Roles of Phe58 residue in stabilizing structure of cytochrome b<Subscript>5</Subscript>

To understand effect of π-stacking interactions between the side chain of aromatic amino acids and the porphyrin ring on structures and properties in cytochrome b₅ (cyt b₅), the Phe58 residue was mutated to tyrosine and tryptophan, respectively by site-directed mutagenesis. The denaturation of cyt b₅ F58W and F58Y toward guanidine hydrochloride was examined by UV-visible and fluorescence spectroscopy. The kinetics of heme transfer reactions between apo-myoglobin and the mutants were studied. The results indicated that the mutation of F58 residue for Y58 or W58 reduced the interaction between of peptide and the heme group, resulting in decrease of the T _m and C _m values of the proteins, increase of the heme transfer reaction rate, and shifts of the redox potential.     

Study on the DME-cytochrome<Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> and its mutants at site of F58

Phenylalanine-58 is one of the conservative residues in the hydrophobic pocket of Cyt bs, which forms aromatic stacking with the heme b. Previous study showed that both the stacking and the property of the aromatic residue affect hydrophobicity of the heme pocket, leading to change of protein‘s property. In order to further reveal the essence we esterify the heme propionate of Cyt bs, F58Y and F58W, and eliminate the hydrogen bond between heme propionate and Ser64 in examining the effect of hydrogen net on the π-π interaction. In this paper thermal denaturation of DME-Cyt b5 and its F58Y and F58W mutants has been studied by UV-visible and CD spectra. The heme transfer reactions between these proteins and apo-myoglobin have been studied as well. The results demonstrate that esterification did not destroy the aromatic stacking; however, it affects the stability of the proteins due to different volumes, hydropho-bicities and hydrogen bonds forming ability of these substituents.     

人工脱色过氧化物酶催化降解苋菜红活性研究

以肌红蛋白(Mb)为骨架构建并纯化了三突变体蛋白F43Y/F138W/P88W Mb,双突变体蛋白F43Y/F138W Mb及单突变体蛋白 F43Y Mb,并对三个突变体蛋白的苋菜红(amaranth)脱色降解活性进行了研究。催化活性及酶动力学研究结果显示,相比于F43Y/F138W Mb和F43Y Mb,三突变体F43Y/F138W/P88W Mb的苋菜红催化效率最高,这表明色氨酸对人工脱色过氧化物酶的活性起了重要作用。该研究不仅为今后的血红素蛋白设计提供了理论依据,同时也表明人工酶在染料废水处理方面具有潜在的应用价值。     

Electrostatic effect on electron transfer between cytochrome b5 and cytochrome c

The binding and electron transfer between wild type, E44A, E56A, E44/56A, E44/48/56A/D60Aand F35Y variants of cytochrome b5 and cytochrome c were studied. When mixed with cytochrome c, the cytochrome b5E44/48/56A/D60A did not show the typical UV-vis difference spectrum of absorption, indicating that the alteration ofthe surface electrostatic potential obviously influenced the spectrum. The electron transfer rates of wild type cytochromeb5, its variants and cytochrome e at different temperature and ionic strength exhibited an order of F35Y > wild type >E56A > E44A > E44/48/56A/D60A. The enthalpy and entropy of the reaction did not change obviously, suggestingthat the mutation did not significantly disturb the electron transfer conformation. The investigation of electron transfer rateconstants at different ionic strength demonstrated that electrostatic interaction obviously affected the electron transfer pro-cess. The significant difference of Cyt b5 F35Y and E44/48/56A/D60A from the wild type protein further confirmed thegreat importance of the electrostatic interaction in the protein electron transfer.     

Electrostatic effect on electron transfer between cytochrome b5 and cytochrome c

The binding and electron transfer between wild type, E44A, E56A, E44/56A, E44/48/56A/D60A and F35Y variants of cytochrome b5 and cytochrome c were studied. When mixed with cytochrome c, the cytochrome b, E44/48/56A/D60A did not show the typical UV-vis difference spectrum of absorption, indicating that the alteration of the surface electrostatic potential obviously influenced the spectrum. The electron transfer rates of wild type cytochrome bj, its variants and cytochrome c at different temperature and ionic strength exhibited an order of F35Y > wild type > E56A > E44A > E44/48/56A/D60A. The enthalpy and entropy of the reaction did not change obviously, suggesting that the mutation did not significantly disturb the electron transfer conformation. The investigation of electron transfer rate constants at different ionic strength demonstrated that electrostatic interaction obviously affected the electron transfer process. The significant difference of Cyt b, F35Y and E44/48/56A/D60A from the wild type protein further confirmed the great importance of the electrostatic interaction in the protein electron transfer.     

Preparation and characterization of some surface negatively charged residue mutants of cytochrome b5

Site-directed mutagenesis was used to obtain seven variants of tryptic fragment of bovine liver cytochrome b_m5 (cyt b₅), in which the negatively charged residues around the heme exposed edge of cyt b₅ were replaced by hydrophobic amino acid alanine. Double-site mutants, triple-site mutants and even quadruple-site mutants were obtained. DNA sequencing and molecular weight measurements of the mutant proteins both confirmed that these site-directed mutagenesises were successfully performed. Spectroelectrochemistry of these mutant proteins revealed that the apparent redox potentials of these mutant proteins caused a positive shift of 2–10 mV. The global structure of these mutant proteins did not show much difference from that of the wild type cyt b₅, providing a solid base for the further study on the roles of the proteins’ surface charges.     

谷氨酸棒杆菌(Corynebacterium glutamicum)海藻糖合成酶的定点突变及其酶学性质研究

以已知晶体结构的Pseudomonas mesoacidophila MX-45菌株海藻酮糖合成酶（MutB）的晶体结构为模板,在SWISS-MODEL模建立谷氨酸棒杆菌（Corynebacterium glutamicum）海藻糖合成酶的立体结构,并对初始结构作能量优化,通过氨基酸序列比对,选择TreS-glu保守区内的氨基酸R245、D247、E289、F244和保守区外的氨基酸A288进行定点突变,并对突变酶F244C、F244L、F244W、F244Y、A288G、R245X、E289X、D247N、D247E进行纯化和酶学性质研究,比较突变子对酶活性和热稳定性的影响。结果表明,R245、E289突变为其它的19个氨基酸后酶活力全部丧失,D247E和D247N也丧失酶活,F244C、F244L、F244W、F244Y和A288G的比活力分别降低到TreS-glu的38%、24%、62%、64%和35%,A288突变成T288后没有酶活。与TreS-glu相比,F244C、F244W、A288G的Km值基本不变,F244L、F244Y对底物麦芽糖的亲和力降低,F244Y的最适反应温度和TreS-glu相同,均为27℃,而F244C、F244L、F244W和A288G的最适温度提高到32℃。与TreS-glu相比,突变酶的最适反应pH值均有所下降,其中F244C、F244Y和A288G的为7.5,比TreS-glu的8.0均下降了约0.5个单位,而F244L和F244W的为6.5,比TreS-glu的8.0均下降了近1.5个单位。与TreS-glu相比,突变酶的热稳定性均有不同程度提高,其中F244Y、F244W和A288G的Tm值比TreS-glu的提高约1℃,F244L提高约2℃,F244C提高了近4℃。     

Eu3+掺杂钨酸钇体系荧光粉的制备及发光性能分析

氧化钇(Y2O3)、 氧化铕(Eu2O3)与三氧化钨(WO3)为原材料,通过调整Y2O3(Eu2O3)与WO3的摩尔比例,采用高温固相法制备钨酸钇体系红色荧光粉,通过X射线衍射(XRD)、扫描电子显微镜(SEM)、光致发光(PL)等表征分析样品的晶相结构、形貌尺寸和光致发光性质。研究结果表明:当Y2O3与WO3的摩尔比例为1∶1和1∶3时,可分别合成纯相的Y2WO6:Eu3+红色荧光粉和Y2W3O12:Eu3+红色荧光粉;该系列红色荧光粉可被近紫外光和蓝光有效激发,发射峰值位于615 nm(Eu3+离子的5D0→7F2跃迁)的红光;Y2WO6:Eu3+红色荧光粉的相对发光强度明显优于Y2W3O12:Eu3+红色荧光粉;Y2WO6:Eu3+红色荧光粉Eu3+的最佳掺杂浓度(摩尔分数)为5%。     

G7代二龄泥蚶生长性状的相关性和通径分析

为了探讨连续选育多代以后泥蚶形态性状对全重和软体部重的影响,采用相关分析、通径分析对330个2龄“乐清湾1号”泥蚶的壳长L、壳高H、壳宽W、全重Y1和软体部重Y2等5个性状指标进行分析,并建立形态性状对全重、软体部重的最优回归方程。结果显示:1）所测5个数量性状之间的相关系数均达到极显著水平（P＜0.01）,壳宽与全重的相关系数最大（0.952）,壳长与软体部重的相关系数最大（0.928）。2）通径分析结果显示,壳宽对全重的直接影响最大（0.479）,是影响全重的主要因素;壳长和壳高主要通过壳宽间接影响全重,是影响全重的次要因素;对软体部重的直接影响最大的是壳长（0.415）,其次是壳宽（0.390）,两者是影响软体部重的主要因素。3）用多元回归分析方法建立壳长、壳高、壳宽估计全重和软体部重的最优回归方程:Y1=-18.798+0.265L+0.294H+0.646W;Y2=-7.194+0.143L+0.088H+0.203W。     

绒螯蟹线粒体细胞色素b基因的RFLP分析

对中国大陆4个水系的8个地理种群绒螯蟹样本的线粒体细胞色素b基因片段进行了PCR扩增，并应用6种限制性内切酶对该PCR产物进行RFLP分析．在应用的6种内切酶中，HinfⅠ，RsaⅠ和EcoRⅤ酶切该PCR片段后，在某些地区绒螯蟹之间表现出限制性片段长度多态性，为多态性的内切酶，但在本研究中尚未发现种群特异的RFLP标记．应用3种多态性的限制性内切酶对8个地理种群的绒鳌蟹个体样本的线粒体细胞色素b基因片段进行RFLP分析，共检测到5种复合限制性酶切类型，即AAA型、BBB型、ABA型、BAB型、ACA型．依据群体间的净遗传距离绘制的UPGMA分子系统树显示，合浦绒螯蟹形成了独立的一支．     

铀酰离子-细胞色素b_5复合物的分子动力学模拟

铀酰离子(UO_2~(2+))的生物学毒性是由于它能与蛋白质形成稳定的复合物而破坏了蛋白的正常功能。通过使用分子建模和分子动力学(molecular dynamics,MD)模拟,对铀酰离子-细胞色素b_5复合物(UO_2~(2+)-cyt b_5)进行了结构预测。模拟结构显示,UO_2~(2+)通过配位和氢键作用结合到Cyt b_5中谷氨酸Glu37和Glu43,导致Cyt b_5的动力学行为发生轻微改变,而对蛋白整体结构几乎不产生影响。这些现象与Cyt b_5的体内形成以及作为电子传递蛋白的生物功能紧密相联。这些原子水平上理论信息的获得,为铀酰离子的毒性机制,尤其是涉及Cyt b_5的细胞凋亡,提供了深刻内涵。     

Influence of site-directed mutation of cytochrome b5 Phe35 on the protein’s structure and properties

Kinetic studies of the heme dissociation from the wild type and Phe35Tyr, Phe35Leu mutants of bovine liver microsomal ferricytochrome b 5 indicate that the oxidized Phe35Tyr mutant is more stable towards denaturant than wild type but Phe35Leu mutant proceeded with a different mechanism compared with wild type cytochrome b 5 and Phe35Tyr mutant protein. Because of the decrease of side chain volume in Phe35Leu mutant, a cavity produced in the interior of the protein may offer a channel for urea molecule to enter the hydrophobic pocket. When urea concentration is larger than 5 mol/L, the urea molecule may compete to coordinate the iron of heme with His39, that results in sharp increase of the rate of heme dissociation. The interaction between cytochrome b 5 and cytochrom c demonstrated that a 1:1 protein complex was formed between the two proteins. The binding constants of cytochrome b 5 with cytochrome c are: wild type K A=4.2(±0.01)×10 6(mol/L) -1 , Phe35Tyr K A=3.7(±0.01)×10 6(mol/L) -1 and Phe35Leu K A=4.7(±0.01)×10 6(mol/L) -1 respectively ( I =1 m mol/L, pH 7.0 soldium phosphate buffer, 25℃). These results clearly show that the mutation at Phe35 has no influence on the binding of cytochrome b 5 with cytochrome c and that the hydrophilic patch residues are not involved in the binding of cytochrome b 5 and cytochrome c.     

细胞色素C修饰电极的制备和性能研究

通过自组装法,把血红蛋白细胞色素C固定于导电聚合物(聚谷氨酸)层,制作成一种微型生物传感器,检测其对过氧化氢和亚硝酸钠的响应.实验发现谷氨酸具有一定的电化学活性,循环伏安图在-0.35 V和-0.05 V出现一对氧化还原峰,而且细胞色素C修饰电极对亚硝酸钠响应所得循环伏安图表明,随着亚硝酸钠加入量增大,还原峰增大.通过吸附法修饰电极,把血红蛋白细胞色素C固定于电极表面,探究修饰电极在对苯二胺中的钝化情况.实验表明:细胞色素C修饰电极可以避免电极钝化的发生.细胞色素C作为一种蛋白酶,使对苯二胺处于氧化状态,从而防止电极表面膜的形成,避免电极钝化的发生.     

Y3Al5O12基质中Dy3+的浓度猝灭机制

采用溶胶-凝胶/燃烧合成法制备不同掺杂浓度的Dy:Y3Al5O12(YAG)发光粉体。分析基质晶体结构、Dy3+掺量对Dy3+光致发光性能的影响,并探讨Dy3+在Y3Al5O12基质中的自身浓度猝灭机制。根据激发光谱,Dy:YAG的主激发峰位置在353 nm,对应Dy3+的6H15/2→6P7/2跃迁。在Dy:YAG晶体结构中,Dy3+取代Y3+的位置具有D2对称性,故Dy:YAG的蓝光发射强度要高于黄光发射强度,且Dy3+最佳摩尔分数为0.02;Dy3+的4F9/2→6H15/2、6H13/2跃迁发射的浓度猝灭机制均为相邻中心的电偶极-电偶极相互作用引起的交叉弛豫(4F9/2+6H15/2→6H9/2+6F3/2)所造成的。     

邻、间、对甲基苯甲酸铽及钇掺杂配合物的合成与荧光性能

合成了6种以邻、间、对甲基苯甲酸(o-,m-,p-MBA)为第1配体,1,10-邻菲口罗啉(phen)为第2配体的铽及钇掺杂铽的三元和四元配合物,通过元素分析和EDTA滴定确定了它们的组成.室温下记录了这6种配合物的荧光光谱,比较了第1配体的改变对配合物荧光强度的影响及非荧光稀土离子Y3+与配合物中的发光离子Tb3+之间的相互作用.结果表明:3种三元配合物和3种四元配合物均具有优良的光致发光性能,各配合物的相应跃迁发射峰位基本上没有变化.3种三元纯铽配合物在489 nm和545 nm处的荧光发射强度均按邻、间、对位的顺序依次增强;3种四元掺杂配合物中掺杂离子Y3+对配合物中Tb(Ⅲ)在489和545 nm两处的强发射均表现为荧光敏化作用.     

不同泥炭替代基质对薄壳山核桃幼苗生长及叶绿素荧光特性的影响

【目的】探讨添加不同配比的泥炭替代基质如菌渣或醋糟等组成的混合基质处理对薄壳山核桃(Carya illinoinensis)容器苗生长以及叶绿素荧光特性的影响,筛选适宜苗木生长的低成本配方基质。【方法】以薄壳山核桃‘绍兴’子代1年生容器苗为材料,设置园土、泥炭、醋糟体积比为4:3:3(A1)、4:2:4(A2)、4:1:5(A3)和园土、泥炭、菌渣体积比为4:3:3(B1)、4:2:4(B2)、4:1:5(B3)配方,园土为对照(CK),共7个处理,分别测定苗高、地径和叶绿素荧光参数等指标,分析不同配比的基质处理下对薄壳山核桃容器苗生长与荧光特性的影响。【结果】不同配比的泥炭替代基质处理下苗木生长指标差异显著,添加菌渣、醋糟较园土均可降低基质容重,提高基质的通气孔隙度,增加有机质含量等,其中添加菌渣的基质配方有利于薄壳山核桃幼苗的生长,且以B3处理效果最佳。该处理苗木的苗高、地径以及叶片各项叶绿素荧光参数均显著高于对照,其中苗高、地径分别比对照增加了65%和73%。【结论】综合苗木的各项指标并考虑各项成本,减少泥炭用量,以园土、泥炭、菌渣体积比4:1:5低成本混配基质适于薄壳山核桃容器苗生长,提高苗木光能利用效率,且育苗成本较低,利于生产推广应用。     

Y/SBA-15的微波水热法合成及其表征分析

研究了微波水热法合成微孔-介孔复合分子筛Y/SBA-15,以XRD为表征手段,考察了pH、二氧化硅与P123物质的量比、微波功率、搅拌速度和晶化温度等因素对复合分子筛结构的影响.经过研究确定了复合分子筛的合成条件:pH值为2.5,SiO2和P123物质的量比为58,微波功率为200W,搅拌速度为250r/min,晶化温度为120℃.采用氮气吸附脱附、透射电镜等分析方法对复合分子筛进行了表征分析,结果表明:合成产物同时具有微孔介孔结构特征,结晶度良好、短棒状大小均匀,微孔、介孔2种孔径分布均匀,平均孔径为4.050 13nm,孔壁厚度为2.2nm,BET孔容为0.350 525cm3/g.     

小粒野生稻型细胞质雄性不育系的创建及其恢保关系鉴定

小野1A是由小粒野生稻(Oryza minuta Presl.)中天然不育株与Y58S/内香B杂种后代优秀单株经多代回交转育而成。由于该雄性不育细胞质来自小粒野生稻,称为XY型雄性不育细胞质。恢保关系鉴定结果表明,XY型细胞质雄性不育系与野败型(WA)、印尼水田谷型(ID)、K型和红莲型(HL)细胞质雄性不育系的恢保关系不同,WA型、ID型、K型和HL型的保持系和恢复系中部分可以作为XY型雄性不育细胞质的保持系,部分可以作为XY型雄性不育细胞质的恢复系。XY型雄性不育细胞质突破了WA型、ID型、K型和HL型等雄性不育细胞质的恢保关系,扩大了保持系育种范围,可以用品质优良的栽培品种培育出高标准优质米不育系。由此可见,XY型雄性不育细胞质的发掘,不仅丰富了杂交稻细胞质遗传多样性,而且为培育高标准优质米不育系、进而实质性提高杂交稻稻米品质开辟了一条崭新的育种途径。     

秸秆生物炭及秸秆对大豆生长及土壤微生物活性的影响

为了明确不同秸秆利用方式对作物生长及土壤微生物的影响,采用盆栽试验,研究秸秆直接添加和秸秆生物炭添加对大豆生长状况、根际土壤有机碳及微生物群落功能多样性的影响.结果表明:秸秆生物炭添加(MB和WB)能有效提高大豆盛花期地下生物量.秸秆直接添加(M和W)能显著增加大豆花期根际土壤有机碳的含量,玉米秸秆直接添加处理(M)下的有机碳含量最高,为21.15 mg/g.大豆成熟期,秸秆生物炭添加处理(MB和WB)下土壤有机碳含量较空白处理(CK)显著增加,玉米秸秆添加处理(M)下根际土壤有机碳含量显著高于小麦秸秆添加处理(W).不同秸秆利用方式下大豆根际土壤平均颜色变化率(AWCD)随时间延长而增加,MB和WB处理较M和W处理能显著提高成熟期大豆根际土壤AWCD值.因此,可利用秸秆生物炭添加改善大豆根际土壤微生物活性,提高土壤碳贮量.     

苯二氮■化合物等对雌性狼山鸡生产性能的影响

本试验研究苯二氮化合物－Ｆ８９以及β－受体激动剂ｃｌｅｎｂｕｔｅｒｏｌ（ＣＬ）对雌性狼山鸡生产性能的影响。２ｐｐｍＦ８９和２ｐｐｍＦ８９＋１ｐｐｍＣＬ添加在６５－１１５日龄雌性狼山鸡日粮中，能分别提高摄食量１１．３９％和７．５８％，提高增重２０．４６％（ｐ＜０．０１）和１５．３３％（ｐ＜０．０５）；降低料重比７．５２％和６．７１％。屠宰测定表明：日粮中添加２ｐｐｍＦ８９对狼山鸡胴体组成没有影响．而添加２ｐｐｍＦ８９＋１ｐｐｍｃＬ却提高了鸡的胸肌率１１．２８％（ｐ＜０．０１）、腿肌率６．７７％（ｐ＜０．０５），从而改善动物胴体组成，提高瘦肉率。添喂含２ｐｐｍＦ８９的日粮后，狼山鸡血糖浓度显著下降，β－脂蛋白含量显著上升。表明Ｆ８９影响鸡的糖代谢脂代谢活动。