苦瓜子蛋白的分离纯化及真性质研究

从苦瓜种子的粗提物苦瓜子蛋白丙酮分级沉淀干粉中,用CM-Spehadex C-50和SephadexG-75分离得到了两个核糖体失活蛋白,α-苦瓜子蛋白(C-momorcharin,α-MMC和β-苦瓜子蛋白(β-momorcharin,β-MMC),其等电点分别为9.10(α-MMC),9.32(β-MMC).用ESI-MS和MALDI-TOF-MS测定它们的分子量,分别为28625(α-MMC),29076(β- MMC)(+Na,29099);,28795(α-MMC),29074.7(β-MMC).它们都是糖蛋白,其生物活性的分析测定表明,都属于RNA N-糖苷酶,本文重点对β-苦瓜子蛋白的分离纯化及其性质进行详细报道,并对其N-端部分的氨基酸顺序进行了测定.     

阴阳离子交换色谱串联分离纯化苦瓜籽核糖体失活蛋白

将阴阳离子交换色谱柱在灌注色谱工作站上串联,使苦瓜籽粗提液中未被阴离子色谱柱吸附的蛋白质直接在阳离子色谱柱上吸附,用盐线性梯度洗脱从阳离子色谱柱上分离出两个具有抗真菌活性的蛋白。两个抗真菌蛋白经鉴定都达到了电泳纯,相对分子质量均为30000左右,N-端氨基酸序列分别为DVSFRLSGADPRSYGMFI与DVNFDLSTATAK,表明所得到的两种蛋白为苦瓜籽中的2个Ⅰ型核糖体失活蛋白α-苦瓜素(α-MMC) 和β-苦瓜素(β-MMC)。抗菌实验表明,α-MMC对香蕉枯萎菌和果腐霉菌均具有较强的抑制作用,β     

奶制品中蛋白质测定的毛细管电泳法研究

本文利用毛细管电泳法研究了奶制品中蛋白成分的分离与测定。采用柠檬酸缓冲体系,对牛奶中的α-乳白蛋白(-αLa)、β-乳球蛋白(-βLg)、α-酷蛋白(-αCN)、β-酪蛋白(-βCN)、酪蛋白(-κCN)五种蛋白进行了很好的分离,其迁移时间和峰面积的相对标准偏差分别小于0.2%和5%;检出限分别为0.01、0.03、0.02、0.02、0.02 mg/mL;加标回收率范围为84%~103%。应用该法测定了多种奶制品中的蛋白质含量。本法简便、快速、准确,为牛奶及奶制品质量的监控提供了一种新的手段。     

苦瓜素的分离纯化与抗肿瘤活性的研究

本文报道了α－苦瓜素和β苦瓜素分离纯化的工艺改进,测定了两种苦瓜素的理化性质。实验证明,两种苦瓜素对小鼠Ｓ－１８０实体瘤均具有显著的抑制作用,抑瘤率分别为７１．２％和６８．６％;两种苦瓜素对人胃癌ＮＫＭ细胞株的ＤＮＡ,ＲＮＡ和蛋白质的合成亦均具有明显的抑制作用。     

毛细管电泳法对南方水牛奶乳清蛋白的分离和定量分析

利用毛细管区带电泳对广东省水牛乳乳清蛋白成分进行了分离和定量分析研究.采用1.2%的十四水合硼酸钠电泳缓冲液,对水牛奶乳清蛋白的四种主要组分α-乳白蛋白(α-La)、β-乳球蛋白(β-Lg)、牛血清白蛋白(BSA)、免疫球蛋白(IgG)进行了很好的分离,其迁移时间和峰面积的RSD分别小于1.5%和0.5%,加标回收率范围91%～102%.建立了基于毛细管区带电泳的分析方法,对牛乳及其乳制品中的乳清蛋白进行了快速分离和定量分析.     

聚合物/磷酸盐双水相分离乳清分离蛋白中α-乳白蛋白和β-乳球蛋白条件的优化

建立了基于聚(乙二醇-ran-丙二醇)单丁基醚(聚合物)与磷酸盐、氯化钠的双水相体系对乳清分离蛋白(WPI)中的α-乳白蛋白(α-LA)和β-乳球蛋白(β-LG)进行分离,并对其分离条件进行了优化.系统地研究了双水相体系pH值、聚合物与KH2PO4溶液体积比、NaC1添加量和WPI浓度对α-LA和β-LG的分离效果的影响,采用液相色谱法对α-LA和β-LG的分离效果进行评价.结果表明,聚合物/KH2PO4双水相体系pH=4.0,40％ (m/m)聚合物与15.5％ (m/m)KH2PO4的体积比为4 mL∶4 mL,NaCl添加量为0.40 g/10 mL,WPI浓度为1 mg/mL时,α-LA和β-LG分离效果最好,上相中α-LA的萃取率为98.2％,下相中β-LG的萃取率为96.6％.     

八棱丝瓜蛋白1的二级结构及其生物活性

测定了八棱丝瓜蛋白1的N端顺序为Asp-Val-Ser-Phe-Ser-,用CD谱测定了八棱丝瓜蛋白 1的α螺旋,β-折叠和无规卷曲含量分别为37.1%,33.4%,29.5%。实验表明,八棱丝瓜蛋白 1具有RNA N-糖苷酶活性,体外抑制肿瘤细胞生长活性表明其对肿瘤细胞株B16,MGC,Bel的半数抑制浓度分别为1.78×10-7mol/L,2.11×10-7mol/L和4.21×10-7mol/L。并在N端顺序,二级结构和生物活性方面对八棱丝瓜蛋白 1和天花粉蛋白进行了比较。     

毛细管区带电泳高效分离和高灵敏检测α-乳清蛋白和β-乳球蛋白

α-乳清蛋白和β-乳球蛋白系由牛乳清提取的乳糖制品中的主要蛋白杂质,是引起食物及药物过敏的主要过敏原。因此,在食品安全及医药安全领域,对二者的分离及痕量检测是至关重要的。本研究建立了一种简单、快速、灵敏、重现性好的毛细管区带电泳(CZE)方法,使用简单的电泳体系和样品处理步骤,实现了α-乳清蛋白和β-乳球蛋白的基线分离和痕量检测。CZE条件如下:25 mmol/L磷酸盐缓冲液(pH 7.0),分析电压+30 kV,毛细管温度25℃,紫外检测波长205 nm,压力进样5 kPa"10 s。在此条件下,样品分析时间为2 min,α-乳清蛋白和β-乳球蛋白的检出限(LOD)分别为3.0和12 mg/L;迁移时间和峰面积的相对标准偏差(RSD,n=6)分别小于1%和6%,符合实际样品检测要求。本方法已成功用于实际乳糖样品的分析,在相关领域也有很好的应用前景。     

阿尔泰狗娃花化学成分研究

本文从阿尔泰狗娃花Heteropappus altaicus的乙醇提取物中分离出12个已知化合物,经过红外光谱(IR),质谱(MS),核磁共振谱(NMR)等分析方法及与标准品对照,鉴定了它们的结构。分别为:木栓酮(1),表木栓醇(2),达玛烷-20,24-二烯-3β-醇(3),达玛烷-20,24-二烯-3β-乙酰基(4),α-波甾醇(5),槲皮素(6),3'-甲氧基槲皮素(7),槲皮素-3-O-β-D-葡萄糖苷(8),阿魏酸(9),β-谷甾醇(10),β-胡萝卜苷(11),没食子酸乙酯(12)。其中化合物9和12为首次从该属植物中分离得到,化合物5,6,9和12为首次从该植物中分离得到。     

金属硫蛋白家族内的结构域拼接

金属硫蛋白(Metallothioneins,MTs)由结构独立且功能明显区别的β,α两个结构域组成。神经生长抑制因子(NeuronalGrowthInhibitoryFactor,GIF)双名金属硫蛋白-Ⅲ(MT-Ⅲ),是神经系统中第一个被鉴定的具有神经元生长抑制功能的蛋白,而β－结构域为其功能结构域。为深入系统地研究MTs,尤其是GIF及其结构域的结构与功能,我们构建了金属硫蛋白家族内结构域拼接体βGIF-αMT-1(βⅢ-αⅠ)和βMT-1-αGIF(βⅠ-αⅢ):PCR扩增得各个结构域的cDNA序列,酶切后克隆入原核表达载体pGEX-4T-1,经发酵、诱导表达、亲和层析、凝血酶切和进一步纯化,得率约为80mg蛋白/L菌液。测其电泳行为、氨基酸组成、质谱、金属巯基含量等,证明得到了目的蛋白。紫外吸收图谱和圆二色性图谱显示,结构域拼接体拥有金属硫蛋白家族成员的特征金属巯基簇结构域,初步功能实验表明,βⅢ－αⅠ也具有抑制神经元生长的功能。     

Local protein backbone folds determined by calculated NMR chemical shifts

NMR chemical shifts (CSs: δN(NH), δC(α), δC(β), δC', δH(NH), and δH(α)) were computed for the amino acid backbone conformers (α(L), β(L), γ(L), δ(L), ε(L), α(D), γ(D), δ(D), and ε(D) [Perczel et al., J Am Chem Soc 1991, 113, 6256]) modeled by oligoalanine structures. Topological differences of the extended fold were investigated on single β-strands, hairpins with type I and II β-turns, as well as double- and triple-stranded β-sheet models. The so-called "capping effect" was analyzed: residues at the termini of a homoconformer sequence unit usually have different CSs than the central residues of an adequately long homoconformer model. In heteroconformer sequences capping effect ruins the direct applicability of several chemical shift types (δH(NH), δC', and δN(NH)) for backbone structure determination of the parent residue. Experimental δH(α), δC(α), and δC(β) values retrieved from protein database are in good agreement with the relevant computed data in the case of the common backbone conformers (α(L), β(L), γ(L), and ε(L)), even though neighboring residue effects were not accounted for. Experimental and computed ΔδH(α)-ΔδC(α), ΔδH(α)-ΔδC(β), and ΔδC(α)-ΔδC(β) maps give qualitatively the same picture, that is, the positions of the backbone conformers relative to each other are very similar. This indicates that the H(α), C(α), and C(β) chemical shifts of alanine depend considerably on the backbone fold of the parent residue also in proteins. We provide tabulated CSs of the chiral amino acids that may predict the various structures of the residues.     

Separation and quantitation of milk whey proteins of close isoelectric points by on-line capillary isoelectric focusing--electrospray ionization mass spectrometry in glycerol-water media

On-line coupling between CIEF and ESI/MS based on the use of bare fused-silica capillaries and glycerol-water media, recently developed in our laboratory, has been investigated for the separation of milk whey proteins that present close pI values. First, a new rinsing procedure, compatible with MS detection, has been developed to desorb these rather hydrophobic proteins (α-casein (α-CN), bovine serum albumin (BSA), lactoferrin (LF)) from the inner capillary wall and to avoid capillary blockages. Common hydrochloric acid washing solution was replaced by a multi-step sequence based on the use of TFA, ammonia and ethanol. To achieve the separation of major whey proteins (β-lactoglobulin A (β-LG A), β-lactoglobulin B (β-LG B), α-lactalbumin (α-LA) and BSA, which possess close pI values (4.5-5.35), CIEF parameters i.e. carrier ampholyte nature, capillary partial filling length with ampholyte/protein mixture and focusing time, have been optimized with respect to total analysis time, sensitivity and precision on pI determination. After optimization of sheath liquid composition (80:20 (v/v) methanol-water+1% HCOOH), quantitation of β-LG A, β-LG B, α-LA and BSA was performed. The limits of detection obtained from extracted ion current (EIC) and single ion monitoring (SIM) modes were in the 57-136 nM and 11-68 nM range, respectively. Finally, first results obtained from biological samples demonstrated the suitability of CIEF-MS as a potential alternative methodology to 2D-PAGE to diagnose milk protein allergies.     

麦芽糖基(α-1→6)β-环糊精的酶法合成和结构鉴定

采用地衣芽孢杆菌普鲁蓝酶合成Mal-β-CD, 反应产物体系成分简单, 易于分离, Mal-β-CD的转化率可达到56%.     

Studies on the Constituents of Paris Formosana Hayata Part II

Methyl palmitate (I), methyl stearate (II), stigmasterol (III), β-sitosterol (IV), (O -acyl)-β-D -glucopyranosyl-(1→3)-stigmasterol (V), (O -acyl)-β-D -glucopyranosyl-(1→3)-β-sitosterol (VI), β-D -glucopyranosyl-(1→3)-stigmasterol (VII), β-D -glucopyranosyl-(1→3)-β-sitosterol (VIII), β-D -ecdysone (IX), diosgenin-3-α-L -rhamopyranosyl-(1→2)-[α-L -arabinofuranosyl-(1→4)]-β-D -glucopyranoside (X), diosgenin-3-O -β-chacotrioside (dioscin) (XI), and diosgenin-3-O -α-L -rhamnopyranosyl-(1→4)-α-L -rhamnopyranosyl-(1→4)-[α-L -rhamnopyranosyl-(1→2)]-β-D -glucopyranoside (XII) were isolated and characterized from the stems of Paris formosana Hayata (Liliaceae).     

A generalised route for the synthesis of β-furyl-α,β-unsaturated aldehydes through Suzuki reactions

A general method for the synthesis of β-(2-furyl)-α,β-unsaturated aldehydes is described using the Suzuki coupling reaction of furan-2-boronic acids and β-bromo-α,β-unsaturated aldehyde derivatives.     

Synthesis of new enantiomerically pure β-amino alcohols of the pinane series

A series of new β-amino alcohols with pinane structure, (+)- and (?)-3α-amino-10β-pinan-4β-ols, 4β-amino-10β-pinan-3α-ol, and 4α-amino-10β-pinan-3α-ol have been synthesized with the goal of using them as organocatalysts in the aldol reaction of isatin with acetone.     

竞争包结法研究β-环糊精及其两种衍生物对一些手性脂肪族客体分子的识别作用

以酚酞作为光谱探针 ,采用紫外 可见光谱滴定法测定了 β 环糊精 (β CD)、单 (6 氧 α 麦芽糖 ) β 环糊精 (6 G2 β CD )和单 [2 氧 (2 羟丙基 ) ] β 环糊精 (2 HP β CD )在 2 5℃时 ,pH =10 5缓冲液中(0 0 2 5mol/L)与几种脂肪族手性客体分子所形成超分子配合物的稳定常数 .结果表明 ,多种弱相互作用力协同作用于环糊精的配位过程 ,主 客体间的尺寸匹配决定所形成配合物的稳定性 .环糊精衍生物的取代基影响主体的配位能力 ,对于尺寸较小的客体分子配位能力的大小一般为 2 HP β CD >β CD >6 G2 β CD .另一方面 ,3种环糊精主体化合物对一些脂肪族客体分子也表现出一定的手性识别能力 ,对 (+ ) 异构体给出相对较强的键合能力 ,其中 ,2 HP β CD对 (+ ) /(- ) 樟脑的配位选择性为 1 2 5 .     

Preparative separation of α- and β-santalenes and (Z)-α- and (Z)-β-santalols using silver nitrate-impregnated silica gel medium pressure liquid chromatography and analysis of sandalwood oil

The major sesquiterpene constituents of East-Indian sandalwood oil (Z)-α- and (Z)-β-santalols have shown to be responsible for most of the biological activities and organoleptic properties of sandalwood oil. The work reported here describes the strategic use of medium pressure liquid chromatography (MPLC) for the separation of both α- and β-santalenes and (Z)-α- and (Z)-β-santalols. Silver nitrate impregnated silica gel was used as the stationary phase in MPLC for quantitative separation of α- and β-santalenes and (Z)-α- and (Z)-β-santalols with mobile phases hexane and dichloromethane, respectively. The purities of α-santalene and (Z)-α-santalol obtained were >96%; however, β-santalene and (Z)-β-santalol were obtained with their respective inseparable epi-isomers. Limits of quantification (LoQ) relative to the FID detector were measured for important sesquiterpene alcohols of heartwood oil of S. album using serial dilutions of the standard stock solutions and demonstrated that the quality of the commercial sandalwood oil can be assessed for the content of individual sesquiterpene alcohols regulated by Australian Standard (AS2112-2003), International Organization for Standardization ISO 3518:2002 (E) and European Union (E. U.).     

Divergent sequential reactions of β-(2-aminophenyl)-α,β-ynones with nitrogen nucleophiles

β-(2-Aminophenyl)-α,β-ynones readily react with nitrogen nucleophiles to give three major types of products, depending on reaction conditions and variation in the nucleophiles. The reaction may lead to simple 1,2-nucleophilic adducts or, at higher temperatures, to a divergent sequential cyclisation giving rise to 2-aryl-4-aminoquinolines by reaction with amines, or to substituted 2-aryl or 2-alkyl-4-alkylidene quinazolines by reaction with amidines. The latter could also be synthesised by reaction of β-(2-aminophenyl)-α,β-ynones with iminochlorides.