteins owing to shielding of the dye. The quantum yields for both damage to chlorin e6 (Φ_d) and for protein oxidation (Φ_ox) were determined under oxygen-free conditions; Φ_d was smaller because of shielding by the protein with respect to the self-quenching of the free dye. The major effects concerning photooxidation in the absence and presence of oxygen are discussed.     

Molecular-Sieve Chromatography of Proteins on Granulated Polyacrylamide Gels

Abstract

A method for molecular-sieve chromatography of proteins on columns of granulated polyacrylamide gels is described in detail. Distribution coefficients (K _D and K _av) are given for the model proteins γ globulin, bovine serum albumin, ovalbumin, ovomucoid, pepsin, myoglobin, and cytochrome c, as well as certain other proteins, between 0.5 M NaCl and polyacrylamide gels of 30 different compositions. The results are interpreted in terms of the Ogston-Laurent-Killander theory of molecular-sieve chromatography, and empirical equations are derived relating the fundamental parameters L and r of this theory to the gel composition. The results are discussed in terms of the gel structure and a method for estimating the average effective pore radius in polyacrylamide gels of different compositions is derived and compared with experimental findings.     

A study of the interaction between fluorescein sodium salt and bovine serum albumin by steady-state fluorescence

The binding of fluorescein sodium salt with three kinds of commercially available bovine serum albumin (BSA) of different grades of purity was investigated at 288, 298 and 313 K by fluorescence and absorption measurements at pH 7.50. The association and dissociation constants K_a and K_d were determined by the quenching of BSA fluorescence in the presence of fluorescein sodium salt. The best results were obtained by fitting raw data by non-linear regression and Lineweaver–Burk equations. The modified Stern–Volmer and Scatchard plots gave less reliable data since the fitting was much more difficult.The agreement of the constants for the three sets of measurements coming from the different BSA was not as good as expected. BSA binding properties differ depending on the different BSA grades of purity. Actually, the binding constants found for the three BSAs used differed in the same set of interactions, even by keeping the experimental conditions constant. These results are a novelty in the field of BSA–ligand binding studies and should be taken into account for future binding studies using BSA. Actually, a large number of aspects should be considered including the grade of purity and the presence of BSA covalent and non-covalent dimers, trimers and oligomers in solution which can affect the goodness of the binding results.     

A thermodynamic study of the binding of human serum albumin onto N,N′‐diethylaminoethyl dextran microbeads

Adsorption of proteins on solid surfaces is widely studied because of its importance in various biotechnological, medical, and technical applications, e.g., biosensors, cardiovascular implants, and chromatography. Adsorption thermodynamics has been studied on the microbeads of N,N′‐diethylaminoethyl (DEAE) Dextran anion exchanger for the human serum albumin (HSA) at 25, 30, 35, 40, and 45°C. As a result, some thermodynamic parameters like Freundlich constants, thermodynamic equilibrium constant (K_D), standard free energy changes (ΔG_assoc), standard entropy changes (ΔS_assoc), and standard enthalpy change (ΔH_assoc) have been evaluated. Using the linear Van't Hoff plot, ΔH_assoc value of the system for the interaction of bovine serum albumin (BSA)‐adsorbed crosslinked DEAE dextran microbeads was determined as 20.650 kJ/mol. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 3942–3947, 2006     

Design and Synthesis of Galactosylated Bifurcated Ligands with Nanomolar Affinity for Lectin LecA from Pseudomonas aeruginosa

Lectin A (LecA) from Pseudomonas aeruginosa is an established virulence factor. Glycoclusters that target LecA and are able to compete with human glycoconjugates present on epithelial cells are promising candidates to treat P. aeruginosa infection. A family of 32 glycodendrimers of generation 0 and 1 based on a bifurcated bis‐galactoside motif have been designed to interact with LecA. The influences both of the central multivalent core and of the aglycon of these glycodendrimers on their affinity toward LecA have been evaluated by use of a microarray technique, both qualitatively for rapid screening of the binding properties and also quantitatively (K_d). This has led to high‐affinity LecA ligands with K_d values in the low nanomolar range (K_d=22 nm for the best one).     

Preparation of bovine serum albumin‐imprinted calcium polyacrylate/alginate hybrid microspheres via Ca2+ crosslinking

Macromolecularly imprinted calcium polyacrylate/alginate hybrid polymer microspheres with the imprinting of bovine serum albumin were prepared with sodium alginate and sodium polyacrylate by using CaCl₂ as a gelling agent in inverse‐phase suspension. Infrared spectrum and conductance titration demonstrated that hybrid components were produced in these microspheres. Rebinding isotherms of the microspheres proved a greater bovine serum albumin affinity for imprinted microspheres relative to nonimprinted ones. Selectivity tests indicated that calcium polyacrylate/alginate hybrid polymer microspheres exhibited good recognition properties for the template protein with the separation factor over 5.30 comparing to the competitive proteins with a similar M_w. The influence of cross‐linker CaCl₂ concentration on the imprinting efficiency (IE) and rebinding capacity was researched. It was found that the cross‐linking density and imprinting efficiency of the hybrid hydrogel could be improved simultaneously. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Hydrophobic charge-induction chromatographic resin with 5-aminobenzimidazol ligand: Effects of ligand density on protein adsorption

Hydrophobic charge-induction chromatography (HCIC) is a highly promising technology for antibody separation. HCIC resins ABI-B-6FF were prepared with 5-aminobenzimidazole (ABI) as the functional ligand. The effects of ligand density on the adsorption of human immunoglobulin G (hIgG) and bovine serum albumin were focused. It was found that the adsorption capacity and dynamic binding capacity (DBC) were improved with the increase of ligand density. The adsorption capacity and DBC of hIgG reached 128.07 mg/g gel and 67.63 mg/g gel. The results indicated that ABI-B-6FF resin has a promising potential for the application of antibody purification.     

Partial purification and characterization of sphingosine N-acyltransferase (ceramide synthase) from bovine liver mitochondrion-rich fraction

Sphingosine N-acyltransferase (ceramide synthase, E.C. 2.3.1.24) was solubilized from bovine liver mitochondrion-rich fraction with n-ocytl β-d-thioglucoside as the detergent and partially purified by sequential chromatography on columns of DE-32, shingosine affinity, and Sepharose CL-6B. The partially purified preparation migrated on SDS-polyacrylamide gel electrophoresis as two major protein bands of 62 and 72 kDa. The molecular mass of the enzyme estimated by gel filtration was 240–260 kDa, suggesting that the partially purified enzyme is present in a subunit form or simply has an aggregative nature. The specific activity of the final preparation for the condensation of sphingosine with stearoyl-CoA increased by 98.7-fold compared with the starting material. The optimal pH value for the ceramide synthesis was 7.5. The partially purified enzyme had an apparent K _m of 146 μM and a V _max of 11.1 nmol/min/mg protein for stearoyl-CoA. The K _m and V _max values toward sphingosine were 171 μM and 11.3 nmol/min/mg protein, respectively. Interestingly, sphinganine was also a good substrate for this enzyme, and the K _m and V _max values were 144 μM and 8.5 nmol/min/mg protein, respectively.     

Synthesis and Structure–Activity Relationship Studies of 2‐(1,3,4‐Oxadiazole‐2(3H)‐thione)‐3‐amino‐5‐arylthieno[2,3‐b]pyridines as Inhibitors of DRAK2

In recent years, DAPK‐related apoptosis‐inducing protein kinase 2 (DRAK2) has emerged as a promising target for the treatment of a variety of autoimmune diseases and for the prevention of graft rejection after organ transplantation. However, medicinal chemistry optimization campaigns for the discovery of novel small‐molecule inhibitors of DRAK2 have not yet been published. Screening of a proprietary compound library led to the discovery of a benzothiophene analogue that displays an affinity constant (K_d) value of 0.25 μM . Variation of the core scaffold and of the substitution pattern afforded a series of 5‐arylthieno[2,3‐b]pyridines with strong binding affinity (K_d=0.008 μM for the most potent representative). These compounds also show promising activity in a functional biochemical DRAK2 enzyme assay, with an IC₅₀ value of 0.029 μM for the most potent congener. Selectivity profiling of the most potent compounds revealed that they lack selectivity within the DAPK family of kinases. However, one of the less potent analogues is a selective ligand for DRAK2 and can be used as starting point for the synthesis of selective and potent DRAK2 inhibitors.     

A Rapid Colorimetric Sensor for Soluble Interleukin-2 Receptor α, Based on Aptamer-Adsorbed AuNP

The soluble interleukin-2 receptor α (sIL-2Rα) is a broad indicator of clinical disease activity in various inflammatory diseases. Here we have developed, for the first time, a rapid, washing-free colorimetric aptasensor based on a sIL-2Rα aptamer (K_d=1.33 nm ). The aptasensor was fabricated with Au nanoparticles (AuNPs) adsorbing sIL-2Rα aptamers. On addition of sIL-2Rα, the aptamers become desorbed from the AuNPs, and this in turn weakens the absorption corresponding to AuNP-catalyzed oxidation of ortho-phenylenediamine (oPD) with H₂O₂. The aptasensor was characterized by TEM imaging, ζ potential measurements, dynamic light scattering (DLS) analysis, and UV/Vis spectrometry, followed by further optimization. The fabricated sensor exhibited great analytical performance, with a linear range of 1 to 100 nm and a detection limit of 1 nm both in buffer and in spiked human serum within 25 min. Other proteins, such as bovine serum albumin (BSA), IL-17Rα, IL-5Rα, IL-13Rα₂, and CD166, showed negligible effects on the aptasensor. Thanks to the great advantages of the aptamers and AuNPs, this aptasensor provides a rapid, simple, and inexpensive process that might offer insights into various diagnostic applications of sIL-2Rα.     

Thermodynamic aspects of the bovine serum albumin adsorption onto N,N′‐ diethylaminoethyl dextran microbeads

Adsorption of proteins on solid surfaces is widely studied because of its importance in various biotechnological, medical, and technical applications, e.g., biosensor cardiovascular implants and chromatography. Adsorption thermodynamics has been studied on the microbeads of N,N′‐diethylaminoethyl (DEAE) dextran anion exchanger for bovine serum albumin at 25, 30, 35 40, and 45°C. As a result some thermodynamic parameters like Freundlich constants, thermodynamic equilibrium constant (K_D), standard free energy changes (ΔG_assoc), standard entropy changes (ΔS_assoc), and standard enthalpy change (ΔH_assoc) have been evaluated. Using the linear Van't Hoff plot, the ΔH_assoc value of the system for the interaction of BSA adsorbed crosslinked DEAE dextran microbeads was determined as 12.5 kJ/mol. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci, 2006     

A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet‐21c expression vector and expressed in E. coli host as soluble protein. A solid‐phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96‐well format for binding to the RKRKRK‐tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK‐tagged GFP with A7C1 emerged as a promising solution (K_a of 2.45×10⁵ M ^?1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.     

Physicochemical characterization of ATP binding to human 5-lipoxygenase

Human 5-lipoxygenase requires ATP as a stimulatory factor. At the two preferred concentrations of the free Ca²⁺, 0.02 μM with a resting cell and 20 μM with a stimulated cell, Scatchard analysis revealed that 5-lipoxygenase has one affinity ATP binding site with aK _d of 4.6 μM at the low Ca²⁺ concentration but has two affinity ATP binding sites with a higherK _d of 4.4 μM and a lowerK _d of 14.5 μM at the high Ca²⁺ concentration. In contrast, in a Tween 20 reaction system, 5-lipoxygenase had similar activation coefficients for ATP at both Ca²⁺ concentrations; these were 12.7 μM at the low Ca²⁺ concentration and 12.0 μM at the high Ca²⁺ concentration. These results showed that 5-lipoxygenase has an ATP binding site and suggest that self-association of 5-lipoxygenase in 20 μM Ca²⁺ may affect ATP binding affinity as measured by Scatchard analysis.     

Lipid-protein interactions

Interaction of human serum albumin with phosphatidylserine was studied by turbidimetric methods. It was found that human serum albu-min will bind phosphatidylserine. Analysis by the law of mass action for multiple equilibria has led to the conclusion that human serum albumin possesses a heterogeneity of binding sites for phos-phatidylserine. The max number of sites and the respective affinity constants were calculated to be : K₁ = 2.0 x 10⁵, n₁ = 2 ; K₂ = 1.3 x 10³, n₂ = 30 Submitted in part as a thesis for the Ph.D. degree, University of Louisville. Carried out under the graduate training program of the U. S. Army Medical Research Laboratory.     

Development of a High-Affinity Antibody-Binding Peptide for Site-Specific Modification

Immunoglobulin G (IgG)-binding peptides such as 15-IgBP are convenient tools for the site-specific modification of antibodies and the preparation of homogeneous antibody–drug conjugates. A peptide such as 15-IgBP can be selectively crosslinked to the fragment crystallizable region of human IgG in an affinity-dependent manner via the ϵ-amino group of Lys8. Previously, we found that the peptide 15-Lys8Leu has a high affinity (K_d=8.19 nM) due to the presence of the γ-dimethyl group in Leu8. The primary amino group required for the crosslinking to the antibodies has, however, been lost. Here, we report the design and synthesis of a novel unnatural amino acid, 4-(2-aminoethylcarbamoyl)leucine (Aecl), which possesses both the γ-dimethyl fragment and a primary amino group. A peptide containing Aecl8 (15-Lys8Aecl) was synthesized and showed a binding affinity ten times higher (K_d=24.3 nM) than that of 15-IgBP (K_d=267 nM). Fluorescein isothiocyanate (FITC)-labeled 15-Lys8Aecl with an N-hydroxy succinimide ester at the side chain of Aecl8 (FITC-15-Lys8Aecl(OSu)) successfully labeled an antibody (trastuzumab, Herceptin^®) with the fluorophore. This peptide scaffold has both strong binding affinity and crosslinking capability, and could be a useful tool for the selective chemical modification of antibodies with molecules of interest such as drugs.     

The use of vegetable oils to recover compounds from aqueous solutions

Corn oil, canola oil, olive oil, sunflower oil, peanut oil and soyabean oil were tested as extractants for the recovery of organic compounds from aqueous solution. Short chain aliphatic alcohols and acids were poorly recovered (K_d < 1.0) while most esters, aldehydes and aromatic compounds tested were satisfactorily recovered (K_d > 2.0) from aqueous solution by vegetable oils. K_d is defined as the ratio of the concentration of the dissolved substance in the extractant to that in the aqueous phase. The exception was caffeine which was poorly extracted from water. The type of oil used appeared to have limited effect on K_d. Increasing the reaction temperature resulted in increased K_d for most readily extractable compounds. Acidulated fatty acid was also tested as an extractant. Although it generally resulted in a greater K_d than the oils or hexane control, acidulated fatty acid was less desirable as an extractant because it tended to create an emulsion with the aqueous phase under the test conditions.     

Competitive interaction of polyanions and anionic dye with bovine serum albumin

The binding of anionic dye, p-(2-amino-6-sulfonyl-8-naphthylazo)benzene sulfonic acid disodium salt (ASANA) to bovine serum albumin (BSA) at pH 7.5 has been studied by spectrophotometric techniques. The values of the dissociation constants were obtained with the use of the Benesi-Hildebrand equation for ASANA. Competitive binding of polyanions, sodium poly(styrene sulfonate) (PSSNa), potassium poly(vinyl sulfonate) (PVSK), poly(acrylic acid) (PAA), and poly(methacrylic acid) (PMAA) and anionic dye to BSA was evaluated through the variations in the different spectra of BSA-dye-polymer systems.