Purification and Characterization of Three Lectins Extracted from Cassia Fistula Seeds and Effect of Various Physical and Chemical Agents on Their Stability

Three lectins designated as CSL‐1, CSL‐2 and CSL‐3 were purified from Cassia fistula seeds by gel filtration on Sephadex G‐50 followed by ion‐exchange chromatography on DEAE cellulose and finally by affinity chromatography on Sepharose 4B. The molecular weights of the lectins CSL‐1, CSL‐2 and CSL‐3, determined by gel filtration on Sephadex G‐75 column were 37,000, 42,400 and 46,000, and by SDS gel electrophoresis were 37500, 42000 and 46500, respectively. All three lectins agglutinated rat red blood cells and the agglutination was inhibited specifically by galactose and galactose containing saccharide. The neutral sugar contents of the lectins, CSL‐1, CSL‐2 and CSL‐3 were estimated to be 3.5, 3.1 and 2.0%, respectively. The sugar composition of the lectins was found to be galactose in CSL‐1, galactose and glucose in CSL‐2, and galactose and mannose in CSL‐3. The lectins exhibited maximum hemagglutinating activities around pH 7.2 to 7.5 and at a temperature range of 20° to 35 °C. Biological activities of the lectins were abolished sequentially with the increase in concentration of acetic acid and denaturant solutions such as urea and guanidine‐HCl.     

Purification,Characterization and Physico‐Chemical Properties of Three Galactose‐Specific Lectins from Pumpkin (Cucurbita Maxima) Seed Kernels

Three galactose‐specific lectins have been isolated and purified from the extract of pumpkin seed kernels by gel filtration on Sephadex G‐75 with 100% ammonium sulfate saturated crude extract, followed by ion exchange chromatography on DEAE‐cellulose and affinity chromatography on Sepharose 4B. All three lectins were found to be homogeneous as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). The molecular weights of lectins, PSL‐1, PSL‐2 and PSL‐3, as estimated by gel filtration on Sephadex G‐75 were 40,000, 42,000 and 46,000, and by SDS‐PAGE about 39,500, 41,000 and 45,000, respectively. The lectins, PSL‐1, PSL‐2 & PSL‐3 were dimer in nature and the molecular weights of their subunits were about 25,500 and 14,000; 26,000 and 15,000; and 30,500 and 15,000, respectively. The lectins are glycoproteins with a neutral sugar content of 3‐5%. The lectins agglutinated rat red blood cells and the hemagglutination was inhibited specifically by galactose and galactose‐containing saccharides. The lectins exhibited a strong cytotoxic effect in a brine shrimp lethality bioassay.     

Purification and Characterization of Lectins from Jute (Chorchorus olitorius) Leaves

Three lectins were isolated from an extract of jute leaves (Chorchorus olitorius) and purified by gel filtration on Sephadex G‐50 of the 100% ammonium sulfate saturated crude extract, followed by ion‐exchange chromatography on DEAE‐cellulose were designated as JLL‐1, JLL‐2 and JLL‐3. All the lectins were homogeneous as judged by SDS‐polyacrylamide slab gel electrophoresis and gave single bands. The molecular weights of the three lectins were estimated by the same method were 35000, 38000 and 42000, respectively. The lectins specifically agglutinated rat red blood cells. The agglutination of JLL‐1 was inhibited by D‐mannose/D‐glucose and their derivatives, whereas D‐galactose was found to be the potent inhibitor for the agglutination of JLL‐2 and JLL‐3. The lectins were glycoprotein in nature with a neutral sugar content of 1.3%, 1.2% and 0.8% for JLL‐1, JLL‐2 and JLL‐3, respectively. The hemagglutinating activity of JLL‐2 was also investigated after the treatment of physico‐chemical agents. The lectin showed maximum activity between the range of pH 7.2–8.0 and the range of temperature of 20‐30 °C. The activity of lectin decreased after treatment with a higher concentration of acetic acid and urea. In the presence EDTA the activity was inhibited while the presence of Ca⁺², Mn⁺² and K⁺ increased the activity of the lectin moderately.     

Combined 1H, 13C and 11B NMR and mass spectral assignments of boronate complexes of D‐(+)‐glucose,D‐(+)‐mannose,methyl‐α‐D‐glucopyranoside,methyl‐β‐D‐galactopyranoside and methyl‐α‐D‐mannopyranoside

Complex formation between N‐butylboronic acid and D ‐(+)‐glucose, D ‐(+)‐mannose, methyl‐α‐D ‐glucopyranoside, methyl‐β‐D ‐galactopyranoside and methyl α‐D ‐mannopyranoside under neutral conditions was investigated by ¹H, ¹³C and ¹¹B NMR spectroscopy and gas chromatography–mass spectrometry (GC–MS) D ‐(+)‐Glucose and D ‐(+)‐mannose formed complexes where the boronates are attached to the 1,2:4,6‐ and 2,3:5,6‐positions of the furanose forms, respectively. On the other hand, the boronic acid binds to the 4,6‐positions of the two methyl derivatives of glucose and galactose. Methyl α‐D ‐mannopyranoside binds two boronates at the 2,3:4,6‐positions. ¹¹B NMR was used to show the ring size of the complexed sugars and the boronate. GC–MS confirmed the assignments. Copyright © 2003 John Wiley & Sons, Ltd.     

Fluorinated Carbohydrates as Lectin Ligands: Dissecting Glycan–Cyanovirin Interactions by Using 19F NMR Spectroscopy

NMR spectroscopy and isothermal titration calorimetry (ITC) are powerful methods to investigate ligand–protein interactions. Here, we present a versatile and sensitive fluorine NMR spectroscopic approach that exploits the ¹⁹F nucleus of ¹⁹F‐labeled carbohydrates as a sensor to study glycan binding to lectins. Our approach is illustrated with the 11 kDa Cyanovirin‐N, a mannose binding anti‐HIV lectin. Two fluoro‐deoxy sugar derivatives, methyl 2‐deoxy‐2‐fluoro‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranoside and methyl 2‐deoxy‐2‐fluoro‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranoside were utilized. Binding was studied by ¹⁹F NMR spectroscopy of the ligand and ¹H–¹⁵N HSQC NMR spectroscopy of the protein. The NMR data agree well with those obtained from the equivalent reciprocal and direct ITC titrations. Our study shows that the strategic design of fluorinated ligands and fluorine NMR spectroscopy for ligand screening holds great promise for easy and fast identification of glycan binding, as well as for their use in reporting structural and/or electronic perturbations that ensue upon interaction with a cognate lectin.     

CHROMATOGRAPHIC SEPERATION AND PURIFICATION OF HUMAN CHORIONIC GONADOTROPIN

Separation and purification of human chorionic gonadotropion(HCG) in the urine sample of early pregnant women by D3520 resin adsorption chromatography is reported.The crude product obtained by DEAE-Cellulose 23 and DEAE-Sephadex A50 column chromatography showed a high activity of HCG. Further purification of the sample by gel filtration chromatography on a Sephadex G75 column gives a final preparation of 6000-6500 IU/mg.The preparation meets the requirements of the pyrogn test in Chinese Law of Pharmacopeia.     

Purification of Two Novel Sugar Acid-binding Lectins from <Emphasis Type="Italic">Haplomitrium Mnioides</Emphasis> (bryophyte,Plantae) and their Preliminary Characterization

Two novel sugar acid-binding lectins were purified from Haplomitrium mnioides (Lindb.) Schust. using a procedure consisting of ammonium sulfate precipitation, G-50 gel filtration, hydroxyapatite chromatography, and HW-50 gel filtration. We reported their partial physicochemical properties: molecular weight, affinity for carbohydrates and organic acids, pH stability, and dependence of their hemagglutination activity on metal ions. We also determined their N-terminal amino acid sequences. H. mnioides lectins (HMLs) were monomers (one with a molecular weight of approximately 27 kDa, and the other with a molecular weight of approximately 105 kDa) under both nonreducing and reducing conditions. They were named HML27 and HML105, respectively. Both HMLs had an affinity for N-acetylneuraminic acid, d-glucuronic acid, d-glucaric acid, bovine submaxillary mucin, heparin, and organic acids, such as citrate, 2-oxoglutaric acid, and d-2-hydroxyglutarate. Furthermore, HML27 had an affinity for α-d-galacturonic acid, d-malate, l-malate, and pyruvate, while HML105 had an affinity for d-gluconic acid. HML27 and HML105 are novel plant lectins: they have an affinity for sugar acids and organic acids and specifically recognize the carboxyl group, and there is no homology between their N-terminal amino acid sequences and those of the previously described lectins and agglutinins.     

Flavonol Glycosides and Iridoids from Asperula lilaciflora

A new flavonol glycoside, quercetin 3‐O‐[6′′′‐O‐3,5‐dihydroxycinnamoyl‐β‐glucopyranosyl‐(1→2)]‐β‐galactopyranoside (named lilacifloroside; 1 ) and a new iridoid 2 (named asperulogenin), were isolated from the aerial parts of Asperula lilaciflora in addition to eight known secondary metabolites, i.e., quercetin, kaempferol, quercetin 3‐O‐β‐glucopyranosyl‐(1→2)‐β‐galactopyranoside, quercetin 3‐O‐β‐glucopyranosyl‐(1→2)‐arabinopyranoside, asperuloside, deacetylasperulosidic acid, asperulosidic acid methyl ester, and chlorogenic acid. The structures were elucidated on the basis of extensive 1D‐ and 2D‐NMR experiments as well as MS data. Compound 1 contains the rare 3,5‐dihydroxycinnamoyl moiety in its structure. This work constitutes the first phytochemical study of the title plant.     

Synthesis and Pairing Properties of Oligodeoxynucleotides Containing N7‐(Purin‐2‐amine Deoxynucleosides)

A general synthesis of the four isomeric N⁷‐α‐D ‐, N⁷‐β‐D ‐, N⁹‐α‐D ‐, and N⁹‐β‐D ‐(purin‐2‐amine deoxynucleoside phosphoramidite) building blocks for DNA synthesis is described (Scheme). The syntheses start with methyl 3′,5′‐di‐O‐acetyl‐2′‐deoxy‐D ‐ribofuranoside ( 2 ) as the sugar component and the N²‐acetyl‐protected 6‐chloropurin‐2‐amine 1 as the base precursor. N⁷‐Selectivity was achieved by kinetic control, and N⁹‐selectivity by thermodynamic control of the nucleosidation reaction. The two N⁷‐(purin‐2‐amine deoxynucleosides) were introduced into the center of a decamer DNA duplex, and their pairing preferences were analyzed by UV‐melting curves. Both the N⁷‐α‐D ‐ and N⁷‐β‐D ‐(purin‐2‐amine nucleotide) units preferentially pair with a guanine base within the Watson‐Crick pairing regime, with ΔT_ms of −6.7 and −8.7 K, respectively, relative to a C⋅G base pair (Fig. 3 and Table 1). Molecular modeling suggests that, in the former base pair, the purinamine base is rotated into the syn‐arrangement and is able to form three H‐bonds with O(6), N(1), and NH₂ of guanine, whereas in the latter base pair, both bases are in the anti‐arrangement with two H‐bonds between the N(3) and NH₂ of guanine, and NH₂ and N(1) of the purin‐2‐amine base (Fig. 4).     

Condensation and Polycondensation of Terephthaldehyde with Methyl D‐Hexopyranosides

The condensation and polycondensations of terephthaldehyde ( 1 ) and methyl D ‐hexopyranosides (gluco‐, galacto‐ and mannopyranoside) are described. Methyl α‐D ‐glucopyranoside and methyl α‐D ‐galactopyranoside react with 1 to give mono‐ 5 a and 6 a and diacetals 5 b and 6 b . Their structures were confirmed by NMR and IR spectroscopy. The polycondensation of methyl α‐D ‐mannopyranoside ( 4 ) with 1 was studied in various solvents within the temperature range of 80–140°C. Regardless of the conversion or the initial comonomer feed ratios the composition of polycondensates depended on the reaction conditions leading to the formation of materials with diverse solubilities, molecular weights and optical properties. The regioselective polycondensation of 1 and 4 was examined by the ¹H NMR spectroscopy of polymer 7 . In the case of five‐membered cyclic acetal units, mixtures of the endo‐H and exo‐H dioxolan‐2‐yl system diastereomers are formed. Experimental examples of functionalization via ester units in polymer molecules 8 are described and the efficiency of the reaction routes and procedures are evaluated. The molecular weight was estimated by size‐exclusion chromatography (SEC) measurements before and after the functionalization.     

Physical properties of diblock methylcellulose derivatives with regioselective functionalization patterns: First direct evidence that a sequence of 2,3,6‐tri‐O‐methyl‐glucopyranosyl units causes thermoreversible gelation of methylcellulose

This article describes detailed structure‐property relationships of 5 regioselectively methylated celluloses and 10 diblock cellulose derivatives with regioselective functionalization patterns: methyl 2,3,6‐tri‐O‐ ( 1 , 236MC), methyl 2,3‐di‐O‐ ( 2 , 23MC), methyl 2,6‐di‐O‐ ( 3 , 26MC), methyl 3‐O‐ ( 4 , 3MC), methyl 6‐O‐methyl‐cellulosides ( 5 , 6MC), methyl β‐D‐glucopyranosyl‐(1→4)‐2,3,6‐tri‐O‐methyl‐ ( 6 , G‐236MC), methyl β‐D‐glucopyranosyl‐(1→4)‐2,3‐di‐O‐methyl‐ ( 7 , G‐23MC), methyl β‐D‐glucopyranosyl‐(1→4)‐2,6‐di‐O‐methyl‐ ( 8 , G‐26MC), methyl β‐D‐glucopyranosyl‐(1→4)‐3‐O‐methyl‐ ( 9 , G‐3MC), methyl β‐D‐glucopyranosyl‐(1→4)‐6‐O‐methyl‐ ( 10 , G‐6MC), methyl β‐D‐glucopyranosyl‐(1→4)‐β‐D‐glucopyranosyl‐(1→4)‐2,3,6‐tri‐O‐methyl‐ ( 11 , GG‐236MC), methyl β‐D‐glucopyranosyl‐(1→4)‐β‐D‐glucopyranosyl‐(1→4)‐2,3‐di‐O‐methyl‐ ( 12 , GG‐23MC), methyl β‐D‐glucopy‐ranosyl‐(1→4)‐β‐D‐glucopyranosyl‐(1→4)‐2,6‐di‐O‐methyl‐ ( 13 , GG‐26MC), methyl β‐D‐glucopyranosyl‐(1→4)‐β‐D‐glucopyranosyl‐(1→4)‐3‐O‐methyl‐ ( 14 , GG‐3MC), and methyl β‐D‐glucopyranosyl‐(1→4)‐β‐D‐glucopyranosyl‐(1→4)‐6‐O‐methyl‐cellulosides ( 15 , GG‐6MC). Surface tension, differential scanning calorimetry, fluorescence, and dynamic light scattering measurements of aqueous solutions of compounds 1 – 15 revealed that there was no relationship between aggregation behaviors and gel formation, gelation occurred only when the hydrophobic environments formed by hydrophobic interactions between the sequences of 2,3,6‐tri‐O‐methyl‐glucopyranosyl units upon heating. The diblock structure consisting of cellobiosyl block and approx. ten 2,3,6‐tri‐O‐methyl‐glucopyranosyl units was of crucial importance for thermoreversible gelation of methylcellulose. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 49: 1539–1546, 2011     

Synthesis and Antigenic Property of a Novel Sialyl 6‐O‐Sulfo Lewis X Neo‐glycolipid Containing Lactamized Neuraminic Acid

Abstract

Synthesis and antigenic property of a novel 6‐O‐sulfated sLe^x neo‐glycolipid containing lactamized neuraminic acid are described. Coupling of methyl (methyl 4,7,8,9‐tetra‐O‐acetyl‐3,5‐dideoxy‐5‐trifluoroacetamido‐D‐glycero‐α‐D‐galacto‐2‐nonulopyranosylonate)‐(2→3)‐4,6‐di‐O‐acetyl‐2‐O‐benzoyl‐1‐thio‐β‐D‐galactopyranoside (3) with 2‐(tetradecyl)hexadecyl (2,3,4‐tri‐O‐benzyl‐α‐L‐fucopyranosyl)‐(1→3)‐2‐acetamido‐2‐deoxy‐6‐O‐4‐methoxyphenyl‐β‐D‐glucopyranoside (7) gave a protected sLe^x tetrasaccharide glycolipid (8). Removal of all the acyl protecting groups and subsequent lactamization afforded the lactamized sLe^x derivative(10), which was converted to the target compound (14) by selective removal of the 4‐methoxyphenyl group and 6‐O‐sulfation of the GlcNAc residue, and removal of all protective groups under the basic conditions furnished the target molecule. The antigenic property of the synthesized neo‐glycolipid was examined by TLC‐immunostaining with G159 monoclonal antibody.     

Novel Carbohydrate‐Appended Metal Complexes for Potential Use in Molecular Imaging

Seven discrete sugar‐pendant diamines were complexed to the {M(CO)₃}⁺ (^99mTc/Re) core: 1,3‐diamino‐2‐propyl β‐D ‐glucopyranoside ( L ¹ ), 1,3‐diamino‐2‐propyl β‐D ‐xylopyranoside ( L ² ), 1,3‐diamino‐2‐propyl α‐D ‐mannopyranoside ( L ³ ), 1,3‐diamino‐2‐propyl α‐D ‐galactopyranoside ( L ⁴ ), 1,3‐diamino‐2‐propyl β‐D ‐galactopyranoside ( L ⁵ ), 1,3‐diamino‐2‐propyl β‐(α‐D ‐glucopyranosyl‐(1,4)‐D ‐glucopyranoside) ( L ⁶ ), and bis(aminomethyl)bis[(β‐D ‐glucopyranosyloxy)methyl]methane ( L ⁷ ). The Re complexes [Re( L ¹ – L ⁷ )(Br)(CO)₃] were characterized by ¹H and ¹³C 1D/2D NMR spectroscopy which confirmed the pendant nature of the carbohydrate moieties in solution. Additional characterization was provided by IR spectroscopy, elemental analysis, and mass spectrometry. Two analogues, [Re( L ² )(CO)₃Br] and [Re( L ³ )(CO)₃Br], were characterized in the solid state by X‐ray crystallography and represent the first reported structures of Re organometallic carbohydrate compounds. Conductivity measurements in H₂O established that the complexes exist as [Re( L ¹ – L ⁷ )(H₂O)(CO)₃]Br in aqueous conditions. Radiolabelling of L ¹ – L ⁷ with [^99mTc(H₂O)₃(CO)₃]⁺ afforded in high yield compounds of identical character to the Re analogues. The radiolabelled compounds were determined to exhibit high in vitro stability towards ligand exchange in the presence of an excess of either cysteine or histidine over a 24 h period.     

Preparation and Evaluation of a Molecular Recognition Bionic Solid Phase Extraction Column for Separation of Glucosides

A molecular recognition bionic solid phase extraction (SPE) column for separation of glucosides has been prepared using a positively charged β‐glucosylamidine as the ligand in which a glycon moiety is connected via an N‐glycoside linkage. β‐Glucosylamidine, highly potent and selective inhibitors of β‐glycosidase, is immobilized through a one‐step synthesize procedure involving the addition of β‐glucosylamine and 2‐iminothiolane. HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give a molecular recognition absorbent for β‐glucosides. N‐octyl‐β‐D‐glucopyranoside and β‐D‐galactopyranoside or α‐D‐mannopyranoside was directly chromatographed through the bionic chromatographic column, resulting in a much stronger retention of β‐D‐glucopyranoside than β‐D‐galactopyranoside and α‐D‐mannopyranoside. The retained glucopyranoside could only be eluted by glucose solution. This indicates that the binding of the glucoside was of specific nature that corresponds to the glycon substrate specificity of the glucoside. The ease of preparation and the selective nature of the molecular recognition bionic chromatography should promise a large‐scale preparation of the molecular recognition adsorbent for the purification and removal of glucosides according to their glycon substrate specificity.     

Cysteine carboxyl O-methylation of human placental 23 kDa protein

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.     

Chromatographic separation and chemical analysis of polymers formed by penicillin G

To elucidate the chemical structures of penicillin polymers that may elicit an allergic reaction, a 25% aqueous solution of penicillin G potassium was kept standing in the dark at room temperature for 14 days and was then separated by gel filtration chromatography on Sephadex G-25. The fractions of Kav 0.0-0.3, 0.3-0.55 and 0.55-0.75 were designated fractions A, B and C, respectively. Chemical and spectral data indicated that fractions A and B had almost similar chemical structures, but differed in molecular weight. They consisted of equimolar phenylacetyl and thiazolidine moieties and showed a C:N:S ratio almost equal to that of penicillin G. Their degrees of polymerization were 10 for A and 3.2 for B. Comparison of 1H NMR and IR spectra and thin-layer chromatographic RF values with those of authentic standards showed that the main components of fraction C were N- formylpenicillamine , benzylpenilloic acid, benzylpenicilloic acid and benzylpenillic acid.     

Structural analysis of pentacyclic triterpenes from the gum resin of Boswellia serrata by NMR spectroscopy

3α‐Acetyl‐β‐boswellic acid ( 1 ), 3α‐acetyl‐α‐boswellic acid ( 2 ), 3α‐acetyl‐9,11‐dehydro‐β‐boswellic acid ( 3 ), 3α‐acetyl‐9,11‐dehydro‐α‐boswellic acid ( 4 ) and 3α‐acetyl‐11‐keto‐β‐boswellic acid ( 5 ) were isolated from the gum resin of Boswellia serrata. 1D and 2D NMR (COSY45, HMQC, HMBC, ROESY) spectra at 500 MHz were used for shift assignments and structure verification. All boswellic acids investigated share the cis conformation at ring D/E and the 3α orientation of the acetyl ester group. Owing to high‐order spectra, NMR could not determine the exact conformation of H‐20/H‐30 of the β‐boswellic acids. 3α‐Acetyl‐β‐boswellic acid methyl ester ( 1 ^′) was synthesized for experiments with a shift reagent, Eu(fod)₃, that enhanced the resolution considerably. The oxygen atoms of the 3α‐acetyl group form the apparent complex binding site for the shift reagent. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis of Substituted Tricyclo[5.3.1.04,9]undecan‐2,6‐dione from 4,4‐Disubstituted Cyclohexanone Enamines and Methacryloyl Chloride

Morpholine enamines 4‐acetyl‐4‐methyl‐1‐morpholinocyclohexene 4a, 4‐acetyl‐4‐phenyl‐1‐morpholinocyclohexene 4b, and 4‐acetyl‐4‐isopropenyl‐1‐morpholinocyclohexene 4c react with methacryloyl chloride to give 1,7‐dimethyl‐4(N‐morpholino) tricyclo[5.3.1.0^4,9]undecan‐2,6‐dione 9a , 1‐phenyl‐7‐methyl‐4(N‐morpholino) tricyclo[5.3.1.0^4,9]undecan‐2,6‐dione 9b , and 1‐ispropenyl‐7‐methyl‐4(N‐morpholino) tricyclo[5.3.1.0^4,9]undecan‐2,6‐dione 9c respectively, along with the corresponding substituted adamandane‐2,4‐diones.     

A Novel α-Amylase from Bacillus mojavensis A21: Purification and Biochemical Characterization

α-Amylase from Bacillus mojavensis A21 (BMA.2) was purified to homogeneity by ultrafiltration, Sephadex G-75 gel filtration and Sepharose mono Q anion exchange chromatography, with a 15.3-fold increase in specific activity and 11% recovery. The molecular weight of the BMA.2 enzyme was estimated to be 58 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration. The optimum temperature and pH were 80?°C and 6.5, respectively. BMA.2 belonged to the EDTA-sensitive α-amylase, but its activity was not stimulated by the presence of Ca²⁺ ions. The major end-products of starch hydrolysis were maltohexaose, maltopentaose and maltotriose. The N-terminal amino acid sequence of the first ten amino acids of the purified α-amylase was ASVNGTLMQY. Compared to sequences of other amylases, the ten amino acid sequence contains Val at position 3, while amylases from Bacillus licheniformis NH1 and Bacillus sp. SG-1 have Leu and Thr at position 3, respectively.     

Two new acylated flavonoid glycosides from Morina nepalensis var. alba Hand.‐Mazz.

From the whole plant of Morina nepalensis var. alba Hand.‐Mazz., two new acylated flavonoid glycosides ( 1 and 2 ), together with four known flavonoid glycosides ( 3–6 ), were isolated. Their structures were determined to be quercetin 3‐O‐[2″′‐O‐(E)‐caffeoyl]‐α‐L ‐arabinopyranosyl‐(1→6)‐β‐D ‐galactopyranoside (monepalin A, 1 ), quercetin 3‐O‐[2″′‐O‐(E)‐caffeoyl]‐α‐L ‐arabinopyranosyl‐(1→6)‐β‐D ‐glucopyranoside (monepalin B, 2 ), quercetin 3‐O‐α‐L ‐arabinopyranosyl‐(1→6)‐β‐D ‐galactopyranoside (rumarin, 3 ), quercetin 3‐O‐β‐D ‐galactopyranoside ( 4 ), quercetin 3‐O‐β‐D ‐glucopyranoside ( 5 ) and apigenin 4^′‐O‐β‐D ‐glucopyranoside ( 6 ). Their structures were determined on the basis of chemical and spectroscopic evidence. Complete assignments of the ¹H and ¹³C NMR spectra of all compounds were achieved from the 2D NMR spectra, including H–H COSY, HMQC, HMBC and 2D HMQC‐TOCSY spectra. Copyright © 2002 John Wiley & Sons, Ltd.