Article

Lectin Coprecipitative Isolation from Crudes by Little Rock Orange Ligand

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Matrix ligands are intended for upstream use with dilute crudes on a large scale, splitting out sought-for proteins by coprecipitating them as dense, protected aggregates. Matrix ligand coprecipitation is rapidly, quantitatively reversible, by pH shifting and trapping matrix ligands on ion exchange resin, releasing the sought-for protein. Four lectins, wheat germ agglutinin, peanut lectin, concanavalin A, and Phaseolus vulgaris (red kidney bean) lectins, were coprecipitated from crude extracts, 0.05 to 0.4% crude protein, in a single step using Little Rock Orange matrix ligand. All were compared in specific activities (erythrocyte agglutination) and in SDS-PAGE analysis with the four corresponding commercial lectins purified by affinity chromatography. All four matrix-coprecipitated ligands were specifically active within range of the corresponding vendor (Sigma Co.) affinity chromatography-purified lectins. The matrix ligand coprecipitative technique requires optimization of ligand-protein (crude) ratios, denoted y, and determination of suitable pH ranges for coprecipitation relative to lectin isoelectric pH. These parameters control electrostatic ion pair association: ligand head anion binding to cationic target proteins. The coprecipitative and protective powers of new ligands like Little Rock Orange, their ability to scavenge sought-for lectins from dilute crudes, depend on ligand organic tail-tail association. After the strong anion heads of ligands bind to cationic proteins, their organic tails stack and draw the ligand-protein complexes together as aggregated coprecipitates.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

Research
Full-text available
The lectins are glycoproteins or sugar binding proteins of non-immune origin but are barred from sugar binding antibodies and enzymes. Lectins are isolated and purified from seeds of Glycine max by soxhlet extraction and dialysis. These collected crude lectins were centrifuged till pH is shifted downward to optimal pH for coprecipitation. Filtration of the same carried out on a Buchner funnel with a pad of Hiflo Supercel on whatman paper. Galactose was added as a ligand to the mixture kept at 250C for 10-20 min. It formed matrix coprecipitation which was centrifuged to remove additional particulates. Supernatant was removed and retained the galactose lectin coprecipitate which finally yields lectins, further purified by dialysis. Encapsulation by spray drying using maltodextrin and lactose along with the Eudragit S100 targeted the drug moiety to colon. Purified Lectins have the binding property of carbohydrate moieties on the surface of erythrocytes which agglutinate the erythrocytes, these lectins were evaluated by the agglutination test using "A" positive blood group. These lectins showed anticancer activity against the colorectal type of cancer cell lines HCT-116; proved as new link for developing anticancer drug specific to colorectal adenomas from Glycine max seeds. Calculated IC 50 value by SRB cytotoxic study which was found 12 compared with capecitabine as standard anticancer drug which was 9.
Chapter
Proteins are most efficiently extracted into dilute solution, whereas subsequent handling is more convenient if the protein is present in a relatively small volume. The first step, following the extraction of the protein into solution is, therefore, usually to concentrate it into a smaller volume. The concentration method may be non-specific, in which case only the water is removed and all non-volatile molecules are concentrated. Alternatively, it may be non-specific with respect to large molecules, i.e. the water and all small molecules are removed and all large molecules. including all the proteins, are concentrated. Finally, the concentration may be more-or-less specific, i.e. a particular protein may be concentrated in relation to the water and other molecules, including some protein molecules.
Article
Ligands are being developed for the upstream isolation-purification of sought-for proteins from dilute crudes by ligand–protein coprecipitation. The ligands are alkane-substituted azoaromatic anions (dyes) with sulfonate heads. Overall coprecipitation is comprised of two main reactions. Ligands first bind electrostatically and stoichiometrically to protein molecule cationic side chains in solution, approximately to a point where the protein net charge ZH is ion-pair titrated with organic anion ligand heads. Organic tail groups cover a sizable portion of the protein molecular surface, triggering the second category of reactions; matrix formation and coprecipitation. Organic tails stack and hydrophobically associate, pulling the complexes together in a host lattice or matrix, enclosing protein molecule guests. Protein molecule structural determinants for coprecipitation of a sought-for protein are protein cationic charge density and location (governed by pH, amino acid composition, and Scatchard–Black reactions). Ligand structural determinants for forcing coprecipitation using 10 to 10 M ligands depend on the ion pairing capacity of the ligands (which determines the stoichiometry) and the details and size of the organic moiety of the ligands. Binding ligands to the target protein in solution contributes the initial part of the overall coprecipitation. However ligand–ligand interactions, in conjunction with ligand placement on proteins to build the host lattice, contribute a large part of the overall coprecipitation. They are sharply dependent on the foregoing factors and on the topology of each lattice to determine the selectivity of matrix ligand coprecipitation. An example is presented of direct coprecipitation of two lectins out of their crudes. Very strongly acting ligands that sweep most proteins and polypeptides out of solution are available. However, use of the maximal coprecipitating power is not necessarily the best strategy. Rather, there needs be struck a balance between coprecipitating power, selectivity, and reversibility for later release of the sought-for protein.
Article
Selected azoaromatic sulfonate anions protect enzymes from inactivation by acid and elevated temperatures. These anionic sulfonate ligands bind to enzyme molecules by forming ion pairs between negatively charged sulfonate groups and positively charged protein groups as demonstrated by the binding stoichiometry determined using isothermal titration calorimetry. When the number of bound sulfonate anions is equal to the total positive charge of the protein, the protein–ligand complexes coprecipitate. Coprecipitation and protection are well correlated, but coprecipitation does not always result in protection. The coprecipitation–protection reactions are reversible. Ligand anions can be removed with anion exchange resins, and full enzymatic activity recovered. Comparison of 29 azoaromatic sulfonate ligands showed that small structural differences in the ligands produce large differences in their abilities to protect enzymes. Some protected enzymes were up to 1000 times more resistant to acid-inactivation, and their inactivation temperatures were over 108C higher compared to nonprotected enzymes. Protection of six sulfhydryl proteases, namely papain, actinidin, chymopapain, bromelain, papaya protease omega, and ficin were compared. These proteases are highly homologous, have almost identical polypeptide chain fold, but differ in the numbers and locations of positive charges, which were crucial factors determining protection. Catalase enzyme, which is larger than papain and of a different class, was also protected by sulfonate ligands from inactivation by acid. q 1999 Elsevier Science B.V. All rights reserved.
Article
Full-text available
Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule.
Article
Full-text available
Crystalline wheat germ agglutinin was prepared from unprocessed wheat germ by a new purification procedure. Its purity and some of its molecular characteristics were examined by a number of criteria. Sedimentation analysis gave a molecular weight of 17,000 ± 1,000 and a sedimentation coefficient of 2.1 S when determined in 0.05 n HCl. At neutral pH, the agglutinin dimerizes with a molecular weight of around 35,000 and a sedimentation coefficient of 3.6 S. Amino acid analyses indicate that the protein contains a high amount of glycine and half-cystine; none of the latter is present as cysteine. Three times crystallized agglutinin is devoid of neutral sugars. Equilibrium dialysis experiments using N-acetyl-[1-¹⁴C]glucosamine indicate that the agglutinin has 2-binding sites for N-acetylglucosamine per mole of the polypeptide chain with a dissociation constant of 7.6 x 10⁻⁴m. This binding is highly specific. The β-1,4 di- and trisaccharides of N-acetylglucosamine showed higher affinities with apparent dissociation constants of 4.9 and 1.2 x 10⁻⁵m, respectively.
Article
Full-text available
1. The purification of wheat-germ agglutinin from commercial wheat germ is described. By ion-exchange chromatography three active proteins (isolectins) were separated, one of which was examined in detail. 2. The amino acid composition is unusual, as 20% of residues are half-cystine and 21% are glycine. Unlike most lectins and contrary to previous reports, this protein is not a glycoprotein. 3. The efficiency of various saccharides as inhibitors of the agglutination reaction was investigated and from this the specificity of the binding site was inferred. Of monosaccharides, only derivatives of glucose with a 2-acetamido group and a free 3-hydroxyl group are effective inhibitors, and glycosides of either anomeric configuration are bound. Oligosaccharides are much more powerful inhibitors of agglutination than are monosaccharides. 4. It is proposed that the binding site consists of three or four subsites with differing specificities, in a cleft in the molecule resembling that proposed for hen's-egg-white lysozyme.
Article
The tautomeric reaction involving a single proton transfer in 1-(phenylazo)-2-naphthol and its 4â²-methoxy, ethoxy, and N,N-dimethylamino derivatives has been investigated with variable-temperature solution and high-resolution solid-state ¹³C NMR spectroscopy. Crystal structures of the parent unsubstituted compound and the 4â²-N,N-dimethylamino derivative have been determined. All of these compounds undergo a fast proton exchange on the NMR time scale between the tautomeric azo and hydrazone forms in both solution and the crystalline phase. Equilibrium compositions in the solid materials are similar to those measured in solution. Crystals of 1-(phenylazo)-2-naphthol are monoclinic, a = 27.875 (7), b = 6.028 (2), c = 14.928 (5) â«, β = 103.57 (2)°, the space group is C2/c with Z = 8, and the structure at 213 K was refined to an R factor of 0.0414 on 1,082 observed reflections. Crystals of 1-((4â²-(dimethylamino)phenyl)azo)-2-naphthol are monoclinic, a = 7.604 (1), b = 7.970 (3), c = 24.381 (7) â«, β = 99.33 (2)°, the space group is P2â/c with Z = 4, and the structure at 193 K was refined to an R factor of 0.0405 on 1,522 observed reflections.
Article
Four chromatographically distinct forms of wheat germ agglutinin were isolated from commercial wheat germ and shown to be similar in amino acid composition, molecular weight, and isoelectric point. Three of these forms were found to undergo subunit exchange with each other or with chemically modified electrophoretic variants to give hybrid agglutinins by exposure to denaturants, pH extremes, or high salt concentrations. One form was not observed to give hybrids, probably due to intersubunit disulfide bonding. Chemical modification studies employed in obtaining electrophoretic variants indicated that acetylation or succinylation of amino groups did not markedly change the lectin dimeric subunit structure or erythrocyte agglutinating ability, but the modified protein was unable to bind to ovomucoid-Sepharose columns. Acetylation of tyrosine residues, in conjunction with amino group acylation, produced a large change in protein conformation, probably involving subunit dissociation. Carbodiimide-mediated carboxyl group modification also produced a conformational change indicative of subunit dissociation, but some binding affinity to ovomucoid-Sepharose columns was retained.
Article
Instead of trying to crystallize or precipitate amino acids and proteins as homogeneous products, often it is easier to coprecipitate or to cocrystallize them. Organic ionic ligands with large apolar groups bind to the solute or compound sought to isolate. The resulting complexes come out of solution as coprecipitates, often as cocrystals. Binding isotherms, Job plot analysis, compositional and calorimetric data give combining stoichiometries for matrix ligands to amino acids, dipeptides and proteins. These are 1:1 or 2:1, for amino acids and dipeptides, in cocrystalline complexes. Coprecipitates of lysozyme and α-chymotrypsin bind anionic ligands strongly in combining ratios very close to protein netproton charge.
Article
A. hypogaea hemagglutinin was purified by ammonium sulfate fractionation and Sepharose 6 B column chromatography. The homogeneity of the purified hemagglutinin was ascertained by ultracentrifugal analysis and polyacrylamide gel electrophoresis. It has a molecular weight of 106.500 and is a tetramer of a subunit with a molecular weight of 27.000. The purified hemagglutinin agglutinated neuraminidase-treated human erythrocytes regardless of their ABO group type, but did not agglutinate intact erythrocytes. In hapten inhibition assays with simple sugars, the so-called Mäkelä's group 2 sugars, which bear the same configuration of hydroxy groups at C-3 and C-4 as D-galactopyranose, were inhibitors for this hemagglutinin. It does not contain any carbohydrate, in contrast to most phytohemagglutinins except concanavalin A and wheat germ agglutinin.
Article
1.1. The Lowry, Rosebrough, Farr, and Randall method for protein assay has been modified so as to give a higher color yield with bovine serum albumin and with five other pure proteins.2.2. Under the new conditions there is direct proportionality between absorbance at 650 nm and weight of protein within the range 15–110 μg.
Article
Crystalline wheat germ agglutinin was prepared from unprocessed wheat germ by a new purification procedure. Its purity and some of its molecular characteristics were examined by a number of criteria. Sedimentation analysis gave a molecular weight of 17,000 ± 1,000 and a sedimentation coefficient of 2.1 S when determined in 0.05 N HCl. At neutral pH, the agglutinin dimerizes with a molecular weight of around 35,000 and a sedimentation coefficient of 3.6 S. Amino acid analyses indicate that the protein contains a high amount of glycine and half cystine; none of the latter is present as cysteine. Three times crystallized agglutinin is devoid of neutral sugars. Equilibrium dialysis experiments using N acetyl [1 14C]glucosamine indicate that the agglutinin has 2 binding sites for N acetylglucosamine per mole of the polypeptide chain with a dissociation constant of 7.6 x 10-4 M. This binding is highly specific. The β 1,4 di and trisaccharides of N acetylglucosamine showed higher affinities with apparent dissociation constants of 4.9 and 1.2 x 10-5 M, respectively.
Article
The cytoskeleton underlying the membrane of erythrocytes is thought to control changes in cell shape such as the diskocyte to the echinocyte. Since the binding of lectins to the transmembrane protein glycophorin blocks the cell shape change, we have proposed that the cytoplasmic end of glycophorin is linked to the cytoskeleton. Here we show that the cytoskeletal protein known as band 4.1 specifically associates with the cytoplasmic domain of glycophorin on inside-out erythrocyte membrane vesicles and also with glycophorin reconstituted into phosphatidylcholine vesicles. The binding of band 4.1 to glycophorin is saturable and inhibitable by antibodies which bind specifically to the cytoplasmic domain of glycophorin. We therefore believe that band 4.1 protein provides the link between glycophorin and the cytoskeleton.
Article
The lectin wheat germ agglutinin (WGA) is an unusually effective agent in controlling both the forward and reverse reactions of the reversible morphology conversion discocyte in equilibrium with echinocyte for the human erythrocyte. Under conditions severe enough to drive the reactions to completion in either direction without the lectin, WGA is able to stabilize both these morphologies and to fully prevent conversion of either morphology. The lectin can quantitatively block both reactions. The ability of WGA to carry out these functions has no obvious rate limitation. Its effectiveness depends mainly on its binding stoichiometry, particularly toward the transmembrane glycoprotein, glycophorin. The critical binding stoichiometries for both the lectin and the echinocytic agent were determined in relation to the binding isotherms using 125I-labeled WGA and 35S-labeled dodecyl sulfate. There appear to be two principal stoichiometries for WGA binding that are important in its control of erythrocyte morphology. The first stoichiometry marks the threshold of obvious protection of the discocyte against strong echinocytic agents such as detergents and, likely, is simply a 1:1 stoichiometry of WGA: glycophorin, assuming currently recognized values of 3--5 x 10(5) copies of glycophorin per cell. The second important stoichiometry, whereby the cell's morphology is protected against extremely severe stress, involves binding of approximately 4--5 WGA molecules per glycophorin. The controls that WGA exerts can be instantly abolished by added N-acetylglucosamine. However, N-acetylglucosamine ligands on the erythrocyte are of less importance than membrane neuraminic acid residues in enabling WGA to control the cell's morphology, as is shown by comparing intact cells with completely desialated cells. WGA can also be used to produce elliptocytes in vitro, but it does this at levels approaching monolayer coverage of the cell with WGA.
Article
Matrix ligands are agents for isolating proteins out of dilute crudes by coprecipitating proteins. The ligands have a strong anion sulfonate head which initiates binding to proteins having a positive net charge, ZH+ approximately 5-20. Initial binding tightens protein conformation and starts to squeeze water from conformationally motile proteins. The tails are stackable hydrophobic organic groups, azoaromatic dyes which draw protein-ligand complexes together. Proteins coprecipitate as guests, in the ligand host matrix. In addition to stacking, ligand tails displace water because of their bulk, and lower the average dielectric constant near charged groups, which reinforces the electrostatic component of binding. Matrix ligands protect proteins, scavenge them from dilute crudes (0.01-0.1 per cent protein), and densify coprecipitates. Detergent ions in low concentrations, 10(-4)-10(-5) M also sometimes serve as coprecipitating agents, entangling their tails but probably not stacking. Divalent metal ions, Zn++, sometimes are useful auxiliary agents. Preparative scaleability from crudes is demonstrated starting from 100-200 g of raw peanuts and raw pineapple to coprecipitate a lectin and bromelain enzyme respectively in 1-2 h with 80-90 per cent activity yields. Ligands are released from coprecipitates by shifting the pH and trapping the ligands with exchange resins. Protein conformation tightening in solution is seen by viscosity measurements.
  • R H Rice
  • M E Etzler
Rice, R. H., and Etzler, M. E. (1975) Biochemistry 14, 4093-4099.
  • D Matulis
  • R Lovrien
  • T I Richardson
Matulis, D., Lovrien, R., and Richardson, T. I. (1996) J. Mol. Recogn. 9, 433– 443.
  • M J Conroy
  • R E And Lovrien
Conroy, M. J., and Lovrien, R. E. (1992) J. Crystal Growth 122, 213–222.
  • R E Lovrien
  • D Matulis
  • J Coligan
  • B H Dunn
  • H L Ploegh
  • D W Speicher
  • P Wingfield
Lovrien, R. E., and Matulis, D. (1997) in Current Protocols in Protein Science (Coligan, J., Dunn, B. H., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T., Eds.), Vol. 1, pp. 4.5.1– 4.5.35. Wiley, New York.
  • A K Allen
  • A Neuberger
Allen, A. K., Neuberger, A., and Sharon, N. (1973) Biochem. J. 131, 163-171.
  • M J Conroy
  • R E Lovrien
Conroy, M. J., and Lovrien, R. E. (1992) J. Crystal Growth 122, 213-222.
  • A C Olivieri
  • R B Wilson
  • I C Paul
  • D Y Curtin
Olivieri, A. C., Wilson, R. B., Paul, I. C., and Curtin, D. Y. (1989) J. Am. Chem. Soc. 111, 5525-5532.
  • I J Goldstein
  • B B Agrawal
Goldstein, I. J., and Agrawal, B. B. (1972) Methods Enzymol. 28, 313-318.