Sodium Boro Research Articles

Lipid composition of Schistosoma mansoni and surface labeling of glycolipid components.

Schistosoma mansoni adult worms obtained from human patients and mice infected with cercariae were compared in lipid composition. Both worms possessed cholesterol ester, free fatty acid and cholesterol as neutral lipids, but the latter worms possessed triacylglyceride as another component. Phospholipid compositions of both worms were similar. Larger amounts of phosphatidyl choline diacylglyceride and smaller amounts of phosphatidyl ethanolamine diacylglyceride were detected. Adult worms from infected mice were surface-labeled by galactose oxidase treatment followed by sodium boro[3H]hydride reduction after or before trypsin treatment. Trypsin treatment was approximately 10 times more effective to label glycolipids than non-treatment. Mono-, di-, tri- and tetra-glycolyl components were labeled with trypsin treatment; however, mainly tri-glycolyl glycolipid was labeled without trypsin treatment. These results indicated that some glycolipid components are really exposed on the surface of the tegumental membrane. But no antigenicity was detected in the purified neutral and acidic glycolipid fractions by the Ouchterlony's double diffusion test using rabbit anti-S. mansoni sera or those obtained from patients with S. mansoni infection, though these sera, each gave two different precipitin lines against homogenate of the adult worms.

Preparation of an affinity chromatographic system for the separation of ADP binding proteins.

[4-(3-Bromoacetylpyridinio)-butyl]adenosine pyrophosphate as a structural analog of NAD+ reacts covalently with the sulfhydryl groups of thiopropyl agarose. 10-20 mumol can be bound to 1 ml gel. Stabilization of the insoluble coenzyme is attained by treatment with sodium boro hydride (NaBH4). This complex when applied to column chromatography, allows the separation of various dehydrogenases as a result of their different complex stability coefficients. Alcohol dehydrogenase from liver, lactate dehydrogenase, and adenylate kinase, which all bind to the ADP-analog residues of the gel matrix, can thus be separated by different salt gradients. Alcohol dehydrogenase from yeast, however, does not form a complex and can easily be eluted from the column with phosphate buffer. Glyceraldehyde-3 phosphate and aldehyde dehydrogenases can be eluted by the addition of NAD+ or NADH to the buffer. The uncharged 1,4-dihydropyridine ring of the reduced coenzyme produces a more stable complex with the dehydrogenases than the oxidized form.

Reduction of iminium ethers with sodium boro[3H] hydride. Preparation of tritium labelled clomiphene and nitromiphene (CI 628)

AbstractThe preparation of [3H]‐clomiphene and [3H]‐nitromiphene from amido precursors in a two‐step reaction sequence utilizing NaB H4 is described. The radiochemical yield and specific activity of the former are 34% and 16 mCi/mmol and those of the latter were 42% and 20 mCi/mmol, respectively. The method described is of micromolar scale designed for maintaining cost economy in the purchase of expensive radiolabelled reagents while yielding final products of sufficient radioactivity for in vitro and in vivo metabolism studies.

Mechanism-based inactivation of mitochondrial monoamine oxidase by N-(1-methylcyclopropyl)benzylamine.

Three different radioactively labeled N-(1-methylcyclopropyl)benzylamines [N-(1-Me)CBA] were synthesized and used to show which atoms of the inactivator remain bound to monoamine oxidase (MAO) after inactivation. Organic chemical reactions were employed to elucidate the structure of the enzyme adduct and clarify the mechanism of inactivation. Following inactivation and dialysis, the benzyl substituent is lost, but the methyl group and cyclopropyl carbons remain attached to the enzyme even after further dialysis against solutions containing 1 mM benzylamine or 8 M urea. Treatment of inactivated enzyme with sodium cyanoborohydride prior to dialysis results in the retention of the benzyl group, suggesting an imine linkage. One hydride from sodium boro[3H]hydride is incorporated into the dialyzed inactivated enzyme consistent with a ketone functional group. When Pronase-digested N-(1-Me)CBA-inactivated MAO is treated with basic potassium triiodide, iodoform is isolated, indicating the presence of a methyl ketone. During inactivation, the optical spectrum of the covalently bound active site flavin changes from that of oxidized to reduced flavin. After urea denaturation, the flavin remains reduced, suggesting covalent linkage of the inactivator to the cofactor. On the basis of previous results [Silverman, R. B., Hoffman, S. J., & Catus, W. B., III (1980) J. Am. Chem. Soc. 102, 7126-7128], it is proposed that the mechanism of inactivation involves transfer of one electron from N-(1-Me)CBA to the flavin, resulting in an amine radical cation and a flavin radical. Then, either the cyclopropyl ring is attacked by the flavin radical or the cyclopropyl ring opens, and the radical generated is captured by the flavin radical. The product of this mechanism is the imine of benzylamine and 4-flavinyl-2-butanone, the proposed enzyme-inactivator adduct.

Localization of epididymal secretory proteins on rat spermatozoa.

Spermatozoa from the testis and cauda epididymidis of the rat were surface labelled with radioactive iodide. Detergent extracts of radioiodinated spermatozoa immunoprecipitated with antisera against specific epididymal proteins, followed by polyacrylamide gel electrophoresis, revealed two proteins (D and E of Mr 27 000 and 28 000, respectively) which became associated with spermatozoa during epididymal transit. These proteins were observed by immunofluorescence microscopy to be located over a restricted area of the head surface. Proteins with similar molecular weight were labelled on spermatozoa from the cauda epididymidis, but not from the testis, by reaction with sodium boro[3H]hydride in the presence of galactose oxidase. However, failure to immunoprecipitate with antibodies to Proteins D and E and non-coincident migration on two-dimensional gel electrophoresis established the non-identity of these proteins. Compared with Proteins D and E, two other major epididymal secretory proteins (Proteins B and C of Mr 16 000) associated with spermatozoa to a relatively minor extent during epididymal transit.

Surface glycoprotein changes in ram spermatozoa during epididymal maturation.

Three radiolabeling procedures were applied to ram spermatozoa obtained at three different stages of posttesticular development: on leaving the testis (testicular sperm); after epididymal transit (cauda epididymal sperm); and after exposure to accessory sex gland secretions (ejaculated sperm). The washed spermatozoa were subjected to three radiolabeling treatments: 1) galactose oxidase and sodium boro [3H]hydride (galactosyl and galactosaminyl residues); 2) sodium metaperiodate and NaBH4 (sialyl residues); and 3) chloroglycoluril and Na125 I (tyrosyl residues). High resolution sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoretic analysis of the surface radiolabeling patterns confirms earlier studies in demonstrating an overall shift in the predominant labeled glycoproteins from the zone 78-115 kd in testicular spermatozoa to relatively low molecular weights of between 15 and 95 kd in cauda epididymal or ejaculated spermatozoa. Labeling procedures specific for glycoproteins and sialoglycoproteins revealed additional complexities in the surface transformation patterns of ram spermatozoa and suggest that cauda epididymal spermatozoa exposed to accessory sex gland secretions adsorb or produce a component of high molecular weight (approx. 350 kd).

A blood group Sd a-active pentasaccharide isolated from Tamm-Horsfall urinary glycoprotein

Human Tamm-Horsfall urinary glycoprotein from an individual of the blood group Sd(a+) phenotype was tritium-labelled by treatment with galactose oxidase and sodium boro[3H]hydride and was then digested with endo-beta-galactosidase. A series of dialysable, labelled fragments was released from which a pentasaccharide was isolated that strongly inhibited the agglutination of Sd(a+) red cells by human anti-Sda serum and hence contained the Sda determinant structure. Reduction, methylation analysis and sequential exo-glycosidase digestion established the structure of the pentasaccharide as: GalNAc beta(1 leads to 4)[NeuAc(2 leads to 3)]Gal beta(1 leads to 4)GlcNAc beta(1 leads to 3)Gal

Identification of galactose as the immunodominant sugar of leishmanial excreted factor and subsequent labeling with galactose oxidase and sodium boro[3H]hydride.

Inhibition by low-molecular-weight sugars of precipitin line formation between a polysaccharide (EF) excreted by Leishmania tropica subsp. major, Leishmania enriettii, and rabbit antileishmanial antibodies on double gel diffusion plates revealed that galactose residues, possibly as components of lactosyl groups, were the critical immunodominant sugars mediating antibody recognition of EF. The galactose residues of the EF of L. tropica subsp. major were specifically labeled with tritium via galactose oxidase and sodium boro[3H]hydride. The radioactive EF had an apparent molecular weight of about 85,000 on sodium dodecyl sulfate-polyacrylamide gels and was precipitated by antileishmanial antibodies as well as Ricinus communis lectins I and II (galactose specific). Lectins specific for glucose-mannose residues, fucose, N-acetylglucosamine, and N-acetylgalactosamine did not precipitate the labeled EF. Treatment of [3H]EF with proteolytic (trypsin, papain, protease) or glycosidic (alpha-amylase, beta-galactosidase) enzymes had no effect on either the electrophoretic pattern of the material or on its recognition by antileishmanial antibodies or R. communis lectin. This resistance to enzyme activity suggests that EF may be a useful marker for the presence of the parasite in vivo if it can be detected in minute quantities.

3H‐labelled bilirubin and biliverdin

AbstractA simple method is described for the preparation of [3H]bilirubin IX‐α by the reduction of biliverdin IX‐α using sodium boro[3H]hydride. The [3H]bilirubin is obtained with a high specific radioactivity and the tritium is located at the methylene bridge (C‐10). Oxidation of the [3H]bilirubin with chloranil results in some loss of tritium from C‐10 and [3H]biliverdin is obtained.

Purification and Mode of Action of a Chitosanase from Penicillium islandicum

Penicillium islandicum produced an inducible extracellular chitosanase when grown on chitosan. Large-scale production of the enzyme was obtained using Rhizopus rhizopodiformis hyphae as substrate. Chitosanase was purified 38-fold to homogeneity by ammonium sulphate fractionation and sequential chromatography on DEAE-Biogel A, Biogel P60 and hydroxyl-apatite. Crude enzyme was unstable at 37 °C, but was stabilized by 1·0 mm-Ca2+. The pH optimum for activity was broad and dependent on the solubility of the chitosan substrate. Various physical and chemical properties of the purified enzyme were determined. Penicillium islandicum chitosanase cleaved chitosan in an endo-splitting manner with maximal activity on polymers of 30 to 60% acetylation. No activity was found on chitin (100% acetylated chitosan) or trimers and tetramers of N-acetylglucosamine. The latter two oligomers and all small oligomers of glucosamine inhibited the activity of chitosanase on 30% acetylated chitosan. The pentamer of N-acetylglucosamine and glucosamine oligomers were slowly cleaved by the enzyme. Analysis of the reaction products from 30% acetylated chitosan indicated that the major oligomeric product was a trimer; with 60% acetylated chitosan as substrate a dimer was also found. The new terminal reducing groups produced by chitosanase hydrolysis of 30% acetylated chitosan were reduced by sodium boro[3H]hydride. The new end residues were found to be N-acetylglucosamine. The analyses strongly indicated that P. islandicum chitosanase cleaved chitosan between N-acetylglucosamine and glucosamine. Both residues were needed for cleavage, and polymers containing equal proportions of acetylated and non-acetylated sugars were optimal for chitosanase activity. The products of reaction depended on the degree of acetylation of the polymer.

Stereospecific Radioimmunoassays for d-Pseudoephedrine in Human Plasma and Their Application to Bioequivalency Studies

Antiserum to d-pseudoephedrine was raised in New Zealand White rabbits in response to immunization with a conjugate of bovine serum albumin and d-pseudoephedrine-N-3-propionic acid. The hapten was prepared by reaction of methyl acrylate with d-pseudoephedrine, followed by ester hydrolysis. Sodium boro[3H]hydride reduction of dl-ephedrone gave [α-3H]-dl-ephedrine, and a Welsh rearrangement with acetic anhydride followed by deacetylation gave [α-3H]-dl-pseudoephedrine, which was used as a radioligand in radioimmunoassay procedures for direct plasma analyses. Three sensitive radioimmunoassay procedures were developed, two using [3H]pseudoephedrine as the radioligand and either adsorption on coated charcoal or polyethylene glycol precipitation for separation of antibody-bound from free radioligand. The third method used an [125I]tyrosine methyl ester analog of pseudo-ephedrine and charcoal separation, preceded by extraction and derivatization of pseudoephedrine with methyl acrylate. All three assays could detect 2.5ng of pseudoephedrine/ml. The antiserum was stereospecific, showing low cross-reactivities with l-pseudoephedrine and d-and l-ephedrines. d-Norpseudoephedrine and some other related compounds also had low cross-reactivity in these radioimmunoassay procedures. Excellent agreement was found between pseudoephedrine concentrations in human plasma determined by radioimmunoassay and by a standard GLC method. The utility of radioimmunoassay was illustrated by application of one of these procedures to an assessment of the bioequivalence of immediate- and sustained-release pseudoephedrine formulations in normal volunteers. A sustained-release preparation containing 120mg of pseudoephedrine hydrochloride given every 12hr was shown by AUC comparisons to be bioequivalent to an immediate-release tablet (containing 60mg of pseudoephedrine hydrochloride) given every 6hr.

Characterization of sperm surface and seminal plasma glycoproteins of the chimpanzee.

Washed ejaculated chimpanzee spermatozoa and a 100 000 g supernatant of seminal plasma were subjected to radiolabelling by sequential treatment with galactose oxidase and sodium boro[3H]hydride or with sodium metaperiodate and NaB3H4. Sperm surface glycoproteins and seminal plasma glycoproteins radiolabelled by these procedures were compared by SDS-polyacrylamide gel electrophoresis. Spermatozoa labelled by galactose oxidase treatment showed a single labelled macromolecular component of 37 000 whereas spermatozoa labelled by sodium metaperiodate-NaB3H4 treatment showed incorporation into macromolecular components of 37 000 and 25 000 mol. wt. Seminal plasma radiolabelled by galactose oxidase-NaB3H4 treatment contained labelled components of 47 000, 37 000, 19 000 and 12 000 mol. wt, whereas seminal plasma radiolabelled with sodium metaperiodate-NaB3H4 contained macromolecular components of 47 000, 37 000, and 19 000 mol. wt.

Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin.

An octasaccharide with high affinity for antithrombin was isolated after partial deaminative cleavage of heparin with nitrous acid. After conversion of the 2,5-anhydro-D-mannose end group to anhydro[1-3H]mannitol, labeled pentasaccharide was released from the octasaccharide by periodate-alkali treatment. Incubation of the pentasaccharide with a recently discovered 3,O-sulfatase from human urine resulted in desulfation, suggesting the occurrence of a 3-sulfate group on the terminal glucosamine residue. The same glucosamine residue was recovered as a 2,5-anhydro[1-3H]mannitol derivative by a procedure involving deamination of the octasaccharide with nitrous acid, reduction of the products with sodium boro[3H]hydride, isolation of 3H-labeled tetrasaccharide by gel chromatography, and release of the labeled end-group by periodate-alkali treatment. Paper electrophoresis indicated disulfated anhydro[3H]mannitol, presumably sulfated at C3 and C6, as a major component, along with smaller amounts of monosulfated (presumably 3-sulfated) anhydro[3H]mannitol. Similar treatment of an analogous tetrasaccharide derived from heparin with low affinity for antithrombin failed to produce any disulfated anhydromannitol. These results suggest that 3-sulfated glucosamine is a unique component of high-affinity heparin, located at a specific position in the antithrombin-binding sequence of the molecule.

Stereospecificity of sodium borohydride reduction of Schiff bases at the active site of aspartate aminotransferase.

Sodium boro[3H]hydride treatment of holoaspartate aminotransferase results in the reduction of the Schiff's base formed between pyridoxal phosphate and Lys 258. Treatment of the reduced enzyme with papain followed by acid hydrolysis liberates epsilon-N-[3H]pyridoxyl lysine which is degraded to [3H]pyridoxamine diHCl and stereochemically analyzed with apoaspartate aminotransferase. Sodium boro[3H]hydride treatment of active site carbamylated aspartate aminotransferase reconstituted with pyridoxyl phosphate and sodium aspartate results in the trapping of an enzyme x substrate complex through the reduction of the Schiff's base formed between pyridoxal phosphate and aspartate. Active site bound N-[3H]pyridoxyl aspartate is liberated by treatment with papain and degraded to [3H]pyridoxamine diHCl for stereochemical analysis. Borohydride reduction of the holoenzyme occurs from the re face of the pyridoxal phosphate Lys 258 Schiff's base. Similarly, reduction of active site carbamylated enzyme x substrate complex occurs from the re face of the pyridoxal phosphate-aspartate Schiff's base. These results indicate that when active site carbamylated enzyme binds substrate to pyridoxal phosphate it does so stereospecifically and without changing the face of the Schiff base that is available for reduction as compared to native enzyme.

Quantitation of the sulfated disaccharides of heparin by high performance liquid chromatography

Heparin was converted by treatment with nitrous acid primarily into sulfated disaccharides. The mixture of disaccharides was reduced with sodium boro[ 3H]hydride and the disaccharides were purified by preparative paper electrophoresis and paper chromatography. Four disaccharides were obtained. On the basis of their paper electrophoretic mobilities and the products formed at intermediate stages of their acid hydrolysis, the disaccharides were identified as 4- O-(2- O-sulfo-α- l-idopyranosyluronic acid)-6- O-sulfo-2,5-anhydro- d-mannitol, 4- O-(2- O-sulfo-α- l-idopyranosyluronic acid)-2,5-anhydro- d-mannitol, 4- O-(α- l-idopyranosyluronic acid)-6- O-sulfo-2,5-anhydro- d-mannitol, and 4- O-(β- d-glucopyranosyluronic acid)-6- O-sulfo-2,5-anhydro- d-mannitol. The purified disaccharides were used as standards in the development of a high-performance liquid chromatography procedure for their separation and quantitation on a Partisil-10 SAX anion-exchange column. The three monosulfated disaccharides were resolved by isocratic elution with 40 m m KH 2PO 4. The KH 2PO 4 concentration was tehn increased to 400 m m to elute the disulfated disaccharide. Column effluents were collected in 1 2 - ml fractions, and the recovery of each 3H-labeled product was determined by scintillation counting. When sodium boro-[ 3H]hydride with a specific activity of 315 mCi/mmol was used in the reduction of the heparin deamination products, the disaccharides gave 28,500 cpm/nmol in the effluent peaks. Quantitative recoveries of the 3H-disaccharides were obtained. It was demonstrated that the method developed using the purified disaccharides gave reproducible and quantitative results in direct assays of aliquots of boro[ 3H]hydride-reduced heparin deamination mixtures.

Externally disposed polypeptides of chick brain mitochondria

Externally disposed polypeptides of chick brain synaptic and non-synaptic mitochondria were identified using lactoperoxidase-catalyzed iodination and galactose oxidase—sodium boro [ 3H]hydride labeling (with or without neurominidase pretreatment). After surface labeling, polypeptides were separated by electrophoresis on sodium dodecyl sulfate—polyacrylamide gels and incorporation was detected by liquid scintillation counting. When intact synaptic or non-synaptic mitochondria were iodinated they gave quite similar labeling patterns with 10 major radioactive peaks. Most of the surface-incorporated iodine was removable by trypsin. Iodination of microsomes or synaptosomes gave incorporation profiles different from that of mitochondria. Without neuraminidase pretreatment, galactose oxidase labeling of mitochondria gave only near background incorporation. After neuraminidase treatment, galactose oxidase and sodium borotritide labeling of both synaptic and non-synaptic mitochondria produced 12 major radioactive polypeptides. Four were not iodinatable by lactoperoxidase while two components iodinated by lactoperoxidase showed only minor radioactivity peaks after galactose oxidase-sodium borotritide labeling. These results suggest that (a) chick brain mitochondrial membranes have a complex mixture of glycoproteins which are highly sialiated, (b) at least 14 polypeptides are exposed on the exterior surface of brain mitochondria, (c) the functional differences between synaptic and non-synaptic mitochondria are not reflected by the similarities of their major externally disposed polypeptides. Thus, surface labeled components may represent structural polypeptides common to both mitochondrial types.

Preparation and characterization of 3H-labeled α-bungarotoxin

α-Bungarotoxin has been labeled with [ 3H]pyridoxamine phosphate, by reaction with pyridoxal phosphate followed by reduction with sodium boro[ 3H]hydride. Specific activities of up to 27 Ci/mmol have been obtained. Mono- and dilabeled toxins bind irreversibly to the acetylcholine receptor from Torpedo electroplax, despite a change in isoelectric point from 9.2 for native toxin to 6.2 for dilabeled toxin. The 3H-labeled α-bungarotoxin is usable for over a year.

ChemInform Abstract: DIAZABENZENES. II. SYNTHESIS, PHOTOLYSIS AND MASS SPECTRA OF SOME TETRAALKYLPYRAZINES

AbstractDie Tetraalkylpyrazine (II) werden durch Schmelzen von Ammoniumacetat mit den entsprechenden Acyloinen (I) oder durch Reduktion der Diketondi‐ oxime (III) mit Zn‐Staub und NaOH dargestellt.

Radioimmunoassay system using a serovar-specific lipopolysaccharide antigen of Leptospira.

The serovar-specific main (TM) antigen of Leptospira interrogans serovar kremastos strain Kyoto was labeled with sodium boro[3H]hydride after 1 h of oxidation with periodate. When 50 ng of the labeled compound was employed, 50% of the compound was bound to the antibodies contained in 7.5 x 10(-2) nl of anti-kremastos Kyoto serum. Under this condition, only 9 ng of the homologous TM antigen was required for 50% inhibition of serovar kremastos Kyoto 3H-TM antigen-anti-kremastos Kyoto serum binding, whereas 5,000 times as much TM antigen of serovar hebdomadis, which belongs to the same Hebdomadis serogroup as serovar kremastos, was required for the same inhibition. The TM antigens from serovars pomona, icterohaemorrhagiae, and copenhageni, which belong to different serogroups, showed no inhibition in an amount of up to 3 x 10(3), 2 x 10(5), and 2 x 10(5) ng, respectively. In the inhibition study using the serovar hebdomadis TM antigen, inhibition values slightly fluctuated due to the antigen's insolubility. When the TM antigens were solubilized with 0.1% sodium taurodeoxycholate, the fluctuation in inhibition values was minimized, and the cross-reactivity of the serovar hebdomadis TM antigen with anti-kremastos Kyoto serum was diminished, while the inhibitory activity of the serovar kremastos Kyoto TM antigen was enhanced.

Structure of the antithrombin-binding site in heparin.

Heparin preparations from pig intestinal mucosa and from bovine lung were separated by chromatography on antithrombin-Sepharose into a high-affinity fraction (with high anticoagulant activity) and a low-affinity fraction (with low anticoagulant). Antithrombin-binding heparin fragments (12-16 monosaccharide units) were prepared, either by digesting a high-affinity heparin-antithrombin complex with bacterial heparinase or by partial deaminative cleavage of the unfractionated polysaccharide with nitrous acid followed by affinity chromatography on immobilized antithrombin. Compositional analysis based on separation and identification of deamination products reduced with sodium boro[3H]hydride showed that nonsulfated L-iduronic acid occurred in larger amounts in high-affinity heparin than in low-affinity heparin; furthermore, this component was concentrated in the antithrombin-binding regions of the high-affinity heparin molecules, amounting to approximately one residue per binding site. It is suggested that nonsulfated L-iduronic acid is essential for the anticoagulant activity of heparin. The location of the non-sulfated uronic acid in the antithrombin-binding site was determined by periodate oxidation of antithrombin-binding fragments containing a terminal 2,5-anhydro-D-[1-3H]mannitol unit. Tentative structures for antithrombin-binding sequences in heparin are proposed, including some structural variants believed to be compatible with, but not required for, activity.