Affinity chromatography of Pseudomonas salicylate hydroxylase

In order to facilitate the purification of salicylate hydroxylase (salicylate 1-monooxygenase, EC 1.14.13.1) from Pseudomonas sp. RPP (ATCC 29351), an affinity chromatography procedure was developed employing immobilized salicylate as the affinity ligand. The immobilization was achieved by reacting p-aminosalicylate with the N-hydroxysuccinimide ester of Sepharose 4B-6-aminohexanoic acid. When the bacterial crude extract was chromatographed with this affinity column, salicylate hydroxylase was absorbed to the gel while the bulk of protein freely passed through. The absorbed enzyme was subsequently eluted from the affinity column by applying a 0–60 mm sodium salicylate gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzymatically most active fraction of the affinity effluent revealed salicylate hydroxylase was by far the most predominant protein but there were also small amounts of contaminating proteins. However, a virtually homogeneous enzyme preparation was obtained when the crude extract was first fractionated with a DE-52 anion-exchange column followed by the affinity step. The enzyme preparation obtained by this two-step procedure showed a specific activity of 14.9 units/mg and an A₄₅₀:A₃₇₂:A₂₈₀ of 1.01:1:10.23. Because most of the enzymes belonging to the class of external flavoprotein monooxygenase utilize salicylate analogs as substrates and share many other common properties, there is a strong possibility that the salicylate column may be useful for the purification of other member monooxygenases.     

Preparative and analytical affinity chromatography of neurophysins on methionyl-tyrosyl-phenylalanyl-aminohexyl-agarose

Methionyl-tyrosyl-phenylalanyl-ω-aminohexyl-agarose was synthesized and shown to be suitable for both the affinity chromatographic purification of neurophysins and the measurement of the ligand binding parameters of these proteins by quantitative affinity chromatography. Bovine neurophysin I binds to the tripeptidyl matrix in 0.4 m ammonium acetate, pH 5.7, conditions under which no binding occurs with the parent ω-aminohexyl-agarose. Subsequent elution can be effected with 0.2 m acetic acid. The affinity matrices obtained have capacities for neurophysin of up to 4 mg/ml gel bed volume and therein provide for the convenient purification of the neurophysins by a two-step buffer-acid elution. [Carbamoyl-¹⁴C]neurophysin I also binds to the ligand-agarose matrix. Using this labeled protein, competitive elution analysis was performed by one-step elution of zones of protein with the binding buffer in the presence of varying amounts of soluble competitive ligand, lysine vasopressin. The characteristic decrease of elution volume of labeled protein with increasing soluble, competing ligand concentration indicates that the affinity matrix interacts biospecifically with neurophysin. This analysis allows the binding affinities for both soluble vasopressin and immobilized tripeptide ligand to be quantitated.     

Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters.     

Quantitative affinity chromatography of alpha-chymotrypsin

Affinity chromatography on a column of 4-phenylbutylamine, immobilized on succinylated polyacrylic hydrazide agarose, has been employed to study binding of ligands to α-chymotrypsin. In contrast to earlier studies of competitive elution phenomena, where an added soluble ligand interferes with enzyme binding to an affinity matrix, benzyloxycarbonyl derivatives of aromatic acids have been shown to facilitate binding of chymotrypsin to this matrix. This behavior has been analyzed in terms of an expanded binding scheme for affinity chromatography including the formation of a ternary complex (α-chymotrypsin · benzyloxycarbonyl-amino acid · 4-phenylbutylamine · matrix) where the soluble ligand and immobilized ligand bind to different sites. Equations to describe the phenonema have been derived and utilized to quantitate equilibrium constants for dissociation of the binary and ternary complexes. Benzyloxycarbonyl-Ala-Ala was found to promote earlier elution of the enzyme from its affinity matrix. Other ligands known to bind to the active site do not alter the binding to the 4-phenylbutylamine affinity matrix. These results illustrate the conclusion that binding of a small molecule can alter affinity retention in positive, negative, or neutral modes. This suggests that affinity chromatography could be “fine-tuned” by appropriate selection of cosolutes and illustrates the value of relatively weakly binding affinity matrices in enzyme studies.     

Measurement of osmotic second virial coefficients by zonal size-exclusion chromatography

Numerical simulation of protein migration reflecting linear concentration dependence of the partition isotherm has been used to invalidate a published procedure for measuring osmotic second virial coefficients (B₂₂) by zonal exclusion chromatography. Failure of the zonal procedure to emulate its frontal chromatographic counterpart reflects ambiguity about the solute concentration that should be used to replace the applied concentration in the rigorous quantitative expression for frontal migration; the recommended use of the peak concentration in the eluted zone is incorrect on theoretical grounds. Furthermore, the claim for its validation on empirical grounds has been traced to the use of inappropriate B₂₂ magnitudes as the standards against which the experimentally derived values were being tested.     

A rapid method for the purification of S-adenosylmethionine: Protein-carboxyl O-methyltransferase by affinity chromatography

A simple method to purify S-adenosylmethionine: protein-carboxyl O-methyltransferase (protein methylase II, EC 2.1.1.24) from calf brain has been developed using affinity chromatography. The product of the reaction, S-adenosyl-l-homocysteine, which is a competitive inhibitor of the enzyme, was covalently linked to Sepharose beads. This gel proved to be an effective binder for protein methylase II at pH 6.2 and allowed for specific removal of the enzyme by the addition of the methyl donor substrate, S-adenosyl-l-methionine to the elution buffer. One step using this affinity chromatography column resulted in 377-fold purification of the enzyme and 71% recovery of the activity. Subsequent Sephadex G-100 chromatography enabled the enzyme to be purified 3000-fold from the calf brain whole homogenate. The purified enzyme showed a number of protein methylase II activity peaks following preparative gel electrophoresis with one major enzyme peak.     

Affinity chromatography of lipoxygenases.

A number of aminohexyl agarose derivatives of unsaturated fatty acids have been prepared and evaluated as materials for the affinity chromatography of soybean and pea lipoxygenases. A practical method for a one-stage purification of soybean lipoxygenase-1, with a purification factor of 16, is described, using either linolenate or docosa-4,7,10,13,16,19-hexaenoate as ligands. Results show that alleged competitive inhibitors do not cause sharp elution from the affinity column, and that there is an increasing specificity of binding and sharpness of elution as the proportion of unsaturation in the ligand is increased. These results are discussed in terms of the relative importance of the types of bonding involved in enzyme-substrate binding.     

Purification of the creatine kinase isozymes of the green sunfish (Lepomis cyanellus) with Blue Sepharose CL-6B.

Three homodimeric creatine kinase isozymes (A₂, B₂, and C₂) of the green sunfish (Lepomis cyanellus) were purified by a combination of affinity chromatography, gel filtration, and preparative starch gel electrophoresis. The final preparations were isozymically pure and were used to elicit antibodies in rabbits. The use of the group-specific adsorbant Blue Sepharose CL-6B (Pharmacia) and specific elution conditions for creatine kinase facilitated purification. Fish creatine kinase isozymes are sensitive to denaturation and cannot be readily purified by procedures routinely used for mammalian creatine kinase isozymes.     

Applications of rapid-scanning multichannel detectors in chromatography : Plenary lecture

A review of progress in the field of multichannel detection in column and thin-layer chromatography is presented, together with some novel applications of a computer-based, linear photodiode array UV—visible spectrophotometer for detection in high-performance liquid chromatography (HPLC). Computer-aided methods for simultaneous monitoring of the elution profile at three wavelengths with automatic peak detection and capture of UV spectra are described. The continuous calculation of absorbance ratios during elution is discussed as an index of peak homogeneity. A novel technique for the enhancement of qualitative identification in HPLC, based on transformation of captured spectra to the second derivative or to the decadic logarithm, is proposed. These developments are exemplified by a model system of diacetylmorphine and its principal metabolites and degradation products, morphine and 6-acetylmorphine. The potential utility of three-dimensional projections of (A,ν,t) data is discussed in the context of pharmaceutical, bioanalytical and forensic applications.     

Quantitation of guanosine 5',3'-bisdiphosphate in extracts from bacterial cells by ion-pair reverse-phase high-performance liquid chromatography

A new method to determine the guanosine 5′,3′-bisdiphosphate (ppGpp) concentration in bacterial cultures has been developed. Cells in which the nucleotide pool was stabilized by treatment with formaldehyde were concentrated from samples of cultures and lysed by alkali. The ppGpp was separated from other nucleotides on a reverse phase C₁₈ column by ion-pair high performance liquid chromatography using an isocratic elution system. The area of the peak corresponding to ppGpp in elution profiles obtained by monitoring the absorbance of the eluant at 254 nm was used to quantitate ppGpp. The method allows detection of as little as 1 pmol of ppGpp per A₄₆₀ of culture, with $\text{2.5}\text{pmol}\text{A}{}_{460}$ being quantitated with greater than 90% accuracy.     

An analysis of the influence of age and school-attendance status on the spread of variola minor.

Definitions are proposed for the independent and joint contributions that the chemical groups A and B make to the free energy of association of the ligand A?B with a receptor. The definitions are independent of the choice of the standard state and are consistent with the basic thermodynamic cycle relating the association of the ligands A?B, A?Y and X?B to the receptor Rappaport 1976. The basic idea is the use of the excess free energy of association of the ligand A?Y over the free energy of association of the reference ligand X?Y as the measure of the “independent” contribution of the group A to the binding. This definition allows the free energy of association of the ligand A?B to be written as the sum of the independent contributions of the groups A and B, their joint contribution, and an invariant free energy of association of the reference ligand with any receptor. With the appropriate definition of the receptor-reference ligand complex, water can be chosen as the reference ligand. Using ΔG(A?OH)?AG(HOH), ΔG(H?B?H)?ΔG(HOH) and ΔG(HO?C)?ΔG(HOH) as the definitions of the “independent” contributions of the chemical groups A, B and C to the binding of the ligand A?B?C, the joint contribution of the groups A and C to the binding is ΔG(A?B?C) ? ΔG(A?B?H) ? ΔG(H-B-C) + ΔG(H?B?H).     

The use of gel chromatography to study rapid equilibria of the type A + B forms and is formed from C + D

A frontal gel chromatographic procedure is illustrated whereby the equilibrium constant for hybridization equilibria (A + B ? C + D) may be obtained provided the relative elution volume situation V_A = V_C <V_B = V_D may be realized. The electron transfer between reduced cytochrome c and ferricyanide is used as a model interaction, with Sephadex G-25 as the gel medium.     

One step purification of D-galactose and L-arabinose kinases from Phaseolus aureus seedlings by ATP-sepharose affinity chromatography.

8-β-Aminoethylthio-adenosine triphosphate has been synthesized as an ATP derivative and was successfully coupled with N-hydroxysuccinate ester sepharose. The bounded ATP analog can be used on affinity chromatography as a specific substrate ligand for the purification of kinases by means of enzyme substrate binding interaction. Both solubilized and membrane-bound d-galactokinase and l-arabinokinase extracted from mung bean seedlings were immobilized on the ATP-sepharose gel and purified by elution either with a high ionic strength solution (1 m NaCl) or high concentration of a second substrate.     

Affinity chromatographic separation of secreted alkaline phosphatase and glucoamylase using reactive dyes

A single-step dye affinity chromatographic separation method was developed to separate secreted alkaline phosphatase (SEAP) and glucoamylase produced in CHO cell culture and Aspergillus niger fermentation, respectively. The reactive dye, Procion^® Green H-E4BD, was found to have a good binding capacity for SEAP, whereas Procion^® Blue H-ERD was the best dye ligand for glucoamylase. However, these dyes have a relatively low selectivity for the target protein. Consequently, elution of the adsorbed proteins by KCl solution resulted in a product with many impurity proteins as evident by the multiple protein bands on SDS-PAGE. However, elution of SEAP by its substrate, phosphate, produced a relatively pure protein with a high specific enzyme activity because of the competition for active site between the substrate and the dye ligand. Also, a high-purity glucoamylase product was obtained by elution with a borate solution. The relatively inexpensive dye affinity chromatography thus can be used for purifying enzymes from cell culture and fermentation broths. The adsorption of SEAP on the dye-ligand affinity resin followed the Langmuir isotherm. An axial dispersion model with external mass transfer limitation was developed to simulate the breakthrough curve in the chromatographic column. This mathematical model can be used to scale up the protein adsorption process.     

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.     

Quantitative affinity chromatography: increased versatility of the technique for studies of ligand binding

The potential of affinity chromatography for the characterization of strong solute-ligand interactions is explored by studying the NADH-dependent elution of rabbit muscle lactate dehydrogenase from a column of trinitrophenyl-Sepharose in 0.067 M phosphate, pH 7.2. An interesting development is the simplification of the general affinity chromatography theory that emanates from the use of affinity matrices with a high concentration of immobilized reactant groups. The resultant expression allows evaluation of the intrinsic association constant for solute-ligand interactions from a single series of either zonal or frontal affinity chromatographic experiments conducted in the presence of a range of free ligand concentrations. Thus, contrary to previous belief, an affinity matrix designed for solute purification work should prove to be an asset for, rather than an impediment to, the study of solute-ligand interactions by quantitative affinity chromatography.     

Characterization of the RNA Required for Biosynthesis of delta-Aminolevulinic Acid from Glutamate : Purification by Anticodon-Based Affinity Chromatography and Determination That the UUC Glutamate Anticodon Is a General Requirement for Function in ALA Biosynthesis

The heme and chlorophyll precursor δ-aminolevulinic acid acid (ALA) is formed in plants and algae from glutamate in a process that requires at least three enzyme components plus a low molecular weight RNA which co-purifies with the tRNA fraction during DEAE-cellulose column chromatography. RNA that is effective in the in vitro ALA biosynthetic system was extracted from several plant and algal species that form ALA via this route. In all cases, the effective RNA contained the UUC glutamate anticodon, as determined by its specific retention on an affinity resin containing an affine ligand directed against this anticodon. Construction of the affinity resin was based on the fact that the UUC glutamate anticodon is complementary to the GAA phenylalanine anticodon. By covalently linking the 3′ terminus of yeast tRNA^Phe(GAA) to hydrazine-activated polyacrylamide gel beads, a resin carrying an affine ligand specific for the anticodon of tRNA^Glu(UUC) was obtained. Column chromatography of plant and algal RNA extracts over this resin yielded a fraction that was highly enriched in the ability to stimulate ALA formation from glutamate when added to enzyme extracts of the unicellular green alga Chlorella vulgaris. Enhancement of ALA formation per A₂₆₀ unit added was as much as 50 times greater with the affinity-purified RNA than with the RNA before affinity purification. The affinity column selectively retained RNA which supported ALA formation upon chromatography of RNA extracts from species of the diverse algal groups Chlorophyta (Chlorella Vulgaris), Euglenophyta (Euglena gracilis), Rhodophyta (Cyanidium caldarium), and Cyanophyta (Synechocystis sp. PCC 6803), and a higher plant (spinach). Other glutamate-accepting tRNAs that were not retained by the affinity column were ineffective in supporting ALA formation. These results indicate that possession of the UUC glutamate anticodon is a general requirement for RNA to participate in the conversion of glutamate to ALA in plants and algae.     

Selective separation of human peripheral platelets,granulocytes and lymphocytes by surface affinity chromatography

Selective separation of human peripheral platelets, granulocytes and lymphocytes was investigated by column liquid chromatography using methoxyethoxymethyl (MEM) bonded-phase columns (25 × 0.9 cm I.D.). Isotonic solutions containing mono- and disaccharides, methyl-α-d-pyranosides and a physiological saline at pH 7.4 were used as the mobile phase. Granulocytes and lymphocytes were separated on the MEM-Cellulofine GH-25 column by elution with 0.3 M d-mannose solution. The isolation of platelets and lymphocytes from human leukocyte-rich plasma was performed with a MEM-Sephadex G25 column and elution with 0.27 M sucrose solution. On the same column platelets could also be collected selectively by elution with 0.31 M methyl-α-d-mannoside at the high recovery of 100%. The isolated cells were viable for more than 90%.     

Predicting the elution behavior of proteins in affinity chromatography on non-porous particles.

Affinity chromatography on non-porous particles of microsize is particularly useful for the rapid analysis and micropreparative separation of proteins. The elution behavior of proteins in an affinity column packed with non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate was investigated both theoretically and experimentally, using the lysozyme-Cibacron Blue 3G-A affinity system. Equations used to predict the elution profiles, resulting from the elution by increasing the ionic strength (NaCl concentration) in the mobile phase, were obtained. The maximum adsorbate concentration, desorption rate constant and equilibrium constant under elution conditions were determined by matching experimental data with predicted elution profiles. Based on the parameters determined at a flow-rate of 0.5 ml/min and with 1 M NaCl in the elution buffer, the model equations could predict the elution profiles for other experimental runs, where different flow-rates and sodium chloride concentrations were used. Both the experimental and predicted results revealed that the affinity interaction kinetics are not significantly influenced by the flow-rate and, hence, the film mass transfer. To elute bound lysozyme from immobilized dye ligand, a higher value of the ionic strength leads to a faster elution and a sharper elution peak. The influence of elution conditions on the kinetic and thermodynamic parameters and, consequently, on the elution peak profiles was evaluated. The model equations can also predict the behavior of protein elution from an affinity column by changing the pH of the mobile phase, according to a previous study.     

Fractionation of Escherichia coli isoaccepting tRNA species by sepharose 4B column chromatography.

We have determined the elution profile on Sepharose 4B chromatographic column ofthe tRNA isoaccepting species of all 20 amino acids from Escherichia coli MRE 600. Further chromatography on a reversed phase column (RPC-5) is sufficient, in some cases, for a complete purification.