Performance of affinity chromatography columns

Performance of affinity chromatography columns was studied by measuring the rates of adsorption and elution of trypsin in a Sepharose 4B-soybean trypsin inhibitor column and a Sepharose 4B-arginine peptides column. The volumetric coefficient for trypsin transfer was evaluated from the break-through curves of trypsin, and elution profiles bed height of Sepharose 4B-STI column was estimated based on these results.     

Design of affinity tags for one-step protein purification from immobilized zinc columns

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.     

The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (K_d) determined were 1.08 ± 0.49 and 0.0086 ± 0.0006 μM, respectively, consistent with previously reported values. Furthermore, binding affinities (K_i) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained K_i values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.     

Some general methods of preparing affinity columns

Some general methods of covalent coupling of nucleotides, especially derivatized nucleotides, polynucleotides and cofactors to insoluble polymers are described in this paper. Wherever necessary individual methods also carry some information on the binding of enzymes to the same polymers to serve as a guide to the efficiency of the coupling methods.     

Liquid chromatographic behavior of ascorbate on amine columns

Quantitation of ascorbate at concentrations normally found in biological samples and foods has previously been shown to be possible by HPLC analysis. Prefilled amine columns from three manufacturers were presently used to evaluate their potential for separating low concentrations of [14C]ascorbic acid from its degradation products, [14C]dehydroascorbic acid and [14C]diketogulonic acid. A successful separation was achieved on some columns with as little as 200 cpm (30 pmol) of total ascorbate injected. On other columns, injection of 30-500 pmol of ascorbate resulted in as much as 80% of [14C]ascorbic acid eluting with an unpredictable retention time. In these instances the inclusion of nonlabeled ascorbic acid (0.5 mg/ml) to the sample resulted in most of the [14C]ascorbic acid activity eluting at the expected retention time of ascorbic acid. The inclusion of ascorbic acid in samples injected onto the column also resulted in a more discrete peak in the elution of dehydroascorbic acid, and more complete recovery of the total [14C]activity (ascorbic acid, dehydroascorbic acid, and diketogulonic acid) injected onto the column.     

Preparation of N2, N2,7-trimethylguanosine affinity columns

2,2,7-trimethylguanosine (TMG) binding proteins from human cells were purified through TMG-affinity columns. TMG synthesis was improved and the TMG obtained was shown to be similar to the TMG in the 5' cap of the UsnRNAs. The eluates obtained with TMG-affinity chromatographies were very different from those isolated with m7G-affinity columns, thus suggesting that specific TMG-binding proteins were obtained. The fraction may be enriched with factors associated with import and/or hypermethylation of UsnRNPs.     

Modelling of the chromatographic separation of xylose-mannose in ion-exchange resin columns

A mathematical model was proposed for the chromatographic separation of xylose and mannose on an ion-exchange resin in the Pb form: dispersion in the mobile phase, external mass transfer around the particles and internal diffusion were taken into account. Small-scale experiments provided an evaluation of the different parameters. Dispersion in the mobile phase was found to be the predominant phenomenon. The Peclet numbers were calculated by identification in the Laplace domain of the elution profiles. Influence of temperature and initial concentration of the sample were studied.     

Lectin purification on affinity columns containing reductively aminated disaccharides.

A method is described for isolating lectins in pure form and quantitative yield in a single step by affinity chromatography on aminoethyl polyacrylamide gels containing reductively aminated disaccharide residues. The affinity columns were prepared in two steps: (a) direct reductive amination of the disaccharide and aminoethyl gel with sodium cyanoborohydride in aqueous solution at pH 9; (b) N-acetylation of excess amino groups. Affinity columns prepared by reductive amination of lactose, melibiose, maltose, and di-N-acetylchitobiose were used to purify the following lectins: lactose, peanut, castor bean; melibiose, Bandeiraea simplicifolia; maltose, jack bean, common lentil; di-N-acetylchitobiose, wheat germ. These columns are extremely stable, have good flow rates, and high binding capacities.     

Improved thin-layer chromatographic detection of diethylstilbestrol and zeranol in plasma and tissues isolated with alumina and ion-exchange membrane columns in tandem

Clean-up procedures for the isolation of zeranol and diethylstilbestrol (DES) were modified to reduce the analysis time and to increase the efficiency of purification. Several dyes (Fast Blue BB, Fast Corinth V, Fast Blue RR, Fast Blue B, Fast Red Violet B and Fast Violet B) were evaluated, and their minimum detectabilities were determined. Conditions for non-instrumental, semi-quantitative thin-layer chromatography were optimized. Zeranol and DES in plasma and tissues were determined using modified procedures. Enzyme digestion brought about significant improvement in detectabilities of zeranol and DES in both fortified and incurred plasma, serum and tissues. Minimum detectabilities for zeranol and DES were 25 ppb in fortified plasma and tissues. The amount of incurred zeranol measured in the serum of an experimental cow was increased four times, i.e. from 50 to 200 ppb, after protease digestion. Glucuronidase digestion showed an eight-fold increase in detection of incurred zeranol levels in bovine liver eight times. These results suggest that digestion releases zeranol and DES from protein and glucuronide complexes, thereby allowing detection of low levels of zeranol and DES which may not be detectable without digestion. Further modification of the purification with an ion-exchange membrane reduced the analysis time by 25%, and the membranes were regenerated up to ten times without loss of activity, allowing an automated process. This method utilizes inexpensive equipment and avoids use of organic solvent, in this case diethyl ether.     

Simple affinity chromatographic procedure to purify beta-galactoside binding lectins

Affinity chromatography based on the commercial resin Sepharose CL-6B was used to isolate new C1-beta-type lectins from crude preparations of snake venoms (Bothrops jararaca, Bothrops jararacussu, Bothrops newiedi, Bothrops moojeni, Lachesis muta rhombeata). Most of the C-type lectins could be eluted with almost 100% recovery using the competitor isopropyl-beta-D-thiogalactoside (IPTG) or through Ca2+ sequestration with EDTA. The lectin yield varied considerably among the different snake species, but B. newiedi venom was a particularly rich source of lectin, retaining 2.7 mg of lectin by milliliter of resin in saturating conditions. C1-alpha-lectins from Crotalus durisus terrificus venom, from the jack fruit (jacalin) and from bread fruit seeds extract (frutalin) had no affinity, either with or without Ca2+ added, for Sepharose CL-6B, showing that the resin is specific for C1-beta-type lectins. Sepharose CL-6B used as galactose-affinity chromatography provides a simple and fast method for isolating C-type beta-galactoside binding lectins from crude sample preparations.     

Partial purification of dopamine D2 receptors using lectin affinity columns

Dopamine D₂ receptors , detected by [³H]spiperone Dopamine D₂ receptors , detected by [³H]spiperone binding, were solubilized from bovine caudate nucleus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard . The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmaco-logical properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D₂ dopamine receptor is therefore a glycoprotein. binding, were solubilized from bovine caudate nucJeus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard . The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmacological properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D₂ dopamine receptor is therefore a glycoprotein.     

A simple model for predicting the performance of affinity chromatography columns

Summary This paper presents a simple model for affinity chromatography, the approach used is based on the equilibrium stage model introduced by Martin and Synge (1941). The current development eliminates the need for an equilibrium assumption by using a simplified rate equation to describe the adsorption process. Although it is not mathematically rigorous this method has proved useful for the design of column experiments in our laboratory.     

Breakthrough performance of linear-DNA on ion-exchange membrane columns

Breakthrough performance of linear-DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves were not significantly affected by flow-rate or inlet solution concentration. In the theoretical analysis a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine coupled to the solution of the partial differential model equations was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant and the forward interaction rate-constant, which are the parameters of the membrane transport model. Through this approach a good prediction of the adsorption phenomena is obtained for inlet concentrations and flow rates greater than 0.2 mg/ml and 0.16 ml/min.