The purification and characterization of wheat-germ agglutinin

The purification of wheat-germ agglutinin by precipitation with ammonium sulphate and by chromatography on Sephadex G-75, Sepharose-ovomucoid and CM-cellulose is described. This procedure gave agglutinin preparations which were homogeneous on polyacrylamide gels under a variety of conditions. Purified wheat-germ agglutinin formed colourless solutions and was relatively insoluble at neutral pH; maximum solubility in 1mm-tris-HCl buffer, pH7.4, was approx. 1mg/ml. The agglutinin was a glycoprotein containing a single polypeptide chain with an approximate molecular weight of 23000. The N-terminus of the oxidized agglutinin was cysteic acid and the C-terminus was glycine. The amino acid composition showed that the protein was extremely rich in cysteine and cystine; there were 15-17 free SH groups/mol. The absorption maximum for the protein was at 272nm and the molar extinction coefficient at 280nm was 1.09x10(5) litre.mol(-1).cm(-1). Equilibrium dialysis indicated that there was only one binding site per molecule for N-acetylglucosamine.     

The purification, composition and specificity of wheat-germ agglutinin

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.     

The reactivity of the disulfide bonds of human serum haemopexin

The reactivity of the disulfide bonds of the specific haeme-binding plasma protein-human haemopexin has been studied with 2-mercaptoethanol. A molecule of haemopexin has six intrachain disulfide bridges (Takahashi et al., 1985) or which four are reactive while the remaining two can be reduced in the presence of greater than or equal to 4M urea. Disruption of the four reactive disulfide bonds in apohaemopexin abolishes the haeme binding ability. In equimolar haeme-haemopexin complex only one disulfide is reactive which suggests a large change in the tertiary structure of this protein on haeme binding.     

The use of dye-ligand affinity chromatography for the purification of non-enzymatic human plasma proteins

Literature data are analysed in this review on the use of immobilized triazine dyes for the characterization, isolation and purification of non-enzymatic human plasma proteins in both conventional and high-pressure liquid chromatography systems. Attention is focused on the mode of interaction between the dyes and these proteins, as well as on the advantages over previously reported techniques. Future developments are discussed.     

The use of dyes in protein purification.

The role of the matrix, ligand and linking mechanism in affinity chromatography is discussed, special emphasis being placed on the use of dyestuff molecules as ligands. Current knowledge of dye-protein interactions is outlined and problems arising from the use of conventional textile dyes as ligands are considered. Work on the synthesis of novel dye-like molecules designed specifically for affinity chromatography is reviewed. This is seen as leading to the development of improved affinity systems capable of advancing the utility of affinity chromatography in protein purification.     

Properties of succinylated wheat-germ agglutinin.

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 +/- 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 microgram/ml with both lectins and the concentrations of N-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins. The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins. The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.     

The use of antigen-coated protein beads as immunoadsorbent for the purification of antibodies.

A procedure is described using antigen-coated beads of calf serum as immunadsorbent for the purification of antibodies against ferritin and against leukemia-sarcoma virus-induced antigens.     

The use of R-looping for structural gene identification and mRNA purification.

A method is presented for the purification of mRNAs and the identification of structural gene sequences in recombinant DNA molecules. RNA is hybridized to double-stranded linear DNA such that R-loops are formed between most DNAs and their complementary RNA sequences. These R-loops are purified from unhybridized RNAs by gel filtration chromatography in the presence of a high concentration of salt. The complementary RNAs are released from the R-loops by heating, and are assayed by gel electrophoresis or cell free translation to determine their purity and to identify the proteins for which they code. We have demonstrated that recombinant DNAs containing sequences for abundant or moderately abundant mRNAs of Saccharomyces cerevisiae can be identified by this means.     

The binding of human liver acid beta-galactosidase to wheat-germ lectin is influenced by aggregation state of the enzyme.

At low ionic strength and pH 6.0 human liver acid beta-galactosidase exists in two aggregation states, monomeric and multimeric. These species can be separated on wheat-germ lectin-Sepharose, Cellogel electrophoresis and gel filtration on Sephadex G-200, and are not normally interconvertible. On re-application of either form to wheat-germ lectin-Sepharose the equilibrium is re-established and the two forms are interconverted.     

Sheep haemopexin: immunological cross-reactivity with other vertebrate sera and evidence for the absence of haemopexin in some mouflon

A monospecific antiserum to sheep haemopexin was produced in rabbits. By using this antiserum, as well as absorbed antisera to sheep and mouflon serum proteins, it was proved that some mouflon sera lack haemopexin. In immunodiffusion, when using the monospecific antiserum, immunoprecipitates were present only in species' belonging to suborder Ruminantia. None of the other mammalian, nor any of the avian, reptilian, amphibian and fish species tested, showed reaction.     

Antioxidant protection by haemopexin of haem-stimulated lipid peroxidation.

Haem (ferrous protoporphyrin IX) is a reactive low-molecular-mass form of iron able to participate in oxygen-radical reactions that can lead to the degradation of proteins, lipids, carbohydrates and DNA. Oxygen-radical reactions are likely to occur upon tissue damage. Extracellular fluids rely on antioxidant mechanisms different from those found inside the cell, and circulating proteins limit radical reactions by converting pro-oxidant forms of iron into less-reactive forms. Of the compounds tested, only apohaemopexin and the chain-breaking antioxidant butylated hydroxytoluene inhibited (by more than 90%) haemin-stimulated peroxidation as measured by formation of conjugated dienes, thiobarbituric acid-reactive material from linolenic acid or peroxidation-induced phospholipid fluorescence. Haptoglobin, the haemoglobin-binding serum protein, was ineffective. Conversely, only haptoglobin significantly inhibited haemoglobin-stimulated lipid peroxidation. Iron-salt-induced lipid peroxidation was inhibited only by apotransferrin and the iron-chelator desferrioxamine. All lipid peroxidations were inhibited by the radical scavengers butylated hydroxytoluene and propyl gallate. These findings support the concept that transport and conservation of body iron stores are closely linked to antioxidant protection.     

The use of monoclonal antibodies for purification of ribonuclease H from E. coli

Monoclonal antibodies to E. coli ribonuclease H were obtained from two hybrid clones. Using the monoclonal antibodies two immunosorbents were synthesized for RNase H which have a slight difference in the capacity and do not differ in the conditions of antigen elution. A homogeneous (according to electrophoresis in PAAG) preparation of the enzyme was obtained using the synthesized immunosorbents.     

Isolation of mammalian tRNAAsp and tRNATyr by lectin-Sepharose affinity column chromatography.

tRNAAsp from rabbit liver, rat liver and rat ascites hepatoma was readily isolated by concanavalin A-Sepharose (Con A-Sepharose) affinity column chromatography. tRNATyr from these sources was extensively purified by Ricinus communis lectin-Sepharose column chromatography. These results, together with the chromatographic behaviour of four tRNAs (tRNATyr, tRNAHis, tRNAAsn and tRNAAsp) on acetylated DBAE-cellulose column chromatography suggested that tRNAAsp contains a Q nucleoside species having a mannose moiety while tRNATyr contains Q nucleoside with galactose. The sugars attached in 4-position of cyclopentene diol in the Q molecule are therefore not present at random in the four tRNAs, but present only in each specific tRNA. This is the first case which shows that plant agglutinin interacts with nucleic Acid as well as polysaccharide and glycoproteins.     

DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin

Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.