Bo Ersson

The phytohemagglutinin from sunn hemp seeds (Crotalaria juncea). Purification by biospecific affinity chromatography.

Abstract The anti-galactose lectin from sunn hemp seeds was isolated from a clarified and dialyzed crude extract by biospecific affinity chromatography on a galactan gel prepared by acid treatment of agarose gel beads. The purified lectin gave a single peak on Sephadex G-200 gel filtration with an elution volume corresponding to a molecular weight of 120 000. The lectin contained about 5% firmly bound carbohydrate (orcinol method). The agglutinating reaction with red cells was inhibited by sugars containing galactose residues.

Protein binding of mercury in milk and plasma from mice and man : a comparison between methylmercury and inorganic mercury

Inorganic mercury has previously been shown to be excreted to milk from plasma to a higher extent than methylmercury. Protein binding of mercury as methylmercury and inorganic mercury in whey and plasma from mouse and man was studied in order to get a better understanding of the transport of mercury into milk. Mice were administered a single i.v. dose of 0.25 mg Hg/kg body weight labelled with (CH3)203HgCl or 203HgCl2, resulting in 11 ng Hg/g milk and 38 ng Hg/g milk after 1 h, respectively. Milk and plasma from mice and man were also incubated with the respective radiolabelled compound (150 ng Hg/g milk or plasma). Casein, fat and whey fractions in milk from methylmercury treated mice were found to contain 11, 39 and 34%, respectively, and from inorganic mercury treated mice 31, 15 and 41%, respectively, of the total amount of mercury in milk. Serum albumin was a major mercury binding protein in whey and plasma from mice for both methylmercury and inorganic mercury, as demonstrated by FPLC gel filtration and anion-exchange chromatography and further characterised by SDS-PAGE for whey. In addition, anion-exchange chromatography indicated that inorganic mercury, but not methylmercury, in whey from mouse milk formed a dimer of serum albumin. The unbound fraction of mercury in whey and plasma from mice was very small (<0.7%), and somewhat higher in plasma and whey from man. It is concluded, that the unbound fraction in plasma cannot be a determining factor for the observed differences in milk excretion between the two mercury compounds. Instead, it is suggested that methylmercury and to some extent inorganic mercury are transferred from plasma into milk using albumin as a passive carrier.

The specificity of the combining site of the lectin from Vicia villosa seeds which reacts with cytotoxic T-lymphoblasts☆

Abstract The combining site of purified Vicia villosa lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin, which specifically hemagglutinated blood group A erythrocytes nevertheless precipitated to different extents with blood group A1, A2, H, B and precursor I substances from saliva and ovarian cysts, and with different blood group substances of the same specificity indicating an interaction of the lectin with a non-blood group determinant site. The agglutinating and precipitating activities of the lectin were not strongly affected by EDTA or bivalent cations. Precipitates of Vicia villosa lectin with certain blood group glycoproteins showed unusually high solubilities as compared to other lectin glycoprotein and antigen-antibody precipitates. Inhibition assays with various monosaccharides, glycosides and oligosaccharides indicate that the Vicia villosa lectin is specific for terminal, non-reducing, α-linked d GalNAc. Of the monosaceharides tested, methyl α d GalNAc, p-NO2-phenyl α d GalNAc and d GalNAc were best. They were about 100 times better inhibitors than the corresponding d Gal compounds. The most potent inhibitor was methyl α d GalNAc which was 10 times more active than d GalNAc and 18 times more potent than the β-anomer. Among disaccharides tested, d GalNAcαl → 3 d Gal was most active, about as potent as methyl α d GalNAc, twice as active as d GalNAcαl → 6 d Gal and 8 times more potent than d GalNAc αl → 6 d GalNAc , indicating the importance of a subterminal α l → 3- linked d Gal in the binding. d GalNAc α l → 3 d Gal β l → 3 d GlcNAc was 7 times less active and the blood group A determinant was less than 1 40 as active as d GalNAc α l → 3 d Gal . These findings indicate that the combining site of the Vicia villosa lectin is at least as large as the disaccharide d GalNAc α l → 3 d Gal and that the αl → 3 linkage is important in the binding. The unique nature of this site is consistent with its high specificity for a glycoprotein on cytotoxic T-lymphocytes.

A phytohemagglutinin from Sunn Hemp seeds (Crotalaria juncea) II. Purification by a high capacity biospecific affinity adsorbent and its physicochemical properties

A galactose-specific lectin from seeds of Sunn Hemp (Crotalaria juncea) has been purified by fractional precipitation with ammonium sulfate followed by biospecific affinity chromatography and preparative isoelectric focusing. The adsorbent was prepared by coupling galactose to Sepharose 6B activated with divinyl sulfone. The lectin was homogeneous as judged by ultracentrifugation and by electrophoresis in cellulose acetate strips and in polyacrylamide gradient gel. Its isoelectric point is pH 8.8 and the molecular weight is about 120 000. It is a glycoprotein containing 9.8% also carbohydrate (mannose, N-acetyl-D-glucosamine, fucose, and xylose). The lectin contains 3.2 mol Ca2+, 2.2 mol Mg2+ and 0.2 mol Mn2+ per 120 000 g. No sulphur-containing amino acids were detected.

Improved Lectin-Mediated Immobilization of Human Red Blood Cells in Superporous Agarose Beads.

A new type of agarose bead, superporous agarose, was used as a gel support for immobilization of human red blood cells (RBCs) mediated by wheat germ lectin. The number of immobilized cells was similar to that obtained with commercial wheat germ lectin-agarose but the cell stability appeared to be superior. This allowed improved frontal affinity chromatographic analyses of cytochalasin B (CB)-binding to the glucose transporter GLUT1 which established a ratio of one CB-binding site per GLUT1 dimer for both plain RBCs or those treated with different poly amino acids. The measured dissociation constants, 70+/-14 nM for CB and 12+/-3 mM for glucose binding to GLUT1, are similar to those reported earlier.

Dependence on cations for the binding activity of lectins as determined by affinity electrophoresis

The metal ion content of eighteen different lectins was determined. The lectins were demetallized and the binding activity of native and demetallized forms were investigated using non-denaturing polyacrylamide affinity gel electrophoresis. The binding activities of all lectins were dependent on their metal ion content; when the cations were removed the lectins lost their carbohydrate binding activity. There was a marked difference in the strength with which lectins bind divalent cations.

Biotechnological Approach to the Synthesis of 9α-Hydroxylated Steroids

Abstract The steroid 9α‐hydroxylase gene has been cloned from Mycobacterium smegmatis into Escherichia coli BL21. Progesterone added to bioreactors was subjected to in vivo transformation into 9α‐hydroxyprogesterone. In 7 days, 43.6 mg 9α‐hydroxyprogesterone was formed from 53.8 mg/L progesterone. The enzyme also has shown evidence of processing 4‐androstene‐3,17‐dione in vivo. An extensive analytical method development, including LLE, HPLC‐DAD, MS, and NMR was performed to verify the product and to enable a quantitative analysis. Protocols for analytical and preparative separation have been developed, using binaphtol as internal standard. Both the growth pattern and the bioconversion rate were unaffected by the presence of binaphtol in the bioreactor. The enzyme was purified by immobilised metal affinity and ion exchange chromatography, resulting in low in vitro activity.

Isolation of calcium-binding proteins on selective adsorbents Application to purification of bovine calmodulin

We report the fractionation of calcium-binding proteins using immobilized metal ion affinity chromatography (IMAC) with hard metal ions. Various hard metal ions (Mn2+, La3+, Nd3+, Eu(3 were immobilized on cross-linked agarose substituted with Tris(carboxymethyl)ethylenediamine (TED) and used as an adsorbent. After systematic studies, europium was selected for further work on the fractionation of calcium-binding proteins. It was found that the presence of Ca2+ in the sample and the solvent strongly promoted the adsorption and selectivity. Selective elution was accomplished in stepwise mode by the addition of calcium chelators such as malonate, citrate and phosphate. Calmodulin of high purity was isolated from a crude extract. Similar behavior of other calcium-binding proteins indicates that the reported chromatographic procedure can be generally applied to such proteins.

Immobilized lectins as adsorbents for serum proteins

The lectins constitute a class of proteins, many of which form complexes with carbohydrate residues. These complex-forming properties can be used to separate carbohydrates or glycoproteins from mixtures, for which purpose the lectins are covalently bound to an inert support, i.e. agarose gel beads. Most serum proteins are glycoproteins, with albumin being one notable exception. Precipitations of certain serum proteins have been carried out with lectins in solution [l-4] . Immobilized lectins should thus be valuable tools in serum fractionation as is indeed shown by matrix-bound concanavalin A which has been used for fractionation of serum proteins [S] . The broad specificity of this adsorbent makes it suitable for group isolation of the glycoprotein fraction from the bulk of the protein. The concanavahn-adsorbent must then be assisted by more specific adsorbents to achieve further fractionation of the glycoprotein components. The lectin from Crotalaria juncea can be used to make an adsorbent of higher specificity with respect to serum proteins than the concanavalin-adsorbent. The Crotalaria lectin is specific against the galactose configuration and thus reacts with galactose-containing carbohydrates and glycoproteins. In this paper we will present some fractionation experiments with these two lectins in immobilized form.