Structural and functional model of methane hydroxylase of membrane-bound methane monooxygenase from Methylococcus capsulatus (Bath)

Computer analysis of a wide range of primary sequences showed that -, -, and -peptides of membrane-bound methane hydroxylase contained 2, 7, and 6 transmembrane helices respectively. Conservative amino acid residues participating in complex formation were revealed. The - and -peptides are suggested to contain mononuclear copper ions with the ligand environment mainly consisting of His residues. The Cu sites are located in the hydrophilic region and are responsible for ESR signals. The active site of -peptide in which the activation of O₂ and oxidation of CH₄ occur is localized in the hydrophobic region close to the membrane surface. This site is formed by the amino acid residues of four transmembrane helices and one loop between them and is suggested to be a binuclear Cu—Fe or Fe—Fe center. The Cu site of -peptide transfers electrons to the active site of -peptide, and the Cu site of -peptide is either involved in this process or only stabilizes the protein structure.     

Improved Resolution of Hydrophobic Penicillin-binding Proteins and Their Covalently Linked Complexes on a Modified C18 Reversed Phase Column

ABSTRACT

Hydrophobic proteins have proven to be difficult to separate chromatographically. The use of a C18 reversed phase column with embedded polar groups showed improved separation and recovery of the hydrophobic, membrane associated, penicillin-binding proteins of Bacillus subtilis and their covalently linked complexes. The covalently linked complexes were generated by a time course reaction of the non-covalently associated complexes in the intact cell with the paired group specific reagent, cyanogen (C₂N₂).     

细胞色素b5-蛋白质相互作用研究

蛋白质-蛋白质相互作用在生命过程中发挥至关重要的作用,特别是血红素类蛋白。细胞色素b5(Cyt b5)是血红素蛋白的一个典型代表,在生物体内通过多种蛋白质-蛋白质相互作用来执行其生物功能。目前所揭示的与Cyt b5相关的蛋白质相互作用包括:细胞色素b5-细胞色素b5还原酶,细胞色素b5-细胞色素P450,细胞色素b5-细胞色素c,细胞色素b5-肌红蛋白或血红蛋白,细胞色素b5-融合蛋白(谷胱甘肽S-转移酶GST和绿色荧光蛋白GFP)和细胞色素b5-转运蛋白(蔗糖转运蛋白SUT1和山梨醇转运蛋白SOT6)等。同一蛋白能与众多不同蛋白相互作用的事实,使我们认识到某些特定蛋白的生物学重要性。另一方面,研究同一蛋白与不同蛋白质间的相互作用将会进一步加深我们对蛋白质结构与功能关系的理解,以及指导新颖蛋白的理性设计与最终应用。     

蒸发器支撑板板孔精密测量系统

建立了蒸发器支撑板板孔精密测量系统,并提出了基于边缘约束条件的轮廓参量测量方法.首先采用图像处理技术将待测目标转化为二维离散坐标点,计算其最小外接矩形并对轮廓进行预定位;然后将轮廓分割成相互重合的扩展邻域轮廓,建立以曲率角为原则的边缘约束算法对各轮廓段精确定界,实现对轮廓参量的精密测量.实验和误差分析表明,该系统测量准确度优于0.02mm,对具有复杂轮廓的零件参量测量有参考价值.     

固载蛋白A交联聚甲基丙烯酸缩水甘油酯连续床亲合色谱柱的制备及性能考察

采用沉淀聚法“原位”（in-situ)聚合合成了交联聚甲基丙烯酸缩水甘油酯连续床色谱柱,对其进行化学改性后,分别得到含有11个碳原子间隔臂以及不含间隔臂键合了蛋白A的高效亲合色谱柱。考察了这两种色谱介质的性能,结果表明含有间隔臂的介质有一定的疏水性;对传统制备蛋白配基的亲合色谱介质的合成路线进行了改进;采用不含间隔臂的亲合柱测定了人血浆中人免疫球蛋白G（HIgG)的含量,所测得的定量标准曲线线性相关系数达到0．999;考察了流速对合成的连续床色谱柱柱压的影响,当流速高达9．0mL/min时,柱压也仅为6．5MPa;采用pH梯度对HIgG的不同亚基进行了分离。     

应用近红外技术测定黄豆粕中水分、蛋白质和粗脂肪

应用近红外分析仪（NIR）快速测定黄豆粕中水分、蛋白质和粗脂肪的含量,通过光谱扫描统计分析,分别找出测量黄豆粕水分、蛋白质、粗脂肪的波长及校准常数值,经数理统计分析结果表明：近红外法与常规水分测定法经典凯氏定氮法、索氏浸提法所测定的结果呈密切的线性相关,检测结果令人满意。     

Elimination profiles of flurbiprofen and its metabolites in equine urine for doping analysis

Flurbiprofen and its main acidic metabolites were detected in equine urine after a single-dose administration of 500 mg flurbiprofen to two 2.5–3.5-years-old mares, in order to be used in equine doping control routine analysis. The urine levels of the parent drug were determined using GC/MS. Five acidic metabolites were found in the urine. The structure of the proposed metabolites was confirmed by HRMS accurate mass measurements. The highest flurbiprofen concentration was 204 μg ml⁻¹ at 1–3 h post administration. Flurbiprofen could be detected for 24–37 h in urine using the standard screening procedure. All metabolites were present 25 h post administration, while 4′-hydroxyflurbiprofen could be traced for more than 48 h and it is regarded as the long-term metabolite of flurbiprofen in horse.     

Lipophilicity in PK design: methyl, ethyl, futile

Lipophilicity, often expressed as distribution coefficients (log D) in octanol/water, is an important physicochemical parameter influencing processes such as oral absorption, brain uptake and various pharmacokinetic (PK) properties. Increasing log D values increases oral absorption, plasma protein binding and volume of distribution. However, more lipophilic compounds also become more vulnerable to P450 metabolism, leading to higher clearance. Molecular size and hydrogen bonding capacity are two other properties often considered as important for membrane permeation and pharmacokinetics. Interrelationships among these physicochemical properties are discussed. Increasing size (molecular weight) often gives higher potency, but inevitably also leads to either higher lipophilicity, and hence poorer dissolution/solubility, or to more hydrogen bonding capacity, which limits oral absorption. Differences in optimal properties between gastrointestinal absorption and uptake into the brain are addressed. Special attention is given to the desired lipophilicity of CNS drugs. In examples using -blockers, Ca channel antagonists and peptidic renin inhibitors we will demonstrate how potency and pharmacokinetic properties need to be balanced.     

The search for a new model structure of β-Factor XIIa

We present the search for a new model of -factor XIIa, a blood coagulation enzyme, with an unknown experimental 3D-structure. We decided to build not one but three different models using different homologous proteins as well as different techniques and different modellers. Additional studies, including extensive molecular dynamics simulations on the solvated state, allowed us to draw several conclusions concerning homology modelling, in general, and -factor XIIa, in particular.     

吸附蛋白质固定相电色谱手性分离的研究

 将牛血清白蛋白 (BSA)吸附于强阴离子交换固定相 (SAX)上用于电色谱手性分离。当SAX吸附BSA后 ,电渗淌度仅仅下降 2 6 3% ,而电渗流的方向没有改变。在该系统中电渗流的方向和阴离子的电泳方向一致 ,因而克服了一般蛋白质固定相不能分离酸性对映体的缺点。 1种中性对映体安息香和 4种阴离子性对映体色氨酸、华法令、非诺洛芬、酮基布洛芬获得了成功分离。当流动相含体积分数为 7%的乙腈时 ,死时间和D 色氨酸、L 色氨酸的迁移时间的相对标准偏差分别为 0 90 % ,0 87%和 0 96 % (n =2 1) ,说明该体系有很好的重现性。