Human alpha-thrombin binding to nonpolymerized fibrin-Sepharose: evidence for an anionic binding region

In order to investigate ligand binding sites in alpha-thrombin that interact with nonpolymerized fibrin, fibrinogen was conjugated (with CNBr) to Sepharose 4B and converted to the nonpolymerized fibrin resin with alpha-thrombin. Human alpha-thrombin was bound to the resin at 22 degrees C and eluted with a linear NaCl gradient [50-300 mM in 50 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.6] with midpeak elution occurring at an ionic strength that corresponds to 170 +/- 5 mM NaCl. Among various ligands examined, ATP and its analogues caused alpha-thrombin to elute with 125 mM or less salt. Apparent dissociation constants were estimated by the dependence of elution volume on ligand concentration. The most potent ligands for desorption from the column were anionic (e.g., adenine nucleotides), which also inhibit thrombin esterolytic/amidolytic and clotting activity [Conery, B. G., & Berliner, L. J. (1983) Biochemistry 22, 369-375]. The desorption series was at 10 mM concentrations: ATP = ADP greater than pyrophosphate greater than citrate greater than oxalate greater than PO4(3-). Contrastingly, serotonin and related apolar compounds did not cause dissociation of alpha-thrombin from the fibrin resin, even though several of these substances inhibit fibrinogen clotting and esterolytic/amidolytic activities of the enzyme. These data imply that independent sites for apolar and anionic binding in alpha-thrombin are required for converting fibrinogen into clottable fibrin and that alpha-thrombin-fibrin binding involves an anionic site.     

Rapid screening of a large number of immobilized textile dyes for the purification of proteins: use of penicillin-binding protein 4 of Escherichia coli as a model enzyme.

A rapid method for screening the affinity of proteins to dye-modified resins is described. Performing the binding and elution of the protein extracts in a batch-wise manner and eluting the bound proteins with SDS-PAGE denaturation buffer speed up the screening process and allow the analysis of large collections of dyes. Penicillin-binding protein 4 of Escherichia coli was used as a model enzyme to determine the influences of pH, metal ions, and ionic strength (0 to 500 mM NaCl) on its binding behavior using a collection of 98 dye-affinity resins.     

Affinity chromatography of Nicotiana tabacum ribonucleases

The purification of tobacco ribonuclease by affinity chromatography is described. 5′-(4-amino-phenylphosphoryl)-guanosine 2′, (3′) phosphate, a ribonuelease inhibitor, has been synthesized and insolubilized onto agarose beads. The resulting adsorbent binds tobacco and some other plant ribonucleases strongly but reversibly at pH 5.4. The bound enzyme can be eluted by changing the pH or ionic strength of the eluting buffer, or by specific elution with substrate or inhibitor. Binding is not due to simple ion-exchange properties of the adsorbent.     

Simultaneous analysis of several antiretroviral nucleosides in rat-plasma by high-performance liquid chromatography with UV using acetic acid/hydroxylamine buffer Test of this new volatile medium-pH for HPLC-ESI-MS/MS

Zalcitabine (ddC), lamivudine (3TC), didanosine (ddI), stavudine (d4T), carbovir (CBV), zidovudine (AZT), tenofovir (PMPA) and its administrated form (tenofovir diisoproxyl fumarate, TDF), are nucleosides currently approved in HIV therapy. To facilitate pharmacokinetics studies, a specific reversed-phase high-performance liquid chromatography (HPLC) method was developed for their analysis in rat plasma. The method involved a quantitative recovery of these drugs from rat plasma by solid-phase extraction on Oasis HLB Waters cartridges followed by optimised HPLC separation on an Atlantis dC18 column with acetic acid-hydroxylamine buffer (ionic strength 5mM, pH 7)-acetonitrile elution gradient. Quantitation was performed by HPLC/UV at 260 nm. Linear calibration curves were obtained within a 30-10,000 ng/mL plasma concentration range. Correlation coefficients (r2) greater than 0.992 were obtained by least-squares regression and limits of quantification were in 30-90 ng/mL concentration range. Quantitative parameters (accuracy, intra-day repeatability and inter-day reproducibility) yielded satisfactory results. Finally, a new buffer, obtained with acetic acid and hydroxylamine, has been tested in HPLC/ESI-MS/MS and appears to be an efficient volatile buffer in the medium 5-7 pH range. Indeed, at pH 7 and low ionic strength (5 mM), its buffer capacity is one hundred times higher to that obtained for the usual acetic acid/ammonia buffer.     

DNA-binding domains of human plasma fibronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin

We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA-cellulose and heparin-Sepharose affinity chromatography columns was performed by NaCl linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaCl concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA-cellulose or heparin-Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaCl, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA-cellulose at 280 mM NaCl and from the heparin-Sepharose column at 210 mM. Furthermore, we have studied the interaction of thermolysin-digested fibronectin both with DNA-cellulose and heparin-Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.     

Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5-8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris-HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved.     

Binding of ATP to Brain Glutamate Decarboxylase as Studied by Affinity Chromatography

Abstract: The interactions of two forms of porcine brain glutamate decarboxylase (β-GAD and γ-GAD) with the effector ATP were studied by affinity chromatography. A third form, γk-GAD, was only slightly retarded by the affinity matrix and was eluted in the buffer wash. The interaction of GAD with the ATP affinity matrix was qualitatively similar to its interaction with free ATP as reported in previous kinetic studies. The rank order of adenine nucleotides as eluting agents and affinity ligands was ATP > ADP > AMP. GAD was also eluted by its cofactor, pyridoxal 5'-phosphate, and this was enhanced by 1 mM P_i In contrast, a high concentration (140 mM) of P_i by itself was required to elute the enzyme. GAD remained active while bound to the affinity column and was eluted in the holoenzyme form by ATP, indicating that the affinity ligand did not bind in the active site and did not displace catalytically active cofactor from the enzyme.     

Erythrocyte isozymes of phosphofructokinase in genetically high- and low-2,3-diphosphoglycerate rats

A major locus (Dpg) with two alleles (d and D) controls erythrocyte 2,3-diphosphoglycerate (DPG) levels in Long-Evans rats and is closely linked to a locus (Hbb) determining a hemoglobin electrophoretic polymorphism. Glycolytic intermediate levels and phosphofructokinase (PFK) kinetic studies suggest that in vivo PFK activity differences underlie the differences in DPG levels. We report here chromatographic and immunologic evidence that rat erythrocyte PFK is composed of two isozymes which elute from DEAE-Sephadex at positions identical to those of the isozymes in platelets and liver, respectively. The percentage of platelet-type PFK is significantly (P less than 0.05) smaller in low-DPG (dd) hemolysates than in DD hemolysates regardless of hemoglobin phenotype. When hemolysates were prepared in a stabilizing buffer, PFK specific activity was significantly (P less than 0.005) higher in DD rats. These data suggest that the PFK kinetic differences may result from alterations in the isozyme composition of active PFK.     

Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography.

1. Lipoamide dehydrogenase NADH: lipoamide oxidoreductase, (EC 1.6.4.3) from pig heart has been separated into two sets of isoenzymes by chromatography on lipoyl- and NAD+-derivatized Sepharose-4B matrices. The first fraction is eluted at 30 mM sodium phosphate buffer (pH 7.2), the other requires a higher ionic strength. The two groups originate from the alpha-ketoglutarate and the pyruvate dehydrogenase complex respectively. 2, Hydrophobic chromatography on a homologous series of alkyl-Sepharoses lead to similar results. The first fraction is eluted with 30 mM phosphate buffer in the case of propyl- and butyl-Sepharose but a high ionic strength is required in the case of an increased chain length (C5--C6). The second fraction is reversibly bound on Sepharose-NC3 and -NC4 but binding becomes irreversible at higher chain lengths. 3. Aminoalkyl-Sepharose behave qualitatively as the alkyl derivatives although elution, particularly in the case of the second fraction, can be realized at lower ionic strength. 4. Matrices which are negatively charged (Sepharose-NCnCOOH, n equal 3--7) have no affinity at pH 7.2. 5. The influence of a neutral polar substituent has been studied by comparing the following matrices: Sepharose-NC6OH, Sepharose-NC6NH2 and Sepharose NC6. Binding of the various isoenzymes is completely reversible in the case of a Sepharose-NC6OH matrix and the elution behaviour is identical to that on propyl- and butyl matrices.     

HPLC analysis of estrogen receptor by a multidimensional approach

Previously we demonstrated the polymorphism of estrogen receptors (ER) in cytosol of various tissues based upon properties of size, shape and surface charge. This study describes the application of a multidimensional approach utilizing HPLC for characterization of ER. Cytosols from human uterus and endometrial carcinomas were characterized sequentially by high performance size exclusion chromatography (HPSEC) on Spherogel TSK-3000 SW, and high performance ion-exchange chromatography (HPIEC) using SynChropak AX-1000 anion exchange columns. Using HPSEC, specific estrogen binding was exhibited by a 30 A isoform and by one appearing after the V0 (approximately 60 A) in human uterus. However, in endometrial carcinoma other smaller binding components with Stoke's radii of less than 20 A were observed also. In buffers containing 400 mM KCl, predominantly a 28-30 A species was observed by HPSEC. Further characterization of the 28-30 A isoform from low and high salt elution from HPSEC was accomplished with an AX-1000 column. With either condition, 2 forms were eluted on HPIEC, 1 in the column wash (retention time 8-9 min), and the other at 50-70 mM phosphate. The elution profile of the larger species (approximately 60 A by HPSEC) on the ion-exchange column was time dependent. Immediate analysis (within 15 min) showed a profile similar to that of the original cytosol which contained minor components eluting in wash buffer and at 50-70 mM phosphate and a major isoform at 180 mM phosphate. However delayed analysis (after 2 h) of the 60 A isoform showed a similar profile (components in buffer wash and at 50-70 mM phosphate) obtained with the 30 A species. This time dependent change was not observed for the 30 A species or for the original cytosol. Estrogen receptors in cytosol sedimented at 10S and 4S in low ionic strength gradients and at 4S in sucrose gradients containing 400 mM KCl. The 28-30 A and 60 A species recovered from HPSEC sedimented at 3.5S. This multidimensional approach indicates that native estrogen receptors dissociated into a number of smaller molecular isoforms, which were distinguishable by different surface charge properties.     

Ion exchange chromatography of proteins-predictions of elution curves and operating conditions. II. Experimental verification

The applicability and validity of the model developed in Part I were confirmed experimentally. In this article, various proteins were eluted both by stepwise and linear gradient elution on DEAE ion exchangers under a variety of experimental conditions. Adsorption isotherms were measured as a function of ionic strength in batch experiments. The moment method was empolyed for the determination of various parameteres such as the gel-phase diffusion coefficient and the longitudinal dispersion coefficient. By use of these parameters and the experimentally measured ionic strength of the peak position, the number opf plates was determined according to the method described in Part I. Theoretical elution curves were calculated with the experimentally measured adsorption eqluilibria and the number of plates. Good agreement was observed between theory an experiments. Various factors affecting the separation were investigated. It was found that the effect of the number of plates for salts, N'(p), was negligible except the case of stepwise elution of high ionic strength buffer. When elution curves were symmetrical, the widths of the elution curves were inversely proportional to the square root of the number of plates of proteins, N(p), as in other chromatographic techniques. A simple graphical method for prediction of the peak position in linear gradient elution described in Part I was found applicable when the elution curves were symmetrical. A useful correlation of prediction of the peak width in a linear gradient elution was proposed on the basis of the approximate solution derived in Part I of this study. This graphical method and correlation permit easy prediction of the peak position and peak width in linear gradient elution in the case of symmetrical elution curves.     

Affinity chromatography of Band 3, the anion transport protein of erythrocyte membranes

Affinity chromatography of Band 3 was performed using a series of affinity matrices synthesized with various inhibitor ligands and spacer arms. Hydrophilic spacer arms greater than four atoms in length were essential for Band 3 binding. An affinity resin prepared by reacting 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (Ki = 10 microM) with Affi-Gel 102 was found to be the most effective resin of the series tested. Solubilized proteins from human erythrocyte membranes were incubated with the affinity resin, and pure Band 3 was recovered by eluting with 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS; Ki = 2 microM). Band 3 bound to the resin specifically in its stilbene disulfonate binding site, and optimal binding was achieved at pH 8 and at high ionic strength. At 4 degrees C, up to 80% of the bound Band 3 could be eluted by 1 mM BADS, whereas the remainder could be eluted under denaturing conditions using 1% lithium dodecyl sulfate. At 22 or 37 degrees C, the amount of BADS-elutable Band 3 was reduced with a concomitant increase of Band 3 in the lithium dodecyl sulfate elute. Thus, for successful affinity chromatography, the experiment must be carried out rapidly at 4 degrees C. This procedure was also used to purify the Band 3 protein from mouse, horse, pig, and chicken erythrocytes.     

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.     

Selectivity of IMAC columns in trypsin inhibitor purification

The properties of an adsorbent and the parameters in an adsorption process affect the resolution of chromatographic purifications. This is reflected in the elution profile, which shows the relative affinity of different proteins for a specific adsorbent. In the work presented here, elution profiles for trypsin inhibitor were used to study the effects of the concentration of trypsin inhibitor, ionic strength of the protein solution, slope of the elution gradient, and the regeneration treatment of the chromatography column on the selectivity of the adsorbent Cellufine Chelate-Cu(II)(ida). Cytochrome c was used as a reference protein. Variations in the concentrations of trypsin inhibitor and in the ionic strength of the buffered solution did not have any effects on the elution profile. On the other hand, changes in the slope of the pH gradient used for elution caused shifting of the elution peaks toward lower values of the elution volume, resulting in the best strategy to modify the elution profile of the system. Finally, using a constant slope pH gradient of elution, the variation of the selectivity of the adsorbent for trypsin inhibitor when subjected to cleaning treatments with 0.5 N NaOH was studied. Appropriate cleaning practices used in industry were followed. The adsorbent showed only a slight tendency for resolution loss in the order of 2 x 10(-4) days(-1). The results presented here show a good stability of the adsorbent when compared to other biospecific adsorbents commonly used.     

Affinity purification of fibrinogen using a ligand from a peptide library.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.     

Ion exchange chromatography of proteins-prediction of elution curves and operating conditions. I. Theoretical considerations

A mathematical model is proposed for the elution of proteins on ion exchange columns by a linear gradient increase and stepwise increase of ionic strength in order to predict relationships between the elution characteristics (the peak position, the peak width, etc.) and the operating conditions (the flow rate, the slope of gradient, etc). This model is in principle based on the continuous-flow plate theory, in which the protein concentration and ionic strength dependent distibution coefficient between proteins and ion exchangers and zone sperading effects are taken into consideration. The advantage of this model is its simplicity since it requires only two parameters: The distribution coefficient and the number of plates. Since the distribution coefficient of proteins depends on both the protein concentration and ionic strength of the elution buffer, the number of plates should vary with time. However, it is extremely difficult to take into consideration the time-dependent number of plates. Therefore, we assume that the number of plates is constant and related to that number derived from a mass balance model which includes longitudinal dispersion and gel phase diffusion. On the basis of these assumptions, a method for determining the number of plates by the moment method is presented. Although the dependencies of the peak position and peak width on the slope of linear gradient are predictable by numerical calculations of the present model, simpler methods for prediction of these dependencies are desirable. A graphical method is proposed for prediction of the peak position. For prediction of the peak width, an asymptotic solution is derived from a quasi-steady-state model.     

Elution countercurrent distribution

The single withdrawal technique, here called elution countercurrent distribution, was applied for the first time on aqueous two-phase systems. The modifications necessary for elution countercurrent distribution of a thin-layer countercurrent distribution apparatus are described. The results from a test run with sulfuric acid showed good agreement between the experimental and the theoretical curves for elution countercurrent distribution. Thylakoid membrane vesicles from spinach chloroplasts were subjected to the elution countercurrent distribution. A gradient of NaCl in the eluting upper phase was used to elute the different fractions in order of their affinity for the moving upper phase. Inside-out thylakoid vesicles were resolved into two major and several minor fractions having different chlorophyll a/b ratios. Sonicated inside-out thylacoid vesicles were resolved into at least five different fractions with different chlorophyll a/b ratios. An increased resolution is obtained with elution countercurrent distribution compared to the fundamental process.     

Association of rapidly-labelled RNAs with actin in nuclear matrix from mouse L5178Y cells

More than 90% of rapidly-labelled nuclear RNA was associated with a nuclear matrix prepared from mouse leukemia L5178Y cells. The binding was not affected with up to 4 M NaCl; however, these RNAs were released from the nuclear matrix by treatment with a low ionic strength buffer (5 mM Tris-HCl buffer, pH 7.5, containing 1 mM ATP, 1 mM dithiothreitol, 0.2 mM ethylenediaminetetraacetic acid (EDTA) and 0.4 mM calcium chloride), without destruction of the sphere of the nuclear matrix. Actin filaments in the nuclear matrix were depolymerized with this buffer accompanied with rapidly-labelled RNAs. When the depolymerization was inhibited by slight modifications of the low ionic strength buffer (replacement of ATP by the same concentration of GTP; replacement of calcium ion by the same concentration of magnesium ion; addition of 20 micrograms/ml of phalloidine, which is a specific inhibitor of actin depolymerization), the release of rapidly-labelled RNAs from the nuclear matrix was also inhibited. The complex containing rapidly-labelled RNAs and matrix proteins was solubilized by a sonication from the nuclear matrix, and subjected to cesium chloride equilibrium centrifugation. Rapidly-labelled RNAs were concentrated on the bottom of the gradient accompanied with a small number of proteins (68K, 60K, 43K and 40K). The 43K protein was identified as actin by immunoblotting. By RNase digestion before equilibrium centrifugation, actin in the bottom fractions disappeared. These results suggest that rapidly-labelled RNAs anchor on the actin filaments in the nuclear matrix.     

Carrier protein effects on DNA-cellulose chromatography of putative steroid receptors.

In the absence of carrier proteins, putative androgen receptors elute from DNA-cellulose in the range of 120 to 190 mM NaCl. However, in the presence of lysozyme, most of the receptor elutes in the range of 200 to 230 mM NaCl. This is the same range in which the lysozyme itself, a basic protein, elutes after being chromatographed in the same manner. Moreover, at low ionic strength, lysozyme also increases the sedimentation velocity of both androgen and estrogen receptors. In contrast, bovine serum albumin neither adheres to DNA-cellulose nor alters the sedimentation properties of these proteins. The lysozyme effects can account for some discrepancies reported in the literature. Thus, for qualitative elution studies, the use of lysozyme as a carrier protein is not advised, although its direct interaction with receptors might facilitate quantitative fractionation.     

Fractionation of plasma proteins using dye-ligand chromatography: elution profiles on immobilized green TSK-AF

The fractionation of human plasma by chromatography on immobilized Green TSK-AF was assessed by immunological analysis of the elution profiles of 27 different plasma proteins. A three-step procedure was used to elute proteins from the column. First a low-molarity buffer (30 mM sodium phosphate, pH 7.0, I = 0.053) was applied; then a linear salt gradient (0-1.0 M NaCl in the above buffer) was followed by an additional wash with four bed volumes of 1.0 M NaCl. Tightly bound proteins were finally stripped with 0.5 M NH4SCN. The elution profile of the proteins using this procedure appears to be very reproducible. Comparison with the profile obtained upon chromatography on Cibacron Blue 3GA [Gianazza, E. and Arnaud, P. (1982) Biochem. J. 201, 129-136] indicates significant differences between the binding properties of the two gels. These differences can be used to design a "tandem-chromatography" system which provides an efficient means for the separation of several plasma proteins.