Mass Spectrometry and X-ray Diffraction Analysis of Two Crystal Types of Dioclea virgata Lectin: An Antinociceptive Protein Candidate to Structure/Function Analysis

The lectin from seeds of Dioclea virgata (DvirL) was purified in a single step affinity chromatography, sequenced by tandem mass spectrometry and submitted to crystallization and biological experiments. DvirL has a molecular mass of 25,412?±?2 Da and the chains β and γ has 12,817 Da?±?2 and 12,612 Da?±?2, respectively. Primary sequence determination was assigned by tandem mass spectrometry and revealed a protein with 237 amino acids and 87% of identify with ConA. The protein crystals were obtained native and complexed with X-Man using vapor-diffusion method at a constant temperature of 293 K. A complete X-ray dataset was collected at 1.8 ? resolution. DvirL crystals were found to be orthorhombic, belonging to the space group I222, with a unit cell parameters a?=?647.5 ?, b?=?86.6 ?, c?=?90.2 ?. Molecular replacement search found a solution with a correlation coefficient of 77.1% and an R(factor) of 44.6%. The present study also demonstrated that D. virgata lectin presents edematogenic and antinociceptive activities in rodents electing this protein as a candidate to structure/function analysis.     

Purification, Characterization, and Preliminary X-Ray Diffraction Analysis of a Lactose-Specific Lectin from Cymbosema roseum Seeds

The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 microg mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A.     

Molecular weight and monosaccharide composition of Astragalus polysaccharides

Two polysaccharides (APS-I and APS-II) were isolated from the water extract of Radix Astragali and purified through ethanol precipitation, deproteination and by ion-exchange and gel-filtration chromatography. Their molecular weight was determined using high performance liquid chromatography and gel permeation chromatography (HPLC-GPC) and their monosaccharide composition was analyzed by TLC and HPLC methods, using a refractive index detector (RID) and an NH(2) column. It was shown that APS-I consisted of arabinose and glucose and APS-II consisted of rhamnose, arabinose and glucose, in a molar ratio of 1:3.45 and 1:6.25:17.86, respectively. The molecular weights (Mw) of APS-I and APS-II were 1,699,100 Da and 1,197,600 Da, respectively.     

Synthesis, crystal structure, and resolution of [10](1,6)pyrenophane: an inherently chiral [N]cyclophane

A synthetic approach to a set of three inherently chiral [n]cyclophanes, [n](1,6)pyrenophanes (29a-c, n = 8-10) was investigated. Progress toward 29a was thwarted by the failure of the key dithiacyclophane-forming reaction. For the next higher homologue, the synthesis was completed, but the desired [9](1,6)pyrenophane (29b) could only be partially separated from an isomeric pyrenophane, [9](1,8)pyrenophane (28b), and an unidentified byproduct. Work aimed at the synthesis of the next higher homologue resulted in the isolation of a 7:4 mixture of [10](1,8)pyrenophane (28c) and [10](1,6)pyrenophane (29c), which could not be separated by column chromatography or crystallization. However, single-crystal X-ray structures of 28c and 29c were obtained after manual separation of two crystals with different morphologies from the same batch of crystals obtained from the 7:4 mixture of 28c and 29c. The pyrene system of 29c was found to have a gentle end-to-end bend as well as a significant longitudinal twist. Short intermolecular C(sp(3))-H···π contacts (2.64 to 2.76 ?) between H-atoms on the bridge and the centroids of three of the four six-membered rings of the pyrene system of a neighboring pyrenophane of like chirality give rise to the formation of single enantiomer columns. From a DNMR study of the mixture of 28c and 29c, the bridge in [10](1,8)pyrenophane (28c) was found to undergo a conformational flip from one side of the pyrene system to the other with ΔG(?) = 14.9 ± 0.2 kcal/mol. A two-stage preparative HPLC protocol was subsequently developed for the separation of 28c and 29c (Chiralpak AD-H column) and then the enantiomers of 29c (Chiralcel OJ-H column). This enabled the measurement of their optical rotations and CD spectra.     

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.     

Single step synthesis of carbohydrate monolithic capillary columns for affinity chromatography of lectins

Carbohydrate monolithic beds were synthesized in a single step in capillary columns to study affinity chromatography of lectins. In this method, carbohydrates (beta-galactose, beta-glucose, and alpha-mannose) with an easy to synthesize alkene terminated tetraethylene glycol spacer were used as functional monomers along the monomer 2-hydroxyethyl methacrylate (HEMA). As crosslinkers (+)-N,N'-diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) were used. SEM showed the successful formation of monolithic beds in the capillary columns. The permeability of the columns was high. The specific interaction of the lectins Con A, Lens culinaris (LCA) and Arachis hypogaea (PNA) with the carbohydrate stationary phase was studied by frontal affinity chromatography (FAC). Con A and LCA were successfully eluted from the column using 0.1 M methyl-alpha-mannopyranoside and PNA with 0.1 M beta-galactose. Dissociation constants (Kd) for carbohydrate-lectin interactions were determined and compared with literature.     

Improved online δ18O measurements of nitrogen- and sulfur-bearing organic materials and a proposed analytical protocol

It is well known that N(2) in the ion source of a mass spectrometer interferes with the CO background during the δ(18)O measurement of carbon monoxide. A similar problem arises with the high-temperature conversion (HTC) analysis of nitrogenous O-bearing samples (e.g. nitrates and keratins) to CO for δ(18)O measurement, where the sample introduces a significant N(2) peak before the CO peak, making determination of accurate oxygen isotope ratios difficult. Although using a gas chromatography (GC) column longer than that commonly provided by manufacturers (0.6 m) can improve the efficiency of separation of CO and N(2) and using a valve to divert nitrogen and prevent it from entering the ion source of a mass spectrometer improved measurement results, biased δ(18)O values could still be obtained. A careful evaluation of the performance of the GC separation column was carried out. With optimal GC columns, the δ(18)O reproducibility of human hair keratins and other keratin materials was better than ± 0.15 ‰ (n=5; for the internal analytical reproducibility), and better than ± 0.10 ‰ (n=4; for the external analytical reproducibility).     

Purification,Partial Characterization,and CNBr-Sepharose Immobilization of a Vasorelaxant Glucose/Mannose Lectin from Canavalia virosa Seeds

A novel mannose/glucose-binding lectin from Canavalia virosa (designated as ConV) has been purified from seeds of C. virosa by affinity chromatography on a mannose-Sepharose 4B column. ConV strongly agglutinates rabbit erythrocytes and was inhibited by monosaccharides (D-mannose, D-glucose, and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). SDS-PAGE revealed three bands corresponding to three subunits (α, β, and γ) confirmed by ESI mass spectrometry with exact mass of 25,480?±?2 Da, 12,864?±?1 Da, and 12,633?±?1 Da, respectively. The purified lectin was more stable in pH ranging from 7.0 to 9.0, supported up to 80?ºC without any loss in activity and unaffected by EDTA. ConV showed no toxicity against Artemia sp. nauplii and relaxed endothelized rat aorta, with the participation of the lectin domain. In our tests, the lectin immobilized on CNBr-Sepharose was capable of binding 0.8 mg of ovalbumin per chromatography, allowing the use of ConV as a tool for capture and purification of glycoproteins.     

Comparative studies of carbohydrate-binding proteins from Xenopus laevis skin and eggs. Sugar-binding specificities and affinity purification

Salt and detergent extracts of acetone-dried powder of Xenopus laevis skin and eggs were fractionated on sugar-Sepharose columns, to which lactose, melibiose, galactose, rhamnose and mannose had been covalently linked, by successive elution with chelating reagent and specific sugars, resulting in separation of the different Ca2(+)-dependent and Ca2(+)-independent carbohydrate-binding proteins. The skin of X. laevis contains a salt-extractable Ca2(+)-dependent lactose-binding lectin of 30 kilodalton (kDa) and the eggs a similar lectin of 43 kDa, but they both lack Ca2(+)-dependent galactose-binding lectins. The 30 kDa lactose-binding lectin which agglutinates human A erythrocytes was isolated by successive affinity chromatography on two linked sugar-Sepharose columns, i.e., a galactose-Sepharose-lactose-Sepharose (GL) column system. Since the 30 kDa lectin was not recovered in the Ca2(+)-dependent lactose-binding protein fraction from the GL column system under the dithiothreitol (DDT)-free conditions, it was concluded that the lectin requires the presence of DTT and calcium for binding to the lactose-Sepharose column.     

New method for the synthesis of N-tert-alkoxyarylaminyl radicals

The reactions of 2,4-diaryl-6-tert-butylnitrosobenzenes with 2,2'-azobis[2-(methoxycarbonyl)propane] (5a), 2,2'-azobis(2-cyano-4-methylpentane) (5b), and 2,2'-azobis(2-cyano-4-methyl-4-methoxypentane) (5c) in refluxing benzene gave stable N-tert-alkoxy-2,4-diaryl-6-tert-butylphenylaminyls, which were successfully isolated as radical crystals in 13-52% yields after column chromatography. The radical yields depended on the reaction time and the molar ratio of azo compounds to nitroso compounds. In the same manner, acetyl- and cyano-group-carrying N-tert-alkoxyarylaminyls were generated by the reaction of 2-phenyl-4-(4-acetylphenyl)-6-tert-butylnitrosobenzene and 2-phenyl-4-(4-cyanophenyl)-6-tert-butylnitrosobenzene with 5a and 5b, and they were isolated as radical crystals. X-ray crystallographic analyses were performed for two radicals, and their molecular structures were discussed in detail. The magnetic properties were measured for the two isolated radicals with SQUID in the temperature range 1.8-300 K. One radical showed a weak ferromagnetic interaction (theta = 0.2 K) between the radicals, and the other showed a weak antiferromagnetic interaction (theta = -3.8 K). The ferromagnetic interaction was analyzed based on the X-ray crystallographic structure.     

Molecular characterization and tandem mass spectrometry of the lectin extracted from the seeds of Dioclea sclerocarpa Ducke

Lectin from the seeds of Dioclea sclerocarpa (DSL) was purified in a single step by affinity chromatography on a Sephadex G-50 column. The primary sequence, as determined by tandem mass spectrometry, revealed a protein with 237 amino acids and 81% of identity with ConA. DSL has a molecular mass of 25,606 Da. The β and γ chains weigh 12,873 Da and 12,752 Da, respectively. DSL hemagglutinated rabbit erythrocytes (both native and treated with proteolytic enzymes), showing stability even after one hour of exposure to a specific pH range. The hemagglutinating activity of DSL was optimal between pH 6.0 and 8.0, but was inhibited after incubation with D-galactose and D-glucose. The pure protein possesses a molecular mass of 25 kDa by SDS-PAGE and 25,606 Da by mass spectrometry. The secondary structure content was estimated using the software SELCON3. The results indicate that b-sheet secondary structures are predominant in DSL (approximately 42.3% antiparallel b-sheet and 6.7% parallel b-sheet). In addition to the b-sheet, the predicted secondary structure of DSL features 4.1% a-helices, 15.8% turns and 31.3% other contributions. Upon thermal denaturation, evaluated by measuring changes in ellipticity at 218 nm induced by a temperature increase from 20 °C to 98 °C, DSL displayed cooperative sigmoidal behavior with transition midpoint at 84 °C and permitted the observation of two-state model (native and denatured).     

Investigating the structure of a monolithic capillary column by means of hydrodynamic and size-exclusion chromatography

The porosity of a monolithic capillary column having a structure optimized for gas chromatographic analysis was investigated by means of hydrodynamic and size-exclusion chromatography. It was found that the total porosity of the column exceeded 90%, and the column had a bimodal pore structure with a micropore diameter of about 1.5 nm and a macropore diameter of about 1.2 μm. The column separated with good selectivity high molecular mass polystyrene standards with molecular masses higher than 100 kDa and low molecular mass solutes of up to 500 Da. The structure of monolithic column has to be optimized for application in hydrodynamic chromatography with an aim to provide selectivity on separation of polymers with molecular mass from 1 to 100 kDa.     

Isolation and characterization of isolectins from Erythrina variegata seeds.

Three isolectins were isolated from seeds of Erythrina variegata (Linn.) var. Orientalis by ion-exchange chromatography, followed by affinity chromatography on lactose-Sepharose 4B and acid-treated Sepharose 4B columns. The purified isolectins (EVLI, EVLII and EVLIII) are all specific for galactopyranosides and N-acetylgalactosamine, and their affinities for simple sugars are EVLIII greater than EVLII greater than EVLI. EVLI and EVLIII are homodimers made up of an A-subunit of molecular mass 36,000 and a B-subunit of molecular mass 33,000, whereas EVLII is a heterodimer composed of the A- and B-subunits. Upon treatment with trifluoromethansulphonic acid, the molecular masses of both subunits decreased to 31,000. Rechromatography of EVLII on the acid-treated Sepharose 4B column again produced the homodimeric lectins (EVLI and EVLIII). It is suggested that the constituent subunits of Erythrina variegata isolectins are eschangeable with each other in vitro.     

Purification and characterization of carbohydrate-binding peptides from Lotus tetragonolobus and Ulex europeus seed lectins using affinity chromatography.

Carbohydrate-binding peptides of several anti-H(O) leguminous lectins were obtained from endoproteinase Asp-N or Lys-C digests of L-fucose-binding Lotus tetragonolobus lectin (LTA) and Ulex europeus lectin I (UEA-I) and from that of a di-N-acetylchitobiose-binding Ulex europeus lectin II (UEA-II) by affinity chromatography on columns of Fuc-Gel (for LTA and UEA-I) and on a column of a mixture of several oligomers of N-acetyl-D-glucosamine (GlcNAc) coupled to Sepharose 4B (GlcNAc oligomer-Sepharose 4B) (for UEA-II). These peptides were retained on the Fuc-Gel or GlcNAc oligomer-Sepharose 4B column and were presumed to have an affinity for the columns. The amino acid sequences of the retarded peptides were determined using a protein sequencer.     

Fast characterization of intact proteins using a high-throughput eight-channel parallel liquid chromatography/mass spectrometry system

The preparation of protein substrates requires that a large number of chromatographic fractions be analyzed for the presence of reactants, products and by-products. Analyses using linear matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or single column liquid chromatography/mass spectrometry (LC/MS) have been inadequate because of mass resolution or throughput. Therefore, a high-throughput method employing an eight-channel parallel reverse-phase LC/MS system was developed. This system is capable of screening fractions from preparative ion-exchange chromatography with the required mass accuracy and throughput so that the protein purification process can be monitored in a relatively short period of time. As an example, the purification and analysis of an acylated protein with a molecular weight of 8.9 kDa is described and the detection of a contaminating by-product that differs in size by less than 20 Da is demonstrated. Using the current instrumentation and approach, it is practical to analyze 50 protein-containing fractions from column chromatography in less than 1 hour using parallel LC/MS.     

光活性酮咯酸衍生物的拆分及绝对构型的测定

酮咯酸与L-脯氨酸苄酯在缩合剂作用下形成酰胺，用柱层析分离了一对非对映异构体并进行了表征.用单晶X衍射测定了酮咯酸脯氨酸苄酯酰胺一个异构体的晶体结构，并用内参法确定了其绝对构型.晶体属正交晶系，空间群P212121，晶胞参数a=0.896 45（1）nm，b=1.271 23（2）nm，c=2.076 21（4）nm， V=2.366 04（6）nm3，Z=4，最终偏离因子R1=0.046 4.     

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.     

正相色谱和反相色谱法分离提纯东北红豆杉中紫杉醇和三尖杉宁碱

以东北红豆杉树叶为原料,采用正相色谱和反相色谱法分离提纯了紫杉醇和三尖杉宁碱。反相色谱中使用了一种新颖的高分子填料。东北红豆杉枝叶经过一次正相色谱分离后,再经两次反相色谱分离,分离得到两种白色针状固体。经1H NMR和13C NMR测试表明,两种物质分别为紫杉醇和三尖杉宁碱,纯度达到98%以上,产率分别为树叶干重的0.0022%和0.0018%,回收率大于70%。     

A rapid method for isolation and purification of an anticoagulant from Whitmania pigra

Whitmania pigra is common in China and has been used as a traditional Chinese anticoagulant medicine for years, but its effective components are unknown to scientists. In this article we report a rapid method for isolation and purification of an anticoagulant from W. pigra for the first time. An acetone-water extract of W. pigra was subjected to anion-exchange chromatography on a Sephadex DEAE A-50 column, and gel permeation chromatography on Sephadex G-25 and Sephadex LH-20 columns successively, which afforded a fraction with potent anticoagulant activity. An anticoagulant was isolated and purified from this fraction by reversed-phase high-performance liquid chromatography (RP-HPLC). It was identified as a single pure substance by RP-HPLC and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This component was named whitmanin and its molecular weight was determined as 8608 Da by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS).     

Characterization of rat liver microsomal and hepatocytal metabolites of brevetoxins by liquid chromatography–electrospray tandem mass spectrometry

Brevetoxins are natural neurotoxins that are produced by “red tide” algae. This class of compounds can cause neurotoxic shellfish poisoning and other health problems. Brevetoxin-2 is the most abundant among the nine brevetoxins that have been characterized, whereas brevetoxin-1 is the most toxic. In this study, brevetoxin-1 and brevetoxin-2 were incubated with rat liver hepatocytes and rat liver microsomes, respectively. After clean-up steps were taken to remove the proteins, samples were analyzed by liquid chromatography (LC) coupled with electrospray mass spectrometry (LC-MS). After incubation of brevetoxin-1, two metabolites were found: brevetoxin-1-M1 (molecular weight = 900 Da), and brevetoxin-1-M2 (molecular weight = 884 Da). The increase in molecular weight combined with evidence from tandem mass spectrometry showing an increased tendency for loss of water molecules, along with considerations of established precedents for chemical transformations led to the conclusion that brevetoxin-1-M1 was formed by converting one double bond in the E or F ring of brevetoxin-1 into a diol. The second metabolite (brevetoxin-1-M2) is proposed to be a hydrolysis product of brevetoxin-1 involving opening of the lactone ring with the addition of a water molecule. The incubation study of the other starting compound, brevetoxin-2, found two metabolites in the LC-ES-MS selected ion chromatogram. Brevetoxin-2-M1 (molecular weight = 912 Da) gave a large [M−H]⁻ peak at m/z 911, and its product ion mass spectrum allowed the deduction that this metabolite was the hydrolysis product of brevetoxin-2 involving conversion of the lactone to a carboxylic acid and an alcohol. The second metabolite (brevetoxin-2-M2, molecular weight = 896 Da) was deduced to have the same structure as that of brevetoxin-3 based on identical chromatographic retention times and similar mass spectra as those obtained for a brevetoxin-3 standard.