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R G Carbonell

Publications and source records attributed to R G Carbonell.

At least 19 recordsLinked to original sources
PubMed2004

A hexamer peptide ligand that binds selectively to staphylococcal enterotoxin B: isolation from a solid phase combinatorial library.

By screening a solid-phase combinatorial peptide library, a short peptide ligand, YYWLHH, has been discovered that binds with high affinity and selectivity to staphylococcal enterotoxin B (SEB), but only weakly to other SEs that share sequence and structural homology with SEB. Using column affinity chromatography with an immobilized YYWLHH stationary phase, it was possible to separate SEB quantitatively from Staphylococcus aureus fermentation broth, a complex mixture of proteins, carbohydrates and other biomolecules. The immobilized peptide was also used to purify native SEB from a mixture containing denatured and hydrolyzed SEB, and showed little cross-reactivity with other SEs. To our knowledge this is the first report of a highly specific short peptide ligand for SEB. Such a ligand is a potential candidate to replace antibodies for detection, removal and purification strategies for SEB.

Amino Acid Sequence↗
PubMed2001

Studies of the binding of alpha-lactalbumin to immobilized peptide ligands.

The present work investigates the mechanism of binding of alpha-lactalbumin to the peptide ligand WHWRKR and its variants HWRKR and acetylated WHWRKR immobilized on a polymethacrylate chromatographic resin. The presence of two temperature-dependent binding mechanisms and one temperature-independent mechanism was demonstrated. Injections of different forms of alpha-lactalbumin (apo-alpha-lactalbumin, D87A mutant alpha-lactalbumin) displayed similar behaviors when compared to native alpha-lactalbumin, while lysozyme showed little or no binding to the WHWRKR and AcWHWRKR resins. An alternative process for isolation of alpha-lactalbumin from WPI was shown, using consecutive injections of WPI with limited elution.

Adsorption↗
PubMed2000

Chromatographic resolution of tryptophan enantiomers with L-Leu-L-Leu-L-Leu peptide effects of mobile phase composition and chromatographic support.

Tryptophan enantiomers have been separated by zwitterion pair chromatography using L-leucine-L-leucine-L-leucine peptide as the zwitterion pairing agent. The peptide ligand is adsorbed onto an octadecylsilane support with excess ligand present in bulk solution. This article examines the roles of the hydrophobic matrix and the mobile phase components on tryptophan enantiomer binding and resolution. Capacity factors and enantioselectivites are given for both hydrophobic and hydrophilic matrices using mobile phases containing Leu-Leu-Leu peptide and/or salt. A decrease in selectivity upon the addition of mobile phase salt suggests that quadrupolar ion-pairing contributes to chiral recognition. Results indicate that binding is significantly reduced and separation is not achieved when Leu-Leu-Leu is coupled onto cross-linked or polymerized hydrophilic resins as well as onto macroporous polystyrene resin. However, resin-immobilized Leu-Leu-Asp-Leu-Leu-Leu, Leu-Leu-Glu-Leu-Leu-Leu, and Leu-Leu-Leu-Glu-Leu-Leu peptides, with ion-pairing sites designed to mimic the Leu-Leu-Leu-saturated C18 support, also do not resolve tryptophan enantiomers. This suggests the Leu-Leu-Leu structure is critical for enantiomer resolution. Because D- and L-tryptophan are separated in the absence of bulk Leu-Leu-Leu, chiral discrimination is believed to occur at the surface of the octadecylsilane support.

Chromatography, High Pressure Liquid↗
PubMed2000

Fractionation of whey proteins with a hexapeptide ligand affinity resin.

The isolation of individual proteins from whey would allow production of more consistent and reliable products by the food industry and possibly would also increase their use in the pharmaceutical industry. Alpha-lactalbumin is the second most prevalent protein in bovine milk whey and has many uses including serving as an excellent protein source in infant formulas, power drinks and other beverages that require soluble, nutritional protein. In this study, we describe two methods for production of alpha-lactalbumin from whey protein isolate using bioselective adsorption. The use of a peptide ligand (WHWRKR) attached to a resin allowed production of an alpha-lactalbumin-rich fraction with a purity of 90.6% and a recovery of 47.9%, while also producing other fractions of commercial interest. The combined use of an amino resin followed by the WHWRKR resin produce a highly purified alpha-lactalbumin (100%) with a yield of 35.2%.

Animals↗
PubMed1999

Affinity chromatographic screening of soluble combinatorial peptide libraries.

Affinity chromatography using immobilized S-protein was used for the screening of affinity peptide ligands from two soluble peptide libraries. Peptide library I consisted of octamers with glycine (G) at both termini of each peptide, i.e. GXXXXXXG. The six center positions were constructed using random sequences of six L-amino acids (Y, N, F, E, V, and L). Peptide library II also consisted of octamers but with glycine and valine (V) at both termini of each peptide (GVZZZZVG). The four variable center positions of peptide library II were random sequences of 18 L-amino acids. Peptides that were retained specifically on the immobilized S-protein column were eluted by 2% acetic acid. The peptides in the acid eluate were further separated using reversed-phase HPLC. Each separated peptide fraction was collected and the peptide sequences deconvoluted by mass spectrometry (MS/MS). The screenings of peptide libraries I and II resulted in 12 and 7 affinity peptides, respectively. Eight out of the twelve peptides from peptide library I contained the clear consensus sequence NFEV. Peptide library II resulted in affinity peptides with the sequences GVNFEVVG, GVNFTVVG and GVFFEL(I)VG. The advantages and limitations of affinity chromatography in peptide library screening are discussed.

Amino Acid Sequence↗
PubMed1998

Screening of combinatorial peptide libraries: identification of ligands for affinity purification of proteins using a radiological approach.

Peptides deduced from peptide libraries may serve as affinity ligands for protein purification. Identification of a ligand that binds the protein of interest depends highly on the screening method used. One approach which offers simple and direct detection involves screening a solid-phase peptide library against a radiolabeled target protein. We have developed a radiological screening method, using 14C as a radioactive label, that offers high resolution and sensitivity. Less than 100 DPM/bead are detectable after a one-day exposure using autoradiography. The validity of the technique was illustrated by screening a solid-phase hexameric-peptide library spiked with YNFEVL-beads against 14C-labeled ribonuclease S-protein. For this particular system, the amount of protein bound to a single bead was estimated to be in the femtomolar range with a peptide:protein ratio of 500:1. Finally, a portion of the library was screened against 14C-labeled fibrinogen. Three peptides deduced from the library, WQEHYN, WQETYQ, and YENYGY, purified fibrinogen from a mixture with albumin.

Autoradiography↗
PubMed1996

Affinity purification of von Willebrand factor using ligands derived from peptide libraries.

The chromatographic purification of vWF (von Willebrand Factor) from human plasma represents a challenge because it consists of multimers with molecular weights ranging from 0.5 to 10 million Daltons. Phage peptide library screening yielded a lead peptide (RLRSFY) that interacts with vWF. Conservative substitutions of terminal residues of the lead peptide led to a second peptide, RVRSFY, which was more efficient in the affinity chromatographic purification of vWF from protein mixtures. Adsorption isotherm measurements indicated multiple interactions between vWF and the immobilized peptide RVRSFY. Increases in peptide density on the chromatographic supports resulted in stronger association constants and higher maximum protein binding capacities. When the peptide density was lower than 32 mg/mL, there was no measurable interaction between vWF and immobilized peptide RVRSFY in HEPES buffer containing 0.5 M NaCl at pH 7. An increase in peptide density from 32 to 60 mg/mL increased the association constants from 0.9 x 10(6) to 2 x 10(6) (M-1). Divalent salts (calcium and magnesium chloride) were used to elute the retained vWF with 82.5% of the activity recovered. The interactions between vWF and the immobilized peptide RVRSFY are dominated by ionic attractions and also involve hydrophobic interactions at close contact. Finally, the purification of vWF from crude material PEG filtrate of a cryoprecipitate of human plasma is demonstrated using affinity chromatography with immobilized N-acetyl-RVRSFYK.

Adsorption↗
PubMed1989

Reversible conversion of octadecyl-bonded silica to ion-exchange surfaces for protein separations.

Octadecyl-bonded silica, commonly used for reverse-phase high-pressure liquid chromatography, was modified using surfactants bearing ionizable groups and the modified packing used in ion-exchange chromatography of proteins. The surfactants 2-(n-hexadecylheptaethoxy)acetic acid, 1-(n-hexadecyloctaethoxy)ethylene-diamine, and N-(n-hexadecyloctaethoxy)pyridinium were adsorbed onto test columns packed with octadecyl-bonded silica particles. The proteins lysozyme, bovine serum albumin, trypsin, horse serum cholinesterase, and bovine liver carboxylesterase were used to study the ion-exchange characteristics of the modified packings. The retention order of the proteins on the surfactant-modified stationary phases were as predicted by the isoelectric point of each protein. In addition, the interaction of enzymes with the packings did not result in significant loss of enzymatic activity. Surfactant removal was possible with the use of organic solvents and this allowed the octadecyl-bonded surface to be used again in the reverse-phase mode. During the course of the experiments, no degradation in the packing's performance was observed due to loss of adsorbed surfactant, even after over 85,000 column volumes of sodium chloride and Tris-HCl buffers were circulated through the column.

Animals↗
PubMed1988

Affinity surfactants as reversibly bound ligands for high-performance affinity chromatography.

Pyridine was coupled covalently to a nonionic ethoxylated alcohol: octaethylene glycol n-hexadecyl ether. This modified surfactant was found to be a reversible, competitive inhibitor of horse serum cholinesterase. The surfactant bound irreversibly, in aqueous media, to octadecyl-bounded reverse phase silica particles commonly used for high-performance liquid chromatography. The amount of ligand bound was found to be 550 mumol/ml of packing, a concentration that is over 100 times higher than what can be normally bound to agarose affinity chromatography supports. With this packing, a 280-fold purification of cholinesterase from horse serum and a 79-fold purification of human serum cholinesterase were accomplished, with yields greater than 80%, using a 2-cm-long column and a 7-min elution time. The affinity surfactant could be eluted from the column using a 6:4 (v/v) mixture of methanol and isopropanol. This technique should be generally applicable in the development of biospecific supports for high-performance affinity chromatography.

Animals↗
PubMed1980

Hydrophobic chromatography of beta-galactosidase.

The hydrophobic interaction of beta-galactosidase with Sepharose 4B substituted with 3,3'-diaminodipropylamine was studied in both batch and column experiments. The equilibrium and the binding rate constants were determined for different phosphate buffer concentrations. The equilibrium constants exhibit a hysteresis effect, i.e., desorption constants are less than adsorption constants, and the higher the ionic strength to start the desorption, the larger the effect. The rate data are not satisfactorily described by a simple reversible first-order model. The column chromatographic data are semiquantitatively described by a local equilibrium theory without axial dispersion or intraparticle diffusion.

Adsorption↗
PubMed1975

Kinetic behavior of microencapsulated beta-galactosidase.

Escherichia coli beta-D-galactosidase (E.C. 3.2.1.23) was immobilized in cellulose nitrate membrane microcapsules and the reaction kinetics with o-nitrophenyl-beta-D-galactopyranoside (ONPG), lactose, and whole milk were studied using both continuous stirred tank and packed bed reactor configurations. The results of the experiments gave effectiveness factors of 0.3 for ONPG, 0.6 to 0.7 for lactose in solution, and close to unity for lactose in milk. Using a coupled mass transfer and kinetic model, it was possible to estimate the permeability of the microcapsule membrane from the reactor data. Membrane permeabilities on the order of 5 X 10(-3) and 3 X 10(-4) cm/sec were estimated for ONPG and lactose, respectively. It was determined that the membrane was the limiting mass transfer resistance for the overall reaction. The analysis showed that within the microcapsule, the reaction is reaction rate limited for lactose and slightly diffusion limited for ONPG.

Cellulose↗
PubMedDate not supplied

Affinity precipitation of proteins by surfactant-solubilized, ligand-modified phospholipids.

The use of ligand-modified phospholipids solubilized in aqueous solution by nonionic surfactant for affinity precipitation of proteins is described. Avidin was precipitated by contact with solutions in which dimyristoylphosphatidylethanolamine (DMPE) functionalized with biotin (DMPE-B) was solubilized in octaethylene glycol mono-n-dodecyl ether (C12E8) solutions. The nonionic surfactant solubilizes the phospholipid in micelles above its critical micelle concentration (CMC) and in small submicellar aggregates below this concentration. At C12E8 concentrations significantly exceeding its CMC, determined to be about 100 microM, precipitation of avidin by solubilized DMPE-B is not observed. In this regime, binding of protein by DMPE-B was monitored by a hyperchroic shift in the protein's UV-visible spectrum at 231.5 nm. The data were analyzed using a model that considers the four binding sites on the protein to be independent and identical in binding strength for DMPE-B. Below the CMC of C12E8, precipitation is observed and is monitored by increasing turbidity of the solution. The kinetics of precipitation and the aggregate size measured by quasielastic light scattering were analyzed using Smoluchowski kinetics and the Mie scattering theory. These results help establish more completely the factors that influence the precipitation of proteins by ligand-modified phospholipids, and they are helpful in specifying conditions for the precipitation of other proteins.

Avidin↗
PubMedDate not supplied

Preparation and characterization of bifunctional unilamellar vesicles for enhanced immunosorbent assays.

Small unilamellar phospholipid vesicles with covalently attached biotin and horseradish peroxidase (HRP) were prepared and characterized in terms of hydrodynamic diameter, amount and activity of immobilized enzyme, and number of biotin molecules on the outer vesicle surface. In addition, the specific adsorption of these bifunctional vesicles and commercially available biotin-labeled horseradish peroxidase (B-HRP) to antibiotin antibody (ABA) coated polystyrene microtiter plate wells was examined. At low antibody surface densities, the signal (delta A/min) generated by the vesicles adsorbed to the surface was approximately 100 times higher than the signal generated by B-HRP. It was also found that the biotin-conjugated vesicles were able to compete effectively with free biotin in solution for surface ABA sites. These results indicate that this type of vesicle may be used in competitive and sandwich-type enzyme-linked immunoassays to improve the detection limits, increase the signal, and decrease the reaction time necessary to detect a given analyte concentration in solution.

Adsorption↗
PubMedDate not supplied

Competitive immunosorbent assays for biotin using bifunctional unilamellar vesicles.

Competitive immunosorbent assays for the model antigen biotin were performed using both unilamellar vesicles with covalently attached biotin and horseradish peroxidase (HBVs) and commercially available biotin-labeled horseradish peroxidase (B-HRP) as the enzyme-labeled antigen. The assays were performed using anti-biotin antibody (ABA) surface densities ranging from one-tenth to full monolayer coverage. It was found that assays using HBVs strongly depended on the antibody surface density, while assays using B-HRP were relatively insensitive to the antibody surface density. The HBV assay dependence on the ABA surface density was most likely due to multiple point attachment of vesicles to the surface. The lowest detectable antigen concentration (least detectable dose) for vesicles (approximately 10(-9) M) was an order of magnitude lower than the value found for B-HRP (approximately 10(-8) M). The sensitivity (slope of the response vs biotin concentration curve) of assays with B-HRP was comparable to the sensitivity of assays with HBVs at low antibody surface density, probably due to less extensive multipoint attachment. It was also found that assays could be performed with vesicles at antibody surface densities that were at least 5 times lower, in terms of the bulk antibody concentrations used to coat the wells, than antibody surface densities at which B-HRP gave comparable signals (approximately 1 delta A/min).

Adsorption↗
PubMedDate not supplied

Noncompetitive immunoassays using bifunctional unilamellar vesicles or liposomes.

Small unilamellar vesicles (SUVs) functionalized with an enzyme label and with specific ligands for biological molecules are useful as signal enhancement vehicles in the development of enzyme-linked immunoadsorbent assays and other biosensor applications. Bifunctional vesicles were prepared by covalently attaching horseradish peroxidase (HRP) and an antibody to the outside of the lipid bilayer of an SUV. The reaction conditions were optimized to obtain 7-12 antibody molecules and 100-200 HRP molecules per vesicle. The enzyme retained 70-80% of its specific activity after immobilization, and the presence of immobilized proteins on the vesicle surface apparently increased the vesicle stability. To minimize the background signal and maximize the specific signal, the immunoassay protocol was optimized with respect to (1) the type and concentration of blocking agent, (2) the diluents for HRP-antibody-vesicles and sample, (3) the incubation period, and (4) the incubation temperature. The bifunctional vesicles were used in a noncompetitive immunoassay to detect d-dimer, a fibrin dimer formed at the early stages of thrombosis. A second conjugate, HRP-antibody, was prepared, characterized, and used as a control against which to compare the assay using vesicles. The assay results using vesicles led to a detection limit for d-dimer in human plasma 9 times lower than what was achieved using the conventional enzyme-antibody conjugate assay.

Antibodies↗
PubMedDate not supplied

Application of antibody and fluorophore-derivatized liposomes to heterogeneous immunoassays for d-dimer.

Small unilamellar liposomes comprised of cholesterol and phospholipids, in which one of the lipids is labeled with a fluorophore, have been covalently functionalized with antibodies. The liposomes were conjugated with thousands of fluorescein molecules and 10-20 monoclonal antibodies per liposome. These bifunctional liposomes were used in a direct (sandwich-type) immunoassay for the detection of thromboembolic disorders by assaying for d-dimer. D-dimer is the final and the smallest proteolytic product in the degradation of cross-linked fibrin by the plasma protein plasmin. The immunoassay using liposomes was compared to a conventional immunoassay that uses a fluor-antibody conjugate. The liposomes, by virtue of having thousands of fluorophores coupled to one liposome in contrast to one or a few reporter molecules in the conventional fluor-antibody conjugate, performed better on two counts: (1) they lowered the detection limit by a factor of 120 and (2) they provided a 1 order of magnitude amplification in signal. The minimum detectable concentration (MDC) of d-dimer was 5.6 ng/mL with the liposomal assay as compared to an MDC of 674 ng/mL with conventional fluor-antibody conjugate. The results of fluorescence assays were also compared with the results obtained by Singh et al. (Biotechnol. Prog. 1995, 11, 333-341) in an enzyme immunoassay developed using liposomes. These results demonstrate the potential of liposomes in lowering detection limits and increasing the sensitivity of immunoassays.

Antibodies, Monoclonal↗