Affinity purification and characterization of a seed lectin from Crotalaria medicaginea

Purification of lectin from the seeds of Crotalaria medicaginea Lamk by affinity chromatography on asialofetuin-linked amino activated silica, yielded a single band on non-denatured PAGE at pH 4.5 and 8.3 and, a single peak on HPLC size exclusion and cation exchange columns. The molecular mass of the native C. medicaginea lectin was determined to be 125 kDa by gel filtration. In SDS-PAGE, the lectin migrated as a single band of M(r) 31.6 kDa under reducing and nonreducing conditions, indicating that it is a tetramer of apparently identical subunits. It agglutinated red blood cells (RBCs) from rabbit and human ABO blood groups. It also reacted with RBCs from rat, sheep, goat and guinea pig but after desialylation with neuraminidase. The hemagglutination activity of the lectin was inhibited by D-galactose and its derivatives. Amino acid analysis showed that lectin was rich in aspartic and glutamic acid and, did not contain sulphur containing amino acids. The lectin is a glycoprotein having 1.41% of neutral sugars. It is labile at temperature above 60 degrees C. It needs divalent cations for its activity, as a loss of activity was observed on removal of Ca2+ and Mn2+. Denaturing agents like urea, thiourea and guanidine-HCl have no effect on its activity.     

Pyrrolizidine alkaloids from seeds of Crotalaria scassellatii

Three pyrrolizidine alkaloids were isolated from the seeds of Crotalaria scassellatii. Axillaridine and axillarine were the two major alkaloids whereas the third minor alkaloid was a new compound. Its structure was determined as desoxyaxillarine.     

A water-soluble galactomannan from the seeds of Phoenix dactylifera L

A water-soluble polysaccharide, isolated from the seeds of dates, has been investigated using methylation, periodate and CrO(3) oxidation, NMR spectroscopy, and reaction with Bandeiraea simplicifolia lectin and alpha-D-galactosidase. The polysaccharide consists of a backbone composed of (1-->4)-beta-D-mannopyranosyl residues and carries a single (1-->6)-alpha-linked D-galactopyranosyl residue.     

A blood group A specific lectin from the seeds of Crotalaria striata

A lectin, monospecific for human blood group A red blood cells was extracted from seeds of Crotalaria striata and purified by molecular sieving on Sephadex G-100 and ion-exchange on DEAE-cellulose. A molecular mass of 30 kDa was determined by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions. Molecular sieving on a Superose 12 column indicated a molecular mass of 110 kDa, suggesting the tetrameric nature of the native protein. Amino-acid composition showed the presence of aminated carbohydrate residues on the lectin. N-terminal amino-acid sequencing showed a striking similarity with the N-terminal sequence of the lectin from Crotalaria juncea, which is blood-group non-specific. The potency order of agglutination inhibition with galactose containing monosaccharides was N-acetyl-D-galactosamine greater than D-galactose greater than D-galactosamine as found for blood-group-A-specific lectins from other species.     

Lectins from seeds of Crotalaria pallida (smooth rattlebox)

A lectin from the seeds of Crotalaria pallida (CPL), with an apparent molecular mass of 30 kDa, determined by SDS-polyacrylamide gel electrophoresis, showed human type A and B erythrocytes agglutination activity, which is inhibited by raffinose and galactose. The lectin requirement for divalent cation was demonstrated with EDTA/EGTA blocking hemagglutination activity. Although the N-terminal amino acid sequence of CPL is identical to another lectin from Crotalaria striata, which is taxonomically synonymous to Crotalaria pallida, these lectins differ in amino acid composition and hemagglutination properties.     

Water-soluble galactomannan from the seeds of Lotus corniculatus L.: structure and properties

Galactomannan, a water-soluble heteropolysaccharide, was isolated from the seed of Far-Eastern population of ground honeysuckle Lotus corniculatus L. (yield, 1.65%). Analysis of this galactomannan showed that is consists of D-mannose and D-galactose residues (molar ratio, 1.22:1). Its aqueous solutions were characterized by specific rotation [alpha]D = +84.10 and characteristic viscosity [eta] = 559 ml/g. Analysis of this heteropolysaccharide using chemical and enzymatic procedures, as well as IR- and 13C-NMR spectroscopy, showed that its main chain comprises 1,4-beta-D-mannopyranose residues, 95.5% of which are substituted at C-6 with single residues of alpha-D-galactopyranose.     

Isolation and characterization of a new trypsin inhibitor from Crotalaria paulina seeds

A new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Purification of the inhibitor was carried out by gel filtration, ion-exchange chromatography, and subsequent reversed-phase HPLC. The presence of a single polypeptide chain, with a molecular mass of 20 kDa and isoelectric point 4.0, was detected. The trypsin inhibitor had a Ki value of 4.5 x 10(-8) M and was capable of acting on human, bovine, and porcine trypsin and weakly on bovine chymotrypsin. Amino acid analysis showed that CPTI has a high content of aspartate, glutamate, leucine, serine, and glycine, having 177 amino acid residues in its composition. These data suggest that the protein belongs to the Kunitz-type trypsin inhibitors.     

Composition and structure of a galactomannan macromolecule from seeds ofAstragalus lehmannianus bunge

The composition and structure of a galactomannan from seeds ofAstragalus lehmannianus, an endemic legume species, is reported for the first time. The purified galactomannan (yield, 4.8%) contained 55% D-mannose and 45%D-galactose and had a molecular weight of 997.03 kDa. Its aqueous solutions were optically active and highly viscous (the specific rotation [α]_D equaled +81.3°; the characteristic viscosity [η], 868.4 ml/g). Chemical, Chromatographic, and spectral (IR and¹³C-NMR spectroscopy) methods were used to demonstrate that the main chain of the molecule is formed by residues of 1,4-β-D-mannopyranose, 78% of which are substituted at position 6 with single α-D-galactopyranose. The distribution of galactose along the chain was calculated from NMR spectra: frequencies of occurrence per pair of neighboring mannose units of ( 1 ) two substituents, (2) one substituent, and (3) no substituents were 65.3%, 31.5, and 3.2, respectively. The specific rotation of the galactomannans was shown to correlate with their content of galactose.     

Large-scale preparation of a lectin from sunn hemp seeds (Crotalaria juncea)

A procedure for large-scale preparation of a lectin from Crotalaria juncea seeds is described. The method involve fractionation by pH- and ammonium sulfate precipitation followed by biospecific affinity chromatography. The adsorbent used for the affinity chromatography was prepared by coupling galactose to Sepharose 6B activated with divinylsulfone. A comparison of different apparatus and techniques involved in the preparation is discussed. The yield and quality of the lectin prepared at a large scale were comparable with laboratory-scale preparation. From 50 kg Crotalaria juncea beans, 14.4 g Crotalaria lectin were obtained.     

Composition and structure of a galactomannan macromolecules from seeds of Astragalus lehmannianus Bunge

The composition and structure of a galactomannan from seeds of Astragalus lehmannianus, an endemic legume species, is reported for the first time. The purified galactomannan (yield, 4.8%) contained 55% D-mannose and 45% D-galactose and had a molecular weight of 997.03 kDa. Its aqueous solutions were optically active and highly viscous (the specific rotation, [alpha]D, equaled +81.3 degrees; the characteristic viscosity, [eta], 868.4 ml/g). Chemical, chromatographic, and spectral (IR and 13C-NMR spectroscopy) methods were used to demonstrate that the main chain of the molecule is formed by residues of 1,4-beta-D-mannopyranose, 78% of which are substituted at position 6 with single alpha-D-galactopyranose. The distribution of galactose along the chain was calculated from NMR spectra: frequencies of occurrence, per pair of neighboring mannose units, of (1) two substituents, (2) one substituent, and (3) no substituents were 65.3, 31.5, and 3.2%, respectively. The specific rotation of galactomannans was shown to correlate with their content of galactose.     

Structure elucidation and properties of a non-ionic galactomannan derived from the Cassia pleurocarpa seeds

A non-ionic water soluble galactomannan made up of d-galactose and d-mannose was isolated from the seed endosperm of Cassia pleurocarpa. Acid catalyzed fragmentation, periodate oxidation, methylation and selective enzymatic hydrolysis revealed that the repeating unit of the heteropolysaccharide had a backbone of beta(1-->4) linked d-mannopyranosyl units to which D-galactopyranosyl units are linked as side chains through alpha(1-->6) linkages. The properties such as viscosity, water/saline retention, gelling behavior, and shelf life of the galactomannan indicated that the material may be exploited in biomedical applications for example drug delivery and tissue engineering fields.     

Determination of the primary and fine structures of galactomannan from seeds of Gleditsia triacanthos f. intermis L

This was the first study to isolate galactomannan, a 660-kDa polysaccharide, from the seeds of Gleditsia triacanthos f. inermis L. (yield 15.4%). Its aqueous solutions were optically active ([alpha] D = +31.0 degrees) and highly viscous ([eta] = 578 ml/g). The analysis of this heteropolysaccharide using chemical, enzymatic, and chromatographic procedures, as well as IR- and 13C-NMR spectroscopy, showed that is consists of D-mannopyranose and D-galactopyranose residues (molar ratio 2.42:1). Its main chain is comprised of 1,4-beta-D-mannopyranose residues, 41% of which is substituted at C-6 with single residues of alpha-D-galactopyranose. The probability of occurrence of differently substituted mannobiose units in the chain, determined experimentally, was 0.16 for the unit Man-Man, 0.50 for the units Gal(Man-Man) and (Man-Man)Gal, and 0.34 for the dissubstitued unit Gal(Man-Man)Gal.     

Anticoagulant activity of low-molecular-weight sulfated derivatives of galactomannan from Cyamopsis tetragonoloba (L.) seeds

Galactomannan from seeds of Cyamopsis tetragonoloba (L.) Taub. (guar) was depolymerized using immobilized enzymatic preparation celloviridin. A set of fragments whose molecular weights varied from 12.6 to 245.6 kDa was obtained. Sulfated derivatives of components of all fractions were synthesized, in which the content of HSO ₃ ^? -groups was 48.05 ± 2.31%. All preparations exhibited anticoagulant activity, which was recorded in vitro in two tests—aIIa and aXa. The antithrombin activity (aIIa) was high (up to 65–87 U/mg) and did not depend on the molecular weight of a sulfated derivative; in the second test (aXa), the effect of molecular weight was observed. Biospecific electrophoresis allowed us to detect the ability of galactomannan sulfates to form complexes with protamine sulfate, a classic antidote to heparin.     

Anticoagulant activity of low-molecular-weight sulfated derivatives of galactomannan from Cyamopsis tetragonoloba (L.) seeds

Galactomannan from seeds of Cyamopsis tetragonoloba (L.) Taub. (guar) was depolymerized using immobilized enzymatic preparation celloviridin. A set of fragments whose molecular weights varied from 12.6 to 245.6 kDa was obtained. Sulfated derivatives of components of all fractions were synthesized, in which the content of HSO3(-) groups was 48.05% +/- 2.31. All preparations exhibited anticoagulant activity, which was recorded in vitro in two tests--aIIa and aXa. The antithrombin activity (aIIa) was high (up to 65-87 U/mg) and did not depend on the molecular weight of a sulfated derivative; in the second test (aXa), the effect of molecular weight was observed. Biospecific electrophoresis allowed us to detect the ability of galactomannan sulfates to form complexes with protamine sulfate, a classic antidote to heparin.     

A reappraisal of the free amino acids in seeds of Crotalaria Juncea (Leguminosae)

The seeds of Crotalaria juncea have been reported at different times to contain β-hydroxy-N-methyl-dl-norvaline and δ-hydroxynorleucine, which are isomers. We detected only one non-protein amino acid in seeds obtained from various sources; it has been isolated its identity as δ-hydroxynorleucine confirmed.     

Fractional isolation and study of the structure of galactomannan from sophora (Styphnolobium japonicum) seeds

Two fractions (1 and 2) of the galactomannan from seeds of sophora (Styphnolobium japonicum) were isolated using cold and hot aqueous extraction with a total yield of 12.88%. The two fractions differed by the ratio between mannose (Man) and galactose (Gal) residues (4.8:1 and 5.3:1, respectively) and molecular weight (1190 and 1400 kDa, respectively). Aqueous solutions of these fractions were optically active ([alpha]D +4.80 degrees and -3.36 degrees, respectively) and highly viscous ([eta] 1028.8 and 1211.2 ml/g). 13C NMR spectra of both fractions were identical with respect to the number and positions of signals, which indicates that their primary structures were identical. Using chemical and spectroscopic (IR and NMR) methods, it was shown that the galactomannan has a main chain consisting of 1,4-beta-D-mannopyranose, some residues of which (16 and 17% in fractions 1 and 2, respectively) are alpha-galactosylated at the C-6 position. Frequencies of differently substituted mannobiose blocks in the chain, calculated for fraction 1 using NMR spectroscopic data, were 0.13 for the disubstitited blocks Gal(Man-Man)Gal, 0.37 for the sum of monosubstituted blocks Gal(Man-Man) and (Man-Man)Gal, and 0.50 for the unsubstituted block Man-Man.     

Antioxidative compounds from Crotalaria sessiliflora

Seven antioxidative compounds were isolated from the EtOAc extract of the aerial part of C. sessiliflora (Japanese name, tanukimame) by activity-guided fractionation with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Among the isolated compounds, hydroxyeucomic acid showed the strongest free radical-scavenging activity, which was almost identical to that of epigallocatechin gallate, against DPPH. Orientin and isoorientin showed strong anti-peroxidative activities toward linoleic acid and protective effects against the bactericidal action of the tert-butyl peroxyl radical. Their activities were nearly equal to that of epigallocatechin gallate.     

Purification and properties of beta-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro.

Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.