The influence of axial ligands on the reduction potential of blue copper proteins

 The reduction potentials of blue copper sites vary between 180 and about 1000 mV. It has been suggested that the reason for this variation is that the proteins constrain the distance between the copper ion and its axial ligands to different values. We have tested this suggestion by performing density functional B3LYP calculations on realistic models of the blue copper proteins, including solvent effects by the polarizable continuum method. Constraining the Cu-S_Met bond length to values between 245 and 310 pm (the range encountered in crystal structures) change the reduction potential by less than 70 mV. Similarly, we have studied five typical blue copper proteins spanning the whole range of reduction potentials: stellacyanin, plastocyanin, azurin, rusticyanin, and ceruloplasmin. These studies included the methionine (or glutamine) ligand as well as the back-bone carbonyl oxygen group that is a ligand in azurin and is found at larger distances in the other proteins. The active-site models of these proteins show a variation in the reduction potential of about 140 mV, i.e., only a minor part of the range observed experimentally (800 mV). Consequently, we can conclude that the axial ligands have a small influence on the reduction potentials of the blue copper proteins. Instead, the large variation in the reduction potentials seems to arise mainly from variations in the solvent accessibility of the copper site and in the orientation of protein dipoles around the copper site. Received: 7 April 1999 / Accepted: 26 July 1999     

Antibody variable region binding by Staphylococcal protein A: thermodynamic analysis and location of the Fv binding site on E-domain.

Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (V(H)), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3-4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5+/-0.5 x 10(5) M(-1). A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0+/-0.1, Ka = 2.0+/-0.3 x 10(5) M(-1), and deltaH = -7.1+/-0.4 kcal mol(-1). Similar binding thermodynamics are obtained for titration of the isolated V(H) domain with E-domain indicating that the E-domain binding site on Fab resides within V(H). E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2+/-0.1, Ka > 1.0 x 10(7) M(-1), and deltaH = -24.6+/-0.6 kcal mol(-1). Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the V(H) domain implicated previously in protein A binding.     

Statistical Analysis of Chemical Release Rates from Soils

Two statistical methods for determining the precision of best-fit model parameters generated from chemical rate of release data are discussed. One method uses the likelihood theory to estimate marginal confidence intervals and joint confidence regions of the release model parameters. The other method uses Monte Carlo simulation to estimate statistical inferences for the release model parameters. Both methods were applied to a set of rate of release data that was generated using a field soil. The results of this evaluation indicate that the precision of F (the fraction of a chemical in a soil that is released quickly) is greater than the precision of k₁ (the rate constant describing fast release), which is greater than the precision of k₂ (the rate constant describing slow release). This occurs because more data are taken during the time period described by F and k₁ than during the time period described by F and k₂. In general, estimates of F will be relatively precise when the ratio of k₁ to k₂ is large, estimates of k₁ for soil/chemical matrices with a high F will be relatively precise, and estimates of k₂ for soil/chemical matrices with a low F will be relatively precise, provided that sufficient time is allowed for full release.     

海洋生理活性物质的研究及发展趋势

本文概述海洋生理活性物质特有的特异结构和药理作用,对研究治疗心脑血管疾病、癌症和艾滋病等方面的意义及发展前景。阐述我国近年来从海洋生物活性物质中发现如三丙酮胺、喹啉酮、柳珊瑚酸及其衍生物等观种显著生理活性新化合物及１６种海洋生理活性物质;８种已投入市场的海洋药物及保健品。提出对海洋生理活性物质研制成新药的可行途径及应采取的决策和有效措施;提出应用如基因工程、细胞工程、发酵工程和酶学工程等生物技术来研制、生产出纯度高、质量高、成本低的海洋药物资源物质。海洋生物技术领域的迅速发展,对海洋生物活性物质的研究及开发海洋药物具有广阔的应用前景。     

黄伞菌丝蛋白质营养价值评价

本文测定了黄伞Pholiotaadiposa菌丝粗蛋白含量和氨基酸组成,采用国际上通用的营养价值评价方法,对其蛋白质的营养价值进行全面评价,并与营养价值较高的白灵侧耳Pleurotusnebrodensis、刺芹侧耳Pleurotuseryngii和金顶侧耳Pleurotrscitrinopileatus进行比较。分析结果表明,黄伞菌丝的必需氨基酸含量最高,占其氨基酸总量的43.9%,蛋白质的氨基酸评分(AAS)、化学评分(CS)、必需氨基酸指数(EAAI)、生物价(BV)、营养指数(NI)和氨基酸比值系数分(SRCAA)分别为92.0、71.3、88.5、84.8、40.4和78.8,六项指标均比参比食用菌高。结果说明黄伞菌丝具有很高的营养价值。     

Site-Directed Mutagenesis Evidence for Arginine-384 Residue at the Active Site of Maize Branching Enzyme II

Essential arginine residues are suggested to be located at the active sites of maize branching enzymes (BE) based on the evidence that two arginine residues are conserved in all BE from various species and that as little as one arginine residue is located at the active site of maize BE by phenylglyoxal (PGO) modification. To determine the exact location of the active-site arginine residue in BE, we employed peptide mapping and site-directed mutagenesis approaches. A single trypsin-digested, [¹⁴C]PGO-labeled peptide was purified from maize BEII by two rounds of HPLC separation, but we failed to obtain amino acid sequencing information. Site-directed mutagenesis was then used to create one mutant (arginine-384 to alanine-384), R384A. Immunoblotting result showed that BEII protein was expressed at a similar level in the wild type and the R384A mutant. However, BE activity in the R384A mutant was only 1.4% of the wild type. These results support the conclusion that the conserved arginine-384 residue is important in BEII catalysis.     

川明参根部的化学成分

从我国特有的单种属药用植物川明参（Ｃｈｕａｎｍｉｎｓｈｅｎ ｖｉｏｌａｃｅｕｍ　 Ｓｈｅｈ ｅｔ Ｓｈａｎ）中分离出８个晶体,经理化性质和波谱分析分别鉴定为：５,８－二甲氧基补骨脂素（晶Ⅰ）,５－异戊烯基－８甲氧基补骨脂素（晶Ⅱ）, ｐｒｏｃｙａｎｉｄｉｎ Ａ－２（晶Ⅲ）,槲皮素－３－Ｏ－葡萄糖醛酸甙（晶Ⅳ）,芦丁（晶Ⅴ）,豆甾醇（晶Ⅵ）,豆甾醇－葡萄糖甙（晶Ⅶ）及棕榈酸,硬脂酸的混合物（晶Ⅷ）。上述化合物均为首次从该植物中分离得到。     

紫杉醇生物合成的研究

抗癌新药紫杉醇是具有萜类环状结构的天然次生代谢产物。研究紫杉醇的生物合成对于人为定向地提高合成效率以及克隆重组合成中的关键酶基因,进而提高紫杉醇的合成量,都是十分有意义的基础工作。本文以植物和微生物的次生代谢及其主要代谢途径为知识背景,介绍了紫杉烷类物质的基本结构及其结构骨架的分类情况,综述了近年来围绕紫杉醇二萜骨架和侧链基团的生物合成途径的研究进展,对合成中的影响因素进行了初步的讨论。     

短茎古当归根的化学成分

从短茎古当归〔Ａｒｃｈａｎｇｅｌｉｃａｂｒｅｖｉｃａｕｌｉｓ（Ｒｕｐｒ．）Ｒｃｈｂ．〕根中分得６个结晶,采用ＩＲ、ＭＳ、ＮＭＲ等方法,鉴定了４个化合物,即甲氧基欧芹素（蛇床子内酯,ｏｓｔｈｏｌ,Ⅰ）、欧前胡素（ｉｍｐｅｒａｔｏｒｉｎ,Ⅱ）、古当归素（ａｒｃｈａｎｇｅｌｉｃｉｎ,Ⅳ）、二十八烷酸（ｏｃｔａｃｏｓａｎｉｃａｃｉｄ,Ⅴ）。结晶Ⅳ和Ⅴ为首次从该植物中分出。古当归素为生物合成中最高阶段产物,是本种的特征性成分     

栗链蚧生物学特性及其防治试验

栗链蚧Asterolecanium castaneae Russell是危害锥粟的主要蚧虫之一。栗链蚧在福建一年发生2代,以受精的雌虫在栗树枝条上越冬。防治试验表明,选用50%快灭磷1：1000进行喷雾．原液进行涂干或注射效果最好．40%氧化乐果次之。农药混合试验表明．50％快灭磷 40%氧化乐果 80％敌敌畏原液混合进行涂干效果最好。