响应曲面法优化毛细管区带电泳分析rhEPO的方法

为了在采用国内常用的60cm毛细管柱检测的条件下,能够完全分离检测重组人促红细胞生成素(rhEPO)8个组分,运用响应面分析法(RSM)对《欧洲药典》中规定的rhEPO的毛细管区带电泳检测法所用缓冲液组成作细微调整,以达到pH值操作允许范围略加宽松,易于控制,又保证所有组分清晰分离的目的。数学分析得到的缓冲液组分为:12 mmol/L氯化钠,12 mmol/L麦黄酮,12 mmol/L醋酸钠,3.0 mmol/L腐胺;用此缓冲液,pH值可拓宽至5.2~5.8范围内。实例检测验证结果表明,采用60 cm毛细管柱,在此缓冲液条件下检测样品,pH值在5.2~5.8范围内,rhEPO的8个糖基化形式在38 min内实现了基线分离,各相邻峰间的分离度达1.63~3.29,柱效达每米1.63×105~3.23×105理论塔板数,峰形良好。本研究提供了一个可行、可靠、高效的rhEPO分析方法。     

芯片毛细管电泳检测痕量酶活性的初步探讨

微芯片技术是基于微机电加工技术(MEMS)工艺,在芯片上完成电泳检测过程的新型技术．利用自制的微流控芯片及激光诱导荧光系统建立了痕量乳酸脱氢酶(LDH)活性的检测方法．以pH 9.4, 75 mmol/L硼酸为芯片电泳缓冲液,9.73 μmol/L乳酸钙为添加剂,整个电泳过程4 min结束,利用该法测得LDH检测限(S/N=3)为6×10^-3 U/L,出峰时间和峰面积的相对标准偏差分别为5.32%和3.17%,该法操作过程简单,检测灵敏度高,在临床痕量酶检测中具有较好的应用前景．     

高效毛细管电泳分离多种植物激素的方法研究

建立了高效毛细管电泳法分离测定茶叶中赤霉素(GA)、吲哚-3-乙酸(IAA)、脱落酸(ABA)、吲哚-3-丁酸(IBA)、细胞分裂素(CTK)等5种植物激素的分析方法。采用正交试验设计对高效毛细管电泳方法中的运行电压、缓冲液pH值和添加剂SDS浓度等分离条件进行优化,结果发现在30 mmol/L H3BO4-KH2PO4、40 mmol/LSDS组成的pH9.0缓冲液中,选择18 kV电压,25℃柱温和200 nm波长,可在11 min以内实现茶叶中5种激素的分离检测。本方法具有较高的灵敏度,5种激素的相关系数r=0.9907～0.9974,加标回收率为78.06%-95.5%,变异系数≤1.8%。利用本方法测定了茶叶不同部位的5种植物激素的含量变化。     

3-氰基吡啶水合酶的反应条件及影响因子

研究了芳腈水合酶催化水合3-氰基吡啶生成尼克酰胺的反应条件及影响因子.酶反应的最适pH为8.0,最适温度为25℃.酶在pH8.5于25℃保温4小时或在25—30℃于pH8.0保温3小时是稳定的.反应液中加入Fe~(3 )(1.5 mmol/L)可使酶活力增加 50%,而加入NH_4~ (300 mmol/L)则使酶活降低了67%.Ag~ 和 Hg(2 )”强烈地抑制酶反应活性,在浓度均为 5mmol/L时,抑制率分别为99.7%和100%.NaCN(50 mmol/L)和苯甲腈(100 mmol/L)对酶活性的抑制率分别为78%和85%.该酶作用于 3-氰基吡啶的Km为62.5 mmol/L,V_(max)为85.8 μmol·min~(-1)·mg~(-1).     

3-氰基吡啶水合酶的反应条件及影响因子

研究了芳腈水合酶催化水合3-氰基吡啶生成尼克酰胺的反应条件及影响因子.酶反应的最适pH为8.0,最适温度为25℃.酶在pH8.5于25℃保温4小时或在25—30℃于pH8.0保温3小时是稳定的.反应液中加入Fe~(3+)(1.5 mmol/L)可使酶活力增加 50%,而加入NH_4~+(300 mmol/L)则使酶活降低了67%.Ag~+和 Hg(2+)”强烈地抑制酶反应活性,在浓度均为 5mmol/L时,抑制率分别为99.7%和100%.NaCN(50 mmol/L)和苯甲腈(100 mmol/L)对酶活性的抑制率分别为78%和85%.该酶作用于 3-氰基吡啶的Km为62.5 mmol/L,V_(max)为85.8 μmol·min~(-1)·mg~(-1).     

发菜藻蓝蛋白分离纯化的研究

以发菜为材料,比较了提取液类型和饱和硫酸铵浓度对藻蓝蛋白提取的影响,并对藻蓝蛋白的提取程序和部分特性进行了研究。结果表明:50 mmol/L KP缓冲液(pH值7.2)是合适的提取液,体积分数为40%~50%饱和硫酸铵盐析效果优于其它浓度。经过DEAE-Toyopeal 650 S离子交换层析和SuperdexTM200凝胶过滤层析后,藻蓝蛋白纯度达6.2,最大吸收峰位于615 nm,荧光发射峰位于649 nm,由α和β2个亚基组成,其分子质量分别为18 051.17和19 142.27 Da。因此,发菜藻蓝蛋白分离纯化较为理想的程序为:藻粉→50 mmol/L KP缓冲液(pH值7.2)浸泡→French pressure(1 500 kg/cm2)破碎细胞→40%~50%饱和硫酸铵盐析→DEAE-Toyopeal 650 S离子交换层析→SuperdexTM200凝胶过滤层析→较纯的藻蓝蛋白。     

应用乙酰短杆菌酶法合成2'-脱氧腺苷

胸苷和腺嘌呤在乙酰短杆菌作用下转化成胸腺嘧啶和目标产物2’-脱氧腺苷。50mL的三角瓶装液10mL,40mmol/L的底物,50mmol/L的磷酸盐缓冲液(pH7)进行反应,5%游离乙酰短杆菌在55℃下,经1h转化率就可达到65.6%。     

螯虾腹屈肌肌原纤维副肌球蛋白和原肌球蛋白的分离和结晶

螯虾Procambarus clarki腹屈肌肌原纤维副肌球蛋白和原肌球蛋白已经分离和结晶。在离子强度近于0.3和pH 6.0的溶液中透析,螯虾副肌球蛋白类晶体析出,与原肌球蛋白可以彼此完全分离。经含200mmol KCl,10mmol磷酸缓冲液,pH 6.0溶液或者50mmol BaCl_2 50mmol Tris.HCl_1 pH7.8溶液透析,可以得到副肌球蛋白针状类晶体,周期为14.5或72.5nm。原肌球蛋白溶液经含2％饱和硫酸铵,10mmol醋酸缓冲液,pH 5.1之溶液透析,呈现针状或梭形结晶,具有39nm周期。     

在非光合条件下外源蔗糖能促进蚕豆的气孔开放

蚕豆植株经暗饥饿处理40h后,取其下表皮,再用超声波“原位分离”下表皮上的保卫细胞对,然后在无菌、非光合条件下,用外源蔗糖处理蚕豆下表皮上的保卫细胞对,考察其对气孔开放的效应。结果发现,在1d的无菌培养过程中,蔗糖显著促进了气孔的开放。100mmol/L的蔗糖在10mmol/L的MES-NaOH/KOH(pH6.1)缓冲液中,开度分别增加2.0/2.6μm;在1μmol/L的气孔开放促进剂fusicoccin(FC)的存在下,100mmol/L的蔗糖在MES-NaOH/KOH(pH6.1)缓冲液中分别增加开度5.0/5.5μm。不同浓度（5～200mmol/L）的蔗糖处理结果表明气孔开度的增加与蔗糖浓度呈一定的正相关,浓度为100mmol/L的蔗糖处理表现出最大促进作用。同时还初步观察到,蔗糖可维持保卫细胞存活率和叶绿体的完整性。     

3-甾酮-Δ1-脱氢酶突变体文库高通量筛选方法的建立

[目的]建立一种快速、高通量筛选3-甾酮-Δ¹-脱氢酶(KstD)突变体库的方法。[方法]基于已发表KstD蛋白晶体结构,设计饱和突变引物,构建突变体文库,优化蛋白表达条件,利用冻融法结合溶菌酶法获得粗酶液;基于分光光度法原理,优化检测反应体系,利用酶标仪检测吸光度的变化来反映酶活力。[结果]利用饱和突变引物成功构建突变体文库,表达KstD最佳条件为：IPTG终浓度0.3 mmol/L,20℃诱导20 h;缓冲液中溶菌酶浓度为0.5 mg/mL时上清目的蛋白含量最高。优化后的酶活检测条件为：Tris-HCl缓冲液50 mmol/L、pH 8.0、0.4 mmol/L DCPIP、1.5 mmol/L PMS、0.7 mmol/L雄烯二酮(4AD)和适量的酶液组成,温度30℃、酶反应时间5 min,于600 nm波长下测定。[结论]成功建立了一种5 min检测96个3-甾酮-Δ¹-脱氢酶(KstD)突变体的高通量筛选方法,重复性变异系数在3%以下。     

Enantioseparation of ofloxacin and its four related substances with ligand exchange–micellar electrokinetic chromatography using copper(II)‐L‐isoleucine complex as chiral selector

A ligand‐exchange micellar electrokinetic capillary electrophoresis system with copper(II)‐L‐isoleucine complexes as the chiral selector incorporated in micelles of sodium dodecyl sulfate (SDS) was developed for the enantioseparation of ofloxacin and its four related substances (impurities A, C, E, and F). The effects of important parameters affecting separation such as buffer pH, SDS concentration, chiral selector concentration, and organic additive were investigated in detail. Under optimum experimental conditions, enantioseparation of ofloxacin, impurities A, C, E, and F enantiomers was accomplished with resolutions of 4.28, 2.83, 3.40, 3.58, and 2.46, respectively. Further, simultaneous separation of impurities A, C, E, and F enantiomers was achieved using 10 mmol/L NH₄OAc as the running buffer containing 4 mmol/L copper sulfate,20 mmol/L L‐isoleucine, 20 mmol/L SDS, and 5% methanol at pH 8.5. To the best of our knowledge, the simultaneous enantioseparation of four impurities of ofloxacin has not been reported previously.     

小麦谷氨酸脱羧酶的纯化及部分性质研究

谷氨酸脱羧酶（ｇｌｕｔａｍａｔｅｄｅｃａｒｂｏｘｙｌａｓｅ,ＧＡＤ,ＥＣ４．１．１．１５）催化谷氨酸脱羧生成γ－氨基丁酸（γ－ａｍｉｎｏｂｕｔｙｒａｔｅ,ＢＡ）,植物中已从南瓜［１］、马铃薯和林生山黧豆［２］纯化了ＧＡＤ．ＧＡＤ活性在禾本科作物中作为...     

Determination of MDMA and MDA in rat urine by semi-micro column HPLC-fluorescence detection with DBD-F and their monitoring after MDMA administration to rat.

A simultaneous semi-micro column HPLC method with fluorescence detection of abused drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in rat urine was examined by using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-1,2,3-benzoxadiazole (DBD-F) as a labelling reagent and alpha-phenylethylamine as an internal standard (IS). A sample (50 microL) of rat urine was added to 5 microL IS and 100 microL 100 mmol/L borate buffer (pH 12) and extracted with 1.5 mL n-hexane. After evaporation, 50 microL 75 mmol/L borate buffer (pH 8.5) and 50 microL 20 mmol/L DBD-F in CH3CN were added to the residue and mixed well. The resultant solution was heated for 20 min at 80 degrees C and then cooled in an ice bath. A good separation of DBD-derivatives could be achieved within 45 min using a semi-micro ODS column with an eluent of CH3CN/CH3OH/10 mmol/L imidazole-HNO3 buffer (pH 7.0) (= 45:5:50, v/v/v %). The DBD derivatives were monitored at 565 nm with an excitation at 470 nm. The calibration curves showed good linearity (r = 0.997) with 0.5-15 ng/mL detection limits at a S/N ratio of 3. MDMA and MDA in rat urine could be monitored for 15 h after a single administration of MDMA to rat (2.0 mg/kg, i.p.). The concentrations for MDMA and MDA (n = 3) were 0.13-160.1 and 0.17-10.9 microg/mL, respectively.     

Stability of firefly luciferase in Tricine buffer and in a commercial enzyme stabilizer

A solution of firefly luciferase in AuthentiZyme Enzyme Stabilizer® retains full activity when stored in an ice bath (0.5°C) during one day. These solutions have the advantage that no additional protein (other than the luciferase) is present, which is desirable for proteolytic digestion and protein dervatization experiments. For longer-term experiments, firefly luciferase solutions in 0.05 mol/l Tricine buffer at pH 7.8, 10 mmol/l MgSO₄, 1 mmol/l EDTA, and 1 mmol/I DTT which contain 100μg/ml of bovine serum albumin are stable for 6 weeks if frozen and thawed only once.     

一种安全高效的血红蛋白纯化方法

目的:建立一种适用于大量制备的,安全、高效的血红蛋白纯化方法。方法: 将压积红细胞装入透析袋,以含有还原剂的Tris缓冲液透析破碎,破碎的上清经两级硫酸铵沉淀后透析至上样缓冲体系,离心后取上清即得血红蛋白提取液;红细胞提取液通过阴离子交换柱层析进一步分离,计算回收率。纯化产物浓缩后以SDS-PAGE及HPLC鉴定纯度,进行紫外-可见光谱扫描并以ABL800血气分析仪分析血气指标,以鲎试剂测定内毒素含量,以磷测定法测定脂质含量。结果: 血红蛋白提取液中脂质去除率98%,容易通过0.45μm滤膜;经阴离子交换层析纯化的血红蛋白经SDS-PAGE(银染法)及WB分析没有杂蛋白条带,HPLC分析纯度>99%、总回收率>85%;内毒素含量<2 EU,高铁血红蛋白含量<5%。结论: 该血红蛋白纯化方法安全高效、成本低廉、易于放大生产,具有较好的应用前景。     

Chemiluminescence determination of pharmacologically active compounds by capillary electrophoresis.

A simple and rapid capillary electrophoresis with direct chemiluminescence method has been developed for the determination of five natural pharmacologically active compounds including rutin, protocatechuic aldehyde, chlorogenic acid, luteolin and protocatechuic acid. The luminol as a component of the separation electrolyte buffer was introduced at the head of the separation capillary. The separation of five compounds was carried out in a fused-silica capillary with 15.0 mmol/L tetraborate, 1.0 mmol/L SDS and 0.42 mmol/L luminol (pH 8.5). The analytes was determined by enhancing the chemiluminescence of luminol with 0.13 mmol/L K3Fe(CN)6 in 0.05 mol/L NaOH, which was introduced at the post-column stage. The voltage applied was 16 kV. Under the optimum conditions, the analytes were separated within 10 min. The excellent linearity was obtained over two to three orders of magnitude with a detection limit (signal:noise = 3) of 0.012-0.055 micromol/L for all five analytes. The method was successfully used in the analysis of pharmaceutical and biological samples, and the assay results were satisfactory.     

类产碱假单胞菌谷氨酸脱氢酶的提纯、鉴定及某些特性的初步研究

从类产碱假单胞菌纯化出电泳纯的谷氨酸脱氢酶,用聚丙烯酰胺梯度凝胶电泳和ＳＤＳ－聚丙烯酰胺凝胶电泳测得分子量为２９０ ｋＤ,亚基分子量为４７ ｋＤ,提示该酶为六聚体．该酶对ＮＡＤＰ（Ｈ）和底物均具有高度专一性,对谷氨酸、α－酮戊二酸及ＮＡＤＰ＋ 的Ｋｍ 值分别为：２８ ｍ ｍ ｏｌ／Ｌ、１．２ｍ ｍ ｏｌ／Ｌ及０．０６３ ｍ ｍ ｏｌ／Ｌ．用Ｈｉｌｌ作图法求得酶对ＮＨ＋４ 和ＮＡＤＰＨ 的［Ｓ］０．５分别为２４ ｍ ｍ ｏｌ／Ｌ和０．０３７ ｍ ｍ ｏｌ／Ｌ．最适反应温度为５０℃,催化氨化反应和脱氨反应的最适ｐＨ 分别为８．０和８．８,在热稳定性方面不及嗜热细菌的谷氨酸脱氢酶稳定．提纯的谷氨酸脱氢酶在低温（４℃）条件下,可在Ｔｒｉｓ－ＨＣｌ缓冲液中贮存半年以上,活力无明显下降,冷冻则可导致纯酶液迅速失活．氮源对菌体谷氨酸脱氢酶水平有显著影响．     

Acid dissociation constants and pH values for standard "bes" and "tricine" buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and -25 degrees C

Information is given concerning two standard buffer solutions suitable as pH references in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H2O mixed solvents at subzero temperatures from -20 to 0 degrees C, with the intention of establishing a pH (designated pH*) scale. The two buffers selected were the ampholytes N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid ("bes") and N-tris(hydroxymethyl)methylglycine ("tricine"), and the reference standard consisted of equal molal quantities of the buffer and its respective sodium salt. The assignment of pH* values was based on measurements of the emf of cells without liquid junction of the type: Pt;H2(g,l atm) /Bes, Na Besate, NaCl / AgCl;Ag and Pt;H2(g,l atm) /Tricine, Na Tricinate, NaCl /AgCl;Ag and the pH* was derived from a determination of K2, the equilibrium constant for the dissociation process (Buffer)+/- in equilibrium with(Buffer)- + H+.     

胶束动电毛细管色谱法测定功能运动饮料中的水溶性维生素

目前,在功能运动饮料中测定水溶性维生素含量的研究相对较少,为此,本研究开发了一种通过胶束动电毛细管色谱法(MEKC)测定几种能量饮料和运动饮料中水溶性维生素的方法,并根据背景电解质组成(硼酸盐含量, p H值,表面活性剂类型等)和其他MEKC参数研究维生素的分离,以及对可能干扰维生素测定的干扰化合物进行了研究。研究显示,背景电解质为pH值8.5,50 mmol/L硼酸盐,50 mmol/L SDS和5%MeOH时,可有效地检测出能量和运动饮料中的水溶性维生素含量。该方法可成功地检测各种能量和运动饮料中的水溶性维生素,并可用于软饮料行业的质量控制。     

粘虫中肠α-淀粉酶活性的敏感性研究

研究了不同酶反应缓冲体系、pH值、氯离子浓度以及噁唑哒嗪对5龄2日粘虫 Pseudaletia separata Walker 中肠α－淀粉酶活性的影响。结果表明,乙酸-乙酸钠缓冲体系（pH 5.8）和磷酸氢二钠-磷酸二氢钠缓冲体系（pH 8.0）有利于增强α-淀粉酶活性,比活力最高分别达到4.49和4.97。在乙酸-乙酸钠缓冲体系（pH 5.8）中,5、10、20、40和80 mmol/L氯离子浓度引起α-淀粉酶活性呈现先减弱后增强的变化规律,而在磷酸氢二钠-磷酸二氢钠缓冲体系（pH 8.0）中仅呈现减弱的趋势。1.4 mmol/L噁唑哒嗪对α-淀粉酶活性的抑制率可达70%,但抑制程度随着反应体系中蛋白含量的增加而逐渐降低。