Unsubstituted Sepharose Research Articles

Effect of accessible immobilized NAD(+) concentration on the bioaffinity chromatographic behavior of NAD(+)-dependent dehydrogenases using the kinetic locking-on strategy.

In preparation for studies aimed at establishing the relationship between immobilized NAD(+) concentration and the concentration of soluble locking-on ligand required to promote biospecific adsorption of NAD(+)-dependent dehydrogenases to immobilized NAD(+) derivatives (the "locking-on" strategy), two approaches were evaluated for varying substitution levels: (i) suitable dilution of the affinity matrix with unsubstituted Sepharose 4B and (ii) direct coupling of the required ligand concentration to the inert matrix. The latter approach was found to be the preferable strategy for evaluation of the locking-on tactic because it produced a more homogeneous distribution of immobilized NAD(+) concentration. Affinity chromatographic studies using S(6)-linked NAD(+) derivatives synthesized to various substitution levels showed that the total accessible immobilized NAD(+) concentration has a direct effect on the locking-on behavior of pyridine nucleotide-dependent dehydrogenases. The one-chromatographic-step bioaffinity purification of l-lactate dehydrogenase (L-LDH, EC 1.1.1.27) from bovine heart illustrates the potential of the locking-on strategy for protein purification applications.

The influence of the degree of cross-linking, type of ligand and support on the chemical stability of chromatography media intended for protein purification

The release of organic compounds from different liquid chromatography media in static conditions has been analysed with a total organic carbon (TOC) analyser. TOC results show that chemical stability increases with the degree of cross-linking in agarose beaded chromatography media and thus extend the working pH-range of the media. Of the unsubstituted chromatography media investigated, Sepharose® 6B, Sepharose CL-6B, Sepharose 4 Fast Flow, Sepharose 6 Fast Flow and Sepharose High Performance, the latter was the most stable medium. Sepharose High Performance releases only about 0·06% of its total carbon content after 1 week in 0·01 m HCl. Agarose beads are more stable to basic conditions (pH 14) compared with acidic conditions (pH 2). From UV spectroscopic and gel filtration results it was found that all Sepharose media release low amounts of 5-(hydroxymethyl)-2-furaldehyde and agarose fragments in acidic conditions. To investigate the effect of different ligands on chemical stability Q Sepharose 6 Fast Flow, DEAE Sepharose 6 Fast Flow, SP Sepharose 6 Fast Flow, CM Sepharose 6 Fast Flow, Phenyl Sepharose 6 Fast Flow, Octyl Sepharose 4 Fast Flow media were also studied under static conditions. In basic conditions it was found that all these chromatography media release carbon compounds to a higher extent than the unsubstituted Sepharose support. In addition, Hofmann elimination of Q and DEAE groups contributes to the decrease in the carbon content of the corresponding anion exchangers. During exposure to acidic conditions (pH 2) the release of carbon compounds was lower than the release from the support to which the ligands were coupled. The exceptions are Octyl Sepharose 4 Fast Flow and SP Sepharose 6 Fast Flow. In the case of Octyl Sepharose 4 Fast Flow, the ligand did not seem to influence chemical stability, whereas the SP group increases the degradation of the Sepharose support. In the case of SP Sepharose 6 Fast Flow the stability in acidic conditions can be improved by increasing the ionic strength. Anion exchangers based on different support polymers (agarose-, polystyrene-, methacrylate- and polyvinyl-based matrixes) were studied under static conditions. Agarose-based anion exchanger was the most stable in basic conditions (pH 14). In acidic conditions (pH 2) the chemical stability was about the same for many different anion exchangers.

Identification and characterization of the major carbohydrate-binding proteins in chicken serum as immunoglobulins.

To isolate the major carbohydrate-binding proteins in pooled normal chicken sera, affinity chromatographies on different affinity gels were carried out. From the results obtained, normal chicken sera were found to contain serum proteins which were Ca(2+)-dependently and -independently reactive with mannose, N-acetylglucosamine, N-acetylgalactosamine, L-fucose, melibiose, lactose, unsubstituted Sepharose 4B, and SeaKem HE agarose. They were electrophoretically and antigenically found to be mainly IgM and IgG.

Immunochemical specificities of the combining sites of bovine immunoglobulins reactive with Sepharose 4B.

Binding specificities of calcium-dependent and -independent bovine IgM and IgG reactive with unsubstituted Sepharose 4B were determined by competitive binding assays. The binding of 125I-labeled calcium-dependent bovine IgM to unsubstituted Sepharose 4B was most effectively inhibited by lactose which was about 100 times more reactive than galactose and melibiose. Other inhibitors were much less potent. With 125I-labeled calcium-independent bovine IgM and IgG, on the other hand, lactose was as potent as galactose. Melibiose was about 10 times less potent than lactose and galactose, whereas other mono- and disaccharides were much less potent. From these findings, the combining site of calcium-dependent bovine IgM reactive with unsubstituted Sepharose 4B may be specific for lactose, whereas those of calcium-independent bovine IgM and IgG specific for galactose.

Identification and isolation of calcium-dependent and -independent bovine immunoglobulins reactive with Sepharose 4B.

Calcium-dependent and -independent Sepharose 4B-binding bovine serum proteins were isolated by affinity chromatography on unsubstituted Sepharose 4B using 2 mM ethylenediamine tetraacetic acid followed by 0.1 M galactose. They appeared in two different protein peaks by gel filtration on Sephacryl S-200. The earlier eluted protein was demonstrated to be immunologically IgM, whereas the retarded one IgG. From these findings, Sepharose 4B-binding bovine serum proteins are suggested to be calcium-dependent and -independent immunoglobulins IgM and IgG.

Salting-out chromatography on unsubstituted Sepharose CL-6B as a convenient method for purifying proteins from dilute crude extracts : Application to horseradish peroxidase

Salting-out chromatography on unsubstituted Sepharose CL-6B as a convenient method for purifying proteins from dilute crude extracts : Application to horseradish peroxidase

Fibronectin binding to a Streptococcus pyogenes strain.

In previous studies, Staphylococcus aureus has been shown to bind fibronectin (P. Kuusela, Nature (London) 276:718-720, 1978), an interaction that may be important in bacterial attachment and opsonization. Recently some strains of streptococci of serological groups A, C, and G were also found to bind fibronectin. The binding to one selected strain of Streptococcus pyogenes has been characterized here. The binding of [125I]fibronectin to streptococcal cells resembles that to staphylococcal cells and was found to be time dependent, functionally irreversible, and specific in the sense that unlabeled proteins other than fibronectin did not block binding. Bacteria incubated with proteases largely lost their ability to bind fibronectin, and material released from the streptococci by a brief trypsin digestion contained active fibronectin receptors. This material inhibited the binding of [125I]fibronectin to the streptococci. The inhibitory activity was adsorbed on a column of fibronectin-Sepharose but not on a column of unsubstituted Sepharose 4B or egg albumin Sepharose. The receptor appeared to be a protein nature since the inhibitory activity of the trypsinate was destroyed by papain and was not absorbed on a column containing monoclonal antibodies directed against lipoteichoic acid bound to protein A-Sepharose. Binding sites in fibronectin for streptococci and staphylococci, respectively, were localized by analyzing the ability of isolated fragments to inhibit [125I]fibronectin binding to bacteria and by adsorbing 125I-labeled tryptic fragments with staphylococcal and streptococcal cells. Both species of bacteria appeared to preferentially bind a fragment (Mr = approximately 25,000) originating from the N-terminal region of the protein. In addition, streptococci also bound a slightly smaller fragment (Mr = approximately 23,000). Fibronectin receptors solubilized from either streptococci or staphylococci inhibited the binding of fibronectin to both species of bacteria.

Hydrophobic interaction chromatography of aliphatic alcohols and carboxylic acids on octyl-sepharose CL-4B: mechanism and thermodynamics

The retenion of homologous aliphatic alchohols and carboxylic acids in dilute phosphate buffer solution was measured on octyl-Sepharose CL-4B and on unsubstituted Sepharose CL-4B as a function f chaing lenghts, pH and temperature. From the retention data the standard thermodynamic function (Δ°, ΔP C p , Δ H°, Δ S°) of the interactio were calculated. The thermodynamics and mechanism of the retention process are discussed.

Isolation and characterization of glycine-rich gelatin-binding protein, a plasma glycoprotein which interacts with gelatin, arginine, heparin, and hyaluronic acid.

A gelatin-binding protein was isolated from porcine blood plasma by affinity chromatography in sequence on columns of Sepharose 4B coupled with gelatin, arginine, and heparin and by gel filtration through Sephadex G-200. The protein was bound strongly to the arginine column and thus could be separated from fibronectin. It reacted with concanavalin A and contained about 5% carbohydrate consisting of neutral sugars (2.6% by weight), hexosamine (1.5%), and sialic acid (0.55%). The glycoprotein also showed an interaction with hyaluronic acid, but not with unsubstituted Sepharose 4B. It consisted of 8 to 10 disulfide-linked subunits of about 47,000 daltons and contained more glycine and less proline than fibronectin. Thus we tentatively named this glycoprotein glycine-rich gelatin-binding protein (GGP). GGP did not react with antifibronectin antiserum. These results indicate that GGP is a glycoprotein which is distinct from fibronectin. However, there remains a possibility that GGP may be derived in vivo from fibronectin.

Isolation of human C-reactive protein and serum amyloid P component

Procedures are described for the isolation in high yield of consistent, highly purified preparations of human C-reactive protein (CRP) and serum amyloid P component (SAP). CRP was obtained from malignant ascitic and pleural fluids by calcium-dependent affinity chromatography on pneumococcal C-polysaccharide covalently coupled to cyanogen bromide-activated Sepharose. It was then gel filtered on Ultrogel AcA44 (acrylamideagarose beads) in the presence of calcium ions, combining molecular sieve chromatography with removal of contaminating SAP by its affinity for agarose. Residual trace contaminants were removed by immunoabsorption with anti-normal human serum and anti-SAP antibodies insolubilised on Sepharose and/or by absorption with Sepharose-Con A to remove glycoproteins and Blue-Sepharose to remove albumin. After a final gel filtration step on Sephacryl S-300 35–44% of the initial CRP was recovered in pure from according to biochemical and immunochemical criteria. SAP was isolated from normal serum by calcium-dependent affinity chromatography on unsubstituted Sepharose beads, followed by solid-phase immunoabsorption of contaminants and finally gel filtration on Sephacryl S-300. At least 50% of the SAP in the starting material was recovered in pure form according to biochemical and immuunochemical criteria. Ready availability of such preparations facilitates biochemical, biophysical and particularly biological studies of these plasma proteins.

A new principle for prevention of diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) possessing colonization factor antigen (CFA/I).

The infant rabbit model, developed to study human ETEC with CFA/I (Evans et al., 1975), was used to evaluate agarose (Sepharose) gel substituted with a hydrophobic ligand for prevention of diarrhoeal symptoms. In a group of rabbits given 0.02 g of palmitoyl Sepharose 1 h after an oral infection with 10(9) ETEC organisms with CFA/I antigen, only 2/11 animals developed diarrhoea, while in control groups given the unsubstituted Sepharose (hydrophilic) gel or ETEC organisms alone, all animals developed diarrhoea. In groups of rabbit given the hydrophobic gel 6 h after the ETEC organisms, only 2/6 animals showed a short period of diarrhoea. The "hydrophobic principle" to prevent ETEC infections will be studied in other animal models and in human volunteers.

Purification of multiple pea ferredoxins by chromatography at high ionic strength on unsubstituted sepharose 4B

At high concentrations of ammonium sulfate (3–4 M) pea ferredoxin (which is soluble under these conditions) can be adsorbed to Sepharose 4B, either by column chromatography or by batchwise treatment. A reverse (3 M to 1 M) ammonium sulfate gradient results in the elution of three peaks of ferredoxin. The spectral ratio A 420/A 280 of 0.47–0.54 indicates that each peak of ferredoxin is highly purified by this single step. A further gel filtration removes residual high molecular weight contaminants from the ferredoxin. The spectrum of the purified pea ferredoxin is typical of other plant ferredoxins in having absorbance peaks at 276 nm, 330 nm, 422 nm and 465 nm. Other chromatographic matrices are capable of adsorbing ferredoxin. Sepharose and Sephacryl were the best adsorbents while Sephadex and cellulose adsorbed ferredoxin less tenaciously. The polyacrylamide-based resins Biogel P-4 and P-200 did not adsorb ferredoxin at high ionic strength.

A novel procedure for the rapid purification of plastocyanin from pea (Pisum sativum) leaves

Plastocyanin is soluble at high concentrations (greater than 3 M) of (NH4)2SO4 but under these conditions will adsorb tightly to unsubstituted Sepharose beads. This observation was utilized to purify plastocyanin from pea (Pisum sativum) in two chromatographic steps. Sepharose-bound plastocyanin was eluted with low-ionic-strength buffer and subsequently purified to homogeneity by DEAE-cellulose chromatography.

The hepatic heparin releasable lipase binds to high density lipoproteins

Received 22 August 1980 1. Introduction Injection of heparin releases two lipases into the circulating blood. One, lipoprotein lipase, originates from extrahepatic tissues [ I]. The physiological role of this lipase is to hydrolyze triacylglycerols and phos- pholipids in chylomicrons and very low density lipo- proteins [ 11. The other lipase comes from the liver [2]. It is generally assumed that also this lipase has an important role in lipoprotein metabolism, but it is now known what its physiological substrate is. We demonstrate here binding of thislipase to high density lipoproteins. 2. Materials and methods Human postheparin plasma was obtained from male donors 10 min after intravenous injection of 100 I.U. heparin/kg body weight. The lipase of hepatic origin was partially purified by adsorption of the postheparin plasma with heparin-Sepharose [3]. It was further purified by adsorption to heparin which had been modified by partialN-desulfation followed by acetyla- tion of exposed amino groups [4]. This step removed antithrombin from the lipase preparation. The specific activity of a typical preparation was 50 pm01 fatty acids released/min X mg (pH 8.5,25’C) against a gum arabic stabilized emulsion of [3H] trioleoylglycerol. Human high density lipoproteins were prepared by ultracentrifugation of normal plasma. Very low density lipoproteins and low density lipoproteins were first flotated by centrifugation at d 1.063 g/cm3 for 24 h at 36 000 rev./min in a Beckman Ti 50 rotor, 15°C. High density lipoproteins were then prepared by cen- trifugation of the infranatant at d 1.21 g/cm3 for 48 h at 36 000 rev./min. In one experiment a subfraction of high density lipoproteins isolated in the density 290 range 1.125-l .21 g/cm3 was used (high density lipo- proteins3). The lipoproteins were dialyzed against 0.1 M NaCl 10 mM Tris/Cl pH 8.5. The concentration of high density lipoproteins used in the experiments is expressed as protein determined by the Lowry method. High density lipoprotein-Sepharose was prepared by mixing CNBr-activated Sepharose (Pharmacia, Uppsala, Sweden) with high density lipoproteins (10 mg protein/ml gel) in 0.1 M NaHC03, 0.5 NaCl at 4°C over night. Remaining activated sites on the Sepharose were blocked by 0.1 M ethanolamine (pH 8). The gel was washed with the carbonate buffer, then with 0.1 M acetate pH 4 and stored in 10 mM phosphate, 0.1 M NaCl, pH 7.4 with 0.2% sodium azide. All experiments were carried out within two weeks after the preparation of the gel. Unsubstituted Sepharose 4B was used as a control. Human apolipo- protein CIIIz was prepared from human very low density lipoproteins as described [5]. Intralipid 10% containing [ 14C] oleic acid-labeled trioleoylglycerol was donated by AB Vitrum, Stockholm, Sweden. The triacylglycerol-rich particles were separated from the excess phospholipids by flo- tation through 20 mM Tris/Cl, 0.1 M NaCl, pH 8.5 by centrifugation for 20 min in a Beckman SW-50: 1 rotor at 25 000 rev./min [5]. The top layer was recovered and dispersed in buffer. Albumin was a fraction V preparation from Sigma, St. Louis, MO., USA. The release of labeled fatty acids was determined as described [5]. Deoxycholate was from Merck, Darmstadt, F. R. G. 3. Results The activity of the hepatic heparin-releasable lipase against a triacylglycerol emulsion was strongly inhib-

Interaction of lipoprotein lipase with heparin-Sepharose. Evaluation of conditions for affinity binding.

Lipoprotein lipases from a variety of sources have been shown previously to bind to heparin and some related polysaccharides. For the present studies lipoprotein lipase purified from bovine milk was used. 1. In batch experiments binding of the enzyme activity to heparin-Sepharose occurred relatively slowly, so that 30min was required for the system to come to near-equilibrium. In contrast, release of the enzyme activity from heparin-Sepharose by addition of salt to the liquid phase occurred rapidly. 2. Some binding was observed also with unsubstituted Sepharose, but this binding had a low capacity compared with that observed with heparin-Sepharose. High salt concentrations, heparin or deoxycholate decreased the binding to unsubstituted Sepharose. These factors also increase the solubility of the enzyme, which is low. 3. Addition of heparin to the liquid phase caused a concentration-dependent release of enzyme activity from the gel. These results suggested that the binding of the enzyme to heparin-Sepharose was mainly through interaction with heparin. 4. The enzyme activity was also quantitatively displaced to the liquid phase at increased concentrations of salt. Among the positive ions tested the following order of effectiveness was noted: Cs(+) approximately K(+)>Na(+)>Li(+); and among the negative the following: SCN(-)>I(-)> NO(3) (-)>Br(-) approximately Cl(-). The differences were quite large. Thus addition of 0.16m-KSCN (in addition to the 0.32m-NaCl originally present) displaced one-half of the enzyme activity to the supernatant, whereas 0.8m-LiCl only displaced one-quarter. 5. The distribution of heparin in the gel also profoundly influenced the binding. Two series of gels were studied. One series was made by mixing heparin-Sepharose with unsubstituted Sepharose. Results obtained with these gels were those expected from a series of decreasing volumes of heparin-Sepharose. In contrast, a series of heparin-Sepharoses made with different degrees of substitution gave quite different results. With these gels the amount of enzyme activity bound per amount of heparin increased markedly, whereas the salt concentration needed to displace the enzyme activity from the gel decreased markedly with decreased concentration of heparin in the gel. 6. On stepwise elution of small columns of heparin-Sepharose the enzyme activity was eluted over a remarkably wide range of salt concentrations. When enzyme eluted at one salt concentration was re-applied, it gave the same elution profile as enzyme previously eluted at other salt concentrations or the entire enzyme preparation. These and other results suggested that, whereas the enzyme preparation was rather homogeneous in its binding to heparin, the heparin preparation was polydisperse in binding of lipoprotein lipase.

Separation of transfer ribonucleic acid by sepharose chromatography using reverse salt gradients.

The transfer ribonucleic acids of Escherichia coli bind to unsubstituted Sepharose in the presence of high concentrations of ammonium sulfate at pH 4.5. Transfer RNA species are eluted individually from the Sepharose by a gradient from high to low concentrations of ammonium sulfate; leucine tRNA is fractionated into five isoaccepting species. The order of elution of these isoaccepting species differs from that seen with reverse phase chromatography. By means of only these two procedures, one isoaccepting species of leucine tRNA can be purified to apparent homogeneity. Isoaccepting tRNA species for 9 amino acids have been resolved. This established the general utility of this chromatographic system for the separation and purification of specific isoaccepting transfer RNAs.

Affinity Chromatography of Cysteine-Containing Histone1

Affinity chromatography based on the reaction between SH groups in protein and +HgC6H4CO groups in the p-mercuribenzoylaminoethyl derivative of Sepharose 4B was examined with a crude preparation of calf thymus cysteine-containing histone. Adsorption of the histone onto the column by specific coupling was found to be optimal in 0.1 M citrate buffer, pH 5.5, containing 5M urea to prevent any aggregation of histones and their non-specific adsorption onto the column, and elution from the column was successfully performed by cleavage of the resulting S-Hg bond with urea-buffer solution containing 0.05 M 2-mercaptoethanol. Under these conditions both the adsorption and elution were quantitative; no adsorption was observed when either SH-blocked histone or unsubstituted Sepharose was used. The cysteine-containing histone thus recovered, after further purification by Bio-Gel P-60 chromatography to remove some cysteine-containing nonhistone proteins contaminating the starting material, showed a single band on polyacrylamide gel electrophoresis and an amino acid composition agreeing with the known sequence of this histone.

Trägergebundene Serotoninderivate und Affinitätschromatographie synaptischer Membranproteine / Resin Linked Derivatives of Serotonin and Affinity Chromatography of Proteins Isolated from Synaptic Membranes

An affinity resin for the isolation of the serotonin receptor from nerve ending membranes was prepared by coupling tryptamine, 5-methoxy-tryptamine or serotonin to CNBr-activated Sepharose 4 B. The tryptamine derivatives, N-6-(aminohexyl)-tryptamines, were obtained by subsequent con­ densation of the corresponding indole derivatives with oxalyl chloride and t-aminocaproamide and reduction with LiAlH4. The 6-aminohexyl-group was used as spacer to link covalently the primary amino-group of tryptamine to agarose. - An extract of membrane proteins isolated from rat brain was resolved on this affinity resin into two fractions as compared with only one fraction using unsubstituted Sepharose 4 B.

Affinity chromatography of a regulatory enzyme based on specific interaction with the effector

A crude extract of yeast was chromatographed on a column of Sepharose containing covalently bound L-tyrosine. The tyrosine-sensitive 3-deoxy-D- arabino -heptulosonate-7-phosphate (DAHP) synthetase was retarded by the column relative to the bulk of the protein and was purified about 100-fold. In contrast, the phenylalanine-sensitive enzyme was not retarded. No retardation was found on an unsubstituted Sepharose column. The purification presumably resulted from the specific interaction between the effector-binding site of the enzyme and the effector attached to Sepharose.