Global Folding of a Na+-Specific DNAzyme Studied by FRET

Recently, a few RNA-cleaving DNAzymes have been isolated with excellent specificity for Na⁺, and some of them contain a Na⁺-binding aptamer. This metal recognition mechanism is different from that of most previously reported DNAzymes. When using 2-aminopurine (2AP) as a probe, interesting local folding induced by Na⁺ was recently observed. In this work, FRET was used to probe the global folding of the Ce13d DNAzyme; one of the Na⁺-specific DNAzymes. FRET pairs were at different locations, which yielded a total of five constructs to probe the three-way junction structure with a large loop. With endlabeled DNAzymes, the global structure appears to be quite rigid with little folding upon adding up to 200 mm monovalent metal ions, although some minor differences were observed between Li⁺, Na⁺, and K⁺. This lack of significant conformational change is also consistent with circular dichroism spectroscopy data. The loop was then labeled with an internal tetramethylrhodamine fluorophore at the G14 position, and its cleavage activity was partially retained. A clear Na⁺-dependent folding was observed with spectral crossover. From a biosensing standpoint, global folding based sensors are unlikely to work due to the overall rigid structure of the DNAzyme. Therefore, the best way to use this DNAzyme to discriminate Na⁺ from K⁺ is based on cleavage activity, followed by probing local folding, whereas global folding is the least effective for metal discrimination.     

Pb2+ as a Substrate and a Cofactor of a Porphyrin Metalation DNAzyme

We herein report a DNAzyme named T30695 (sequence: (G₃T)₄) that can catalyze Zn²⁺ insertion into three different porphyrins in the presence of Pb²⁺ as a cofactor. Meanwhile, T30695 with Pb²⁺ alone was found to cause a shift in both the fluorescence and UV-vis spectra of protoporphyrin IX (PPIX), thus suggesting that metalation of Pb²⁺ was also achieved at room temperature. From kinetic measurements, the reaction required two Pb²⁺ ions; this is consistent with one being a cofactor and the other being a substrate. No previous reports inserted Pb²⁺ into porphyrins by using DNAzymes or protein-based enzymes. This reaction was most significantly inhibited in the presence of K⁺ followed by Na⁺ and Li⁺, suggesting the importance of the Pb²⁺-stabilized G-quadruplex. When Pb²⁺ is inserted into PPIX, its emission blue shifts from 635 to 590 nm, thus allowing simple ratiometric fluorescent sensing with a detection limit of 1.2 nM Pb²⁺.     

Biochemical Characterization of a Uranyl Ion‐Specific DNAzyme

Uranyl ion‐specific DNAzyme : A DNAzyme (lower strand) cleaves the substrate (upper strand) in the presence of the uranyl ion. The enzyme folds into a bulged three‐way‐junction structure with catalytically important nucleotides residing in the bulge. A highly conserved G?A mismatch is also crucial for the enzyme's activity.

     

The importance of peripheral sequences in determining the metal selectivity of an in vitro-selected Co(2+) -dependent DNAzyme

DNAzymes are catalytically active DNA molecules that use metal cofactors for their enzymatic functions. While a growing number of DNAzymes with diverse functions and metal selectivities have been reported, the relationships between metal ion selectivity, conserved sequences and structures responsible for selectivity remain to be elucidated. To address this issue, we report biochemical assays of a family of previously reported in vitro selected DNAzymes. This family includes the clone 11 DNAzyme, which was isolated by positive and negative selection, and the clone 18 DNAzyme, which was isolated by positive selection alone. The clone 11 DNAzyme has a higher selectivity for Co(2+) over Pb(2+) compared with clone 18. The reasons for this difference are explored here through phylogenetic comparison, mutational analysis and stepwise truncation. A novel DNAzyme truncation method incorporated a nick in the middle of the DNAzyme to allow for truncation close to the nicked site while preserving peripheral sequences at both ends of the DNAzyme. The results demonstrate that peripheral sequences within the substrate binding arms, most notably the stem loop, loop II, are sufficient to restore its selectivity for Co(2+) over Pb(2+) to levels observed in clone 11. A comparison of these sequences' secondary structures and Co(2+) selectivities suggested that metastable structures affect metal ion selectivity. The Co(2+) selectivity of the clone 11 DNAzyme showed that the metal ion binding and selectivities of small, in vitro selected DNAzymes may be more complex than previously appreciated, and that clone 11 may be more similar to larger ribozymes than to other small DNAzymes in its structural complexity and behavior. These factors should be taken into account when metal-ion selectivity is required in rationally designed DNAzymes and DNAzyme-based biosensors.     

Structural Studies on the Reaction of Isopenicillin N Synthase with a Sterically Demanding Depsipeptide Substrate Analogue

Isopenicillin N synthase (IPNS) is a nonheme iron(II)‐dependent oxidase that catalyses the central step in penicillin biosynthesis, conversion of the tripeptide δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐valine (ACV) to isopenicillin N (IPN). This report describes mechanistic studies using the analogue δ‐(L ‐α‐aminoadipoyl)‐(3S‐methyl)‐L ‐cysteine D ‐α‐hydroxyisovaleryl ester (A^SmCOV), designed to intercept the catalytic cycle at an early stage. A^SmCOV incorporates two modifications from the natural substrate: the second and third residues are joined by an ester, so this analogue lacks the key amide of ACV and cannot form a β‐lactam; and the cysteinyl residue is substituted at its β‐carbon, bearing a (3S)‐methyl group. It was anticipated that this methyl group will impinge directly on the site in which the co‐substrate dioxygen binds. The novel depsipeptide A^SmCOV was prepared in 13 steps and crystallised with IPNS anaerobically. The 1.65 Å structure of the IPNS–Fe^II–A^SmCOV complex reveals that the additional β‐methyl group is not oriented directly into the oxygen binding site, but does increase steric demand in the active site and increases disorder in the position of the isovaleryl side chain. Crystals of IPNS–Fe^II–A^SmCOV were incubated with high‐pressure oxygen gas, driving substrate turnover to a single product, an ene‐thiol/C‐hydroxylated depsipeptide. A mechanism is proposed for the reaction of A^SmCOV with IPNS, linking this result to previous crystallographic studies with related depsipeptides and solution‐phase experiments with cysteine‐methylated tripeptides. This result demonstrates that a (3S)‐methyl group at the substrate cysteinyl β‐carbon is not in itself a block to IPNS activity as previously proposed, and sheds further light on the steric complexities of IPNS catalysis.     

NO Formation by Neuronal NO‐Synthase can be Controlled by Ultrafast Electron Injection from a Nanotrigger

Synchronized catalysis in native enzyme : We used a photoactive nanotrigger (NT) to study the initial electron transfer to FAD in native neuronal NOS catalysis. Modeling and fluorescence spectroscopy showed that selective NT binding to NADPH sites is able to override Phe1395 regulation, thus permitting ultrafast injection of electrons into the protein electron pathway. That NT initiation of flavoenzyme catalysis led to the formation of NO is promising for time‐resolved X‐ray and other cellular applications.

     

Unexpected oxidation of a depsipeptide substrate analogue in crystalline isopenicillin N synthase

Isopenicillin N synthase (IPNS) is a non-heme iron(ii)-dependent oxidase that is central to penicillin biosynthesis. Herein, we report mechanistic studies of the IPNS reaction in the crystalline state, using the substrate analogue delta-(L-alpha-aminoadipoyl)-(3R)-methyl-L-cysteine D-alpha-hydroxyisovaleryl ester (AmCOV) to probe the early stages of the catalytic cycle. The X-ray crystal structure of the anaerobic IPNS:Fe(II):AmCOV complex was solved to 1.40 A resolution, and it reveals several subtle differences in the active site relative to the complex of the enzyme with its natural substrate. The crystalline IPNS:Fe(II):AmCOV complex was then exposed to oxygen gas at high pressure; this brought about reaction to give what appears to be a hydroxymethyl/ene-thiol product. A mechanism for this reaction is proposed. These results offer further insight into the delicate interplay of steric and electronic effects in the IPNS active site and the mechanistic intricacies of this remarkable enzyme.     

聚苯乙烯在空气中热降解的化学动力学研究

研究了聚苯乙烯在空气中的热降解化学动力学机理。实验结果显示,聚苯乙烯在空气中热降解存在两个失重阶段:第一阶段,温度范围从250.59℃开始至397.41℃,在此范围内,大约有85%的聚苯乙烯发生了分解;第二阶段为397.41～515.29℃,大约有15%的聚苯乙烯发生分解。动力学计算拟合结果表明,聚苯乙烯的主要失重阶段(第一阶段)的热解符合二级化学反应方程,其平均表观活化能Ea值为166.19kJ/mol,lnA值为32.15。     

结晶动力学研究进展

综述了溶液结晶动力学及聚合物结晶动力学的现状及进展,并对其研究中存在的缺陷与不足进行了评述.     

The effects of ligand exchange and mobility on the peroxidase activity of a bacterial cytochrome c upon unfolding

The effect on the heme environment upon unfolding Paracoccus versutus ferricytochrome c-550 and two site-directed variants, K99E and H118Q, has been assessed through a combination of peroxidase activity increase and one-dimensional NMR spectroscopy. At pH 4.5, the data are consistent with a low- to high-spin heme transition, with the K99E mutation resulting in a protein with increased peroxidase activity in the absence of or at low concentrations of denaturant. Furthermore, the mobility of the polypeptide chain at pH 4.5 for the wild-type protein has been monitored in the absence and presence of denaturant through heteronuclear NMR experiments. The results are discussed in terms of local stability differences between bacterial and mitochondrial cytochromes c that are inferred from peroxidase activity assays. At pH 7.0, a mixture of misligated heme states arising from protein-based ligands assigned to lysine and histidine is detected. At low denaturant concentrations, these partially unfolded misligated heme forms inhibit the peroxidase activity. Data from the K99E mutation at pH 7.0 indicate that K99 is not involved in heme misligation, whereas histidine coordination is proven by the data from the H118Q variant.     

The kinetics and mechanism of polyethylene photo-oxidation

The kinetics of the natural and accelerated photo-oxidation of low-denisty polyethylene (LDPE) films have been studied; different geographical locations have been selected for the natural tests, and a range of temperatures used in the accelerated experiments in a specially built temperature-controlled ultraviolet radiation enclosure. A meaningful correlation between natural and accelerated weathering results was established, by means of an adequate superposition of the effects of UV radiation exposure and temperature. Reasonably detailed and accurate, lumped-parameter, kinetic models of the photo-oxidation process have also been developed, to interpret and predict the results of measurements of carbonyl, hydroperoxide and vinyl absorbances as functions of time and temperature; the models predict the general experimental behaviour, and also that both the formation of hydroperoxides and carbonyl Norrish-I reactions are important initiation steps. More complex models have the potential of interpreting other fine details of the degradation behaviour, namely the generation of other chemical species, and the chain scission and cross-linking process which are directly related to changes in the mechanical properties.     

The [4Fe-4S]-Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]-Hydrogenases

The active site of [FeFe]-hydrogenases contains a cubane [4Fe-4S]-cluster and a unique diiron cluster with biologically unusual CO and CN⁻ ligands. The biogenesis of this diiron site, termed [2Fe_H], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2Fe_H] precursor, termed [2Fe_P], to the [FeFe]-hydrogenase. The binding site of [2Fe_P] in HydF has not been elucidated, however, the [4Fe-4S]-cluster of HydF was considered as a possible binding partner of [2Fe_P]. By targeting individual amino acids in HydF from Thermosipho melanesiensis using site directed mutagenesis, we examined the postulated binding mechanism as well as the importance and putative involvement of the [4Fe-4S]-cluster for binding and transferring [2Fe_P]. Surprisingly, our results suggest that binding or transfer of [2Fe_P] does not involve the proposed binding mechanism or the presence of a [4Fe-4S]-cluster at all.     

纳米氧化锌晶体生长宏观动力学研究

采用均匀沉淀法制备了纳米氧化锌晶体,考察了影响纳米氧化锌晶粒大小的因素及晶粒生长特性,利用晶体生长动力学理论得到纳米氧化锌晶体生长动力学方程,激活能E=27.87 kJ/mol,指前因子A=6.31×106 nm6·h-1,并对氧化锌晶粒生长机制进行了初步探讨.     

Study on reaction kinetics of epoxy resin cured by a modified dicyandiamide

To improve the disadvantage of the low reactivity and reduce the high curing temperature of epoxy resin cured by dicyandiamide (DICY), DICY is chemically modified with phenyl hydrazine and a new curing agent, LB‐A, is developed in this research. The structure, the curing behavior, and reaction kinetics of LB‐A curing epoxy resin are investigated. Results show that the DICY is modified successfully and the well‐defined structure of DICY is destroyed after modification. Consequently, a new curing agent in a noncrystal form is resulted. Thereupon, the reactivity and compatibility between the epoxy resin and the curing agent are improved appreciably using LB‐A instead of DICY. Meanwhile, the curing temperature and activation energy of the curing reaction decrease outstanding, whereas the rate constant increases remarkably. In addition, the compressive strength and the adhesive strength in shear by tension loading of the resulting epoxy resins have been increased using LB‐A instead of DICY as the curing agent. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

On the mechanism of the copper‐catalysed hydro‐genation; a reinterpretation of published data

The copper‐catalysed hydrogenation of triglyceride oils differs from the nickel‐catalysed reaction in that copper catalysts only hydrogenate double bonds in methylene interrupted polyunsaturated fatty acid moieties. Accordingly, the copper‐catalysed reaction stops when the triglycerides present in the reaction mixture are only monounsaturated fatty acids and polyunsaturated fatty acids in which the double bonds are separated by more than one methylene group. Copper catalysts thus exhibit a very high oleic acid selectivity. This observation has been explained in the literature by assuming that copper catalysts can only catalyse the hydrogenation of conjugated polyenes and that they are also capable of catalysing the conjugation of methylene interrupted polyenes. Accordingly, the hydrogenation of linolenic acid and linoleic acid moieties starts with their conjugation which is then followed by hydrogen addition to these conjugated acids. For both reactions (conjugation and hydrogenation) the literature assumes the Horiuti‐Polanyi mechanism stipulating the addition of a hydrogen atom to a double bond as the first step. Reinterpretation of the literature data now leads to the hypothesis that the first step in the conjugation mechanism could well be the abstraction of a hydrogen atom from an allylic methylene group rather than the addition of a hydrogen atom to a doubly bonded carbon atom. A conjugated double bond system then results from the addition of a hydrogen atom to the allylic radical formed by the foregoing hydrogen abstraction.     

TiO2光催化降解2,4-二氯酚动力学及机理的研究

以TiO2作为催化剂,500 W汞灯作为光源,对水体中2,4-二氯酚（2,4-DCP）的光催化降解特性进行了研究。实验结果表明,TiO2光催化降解2,4-DCP效果较好,降解的最佳pH为7,催化剂的最佳投加量为200 mg·L-1。2,4-DCP的初始浓度越小,光催化的效率越高。在初始浓度为5 mg·L-1时,光催化1 h的降解率达90%。在实验的四种不同初始浓度（5 mg·L-1、10 mg·L-1、20 mg·L-1、40 mg·L-1）下,2,4-DCP的光降解速率常数近似与其初始质量浓度成一级反应动力学关系。结合高效液相色谱、气相色谱-质谱的测定结果以及Cl-、COD的变化,认为2,4-DCP的主要降解过程为：脱氯-开环-矿化、中间产物相互作用-进一步矿化。     

Curing characteristics of epoxy resin systems that include a biphenyl moiety

The curing characteristics of epoxy resin systems that include a biphenyl moiety were investigated according to the change of curing agents. Their curing kinetics mainly depend on the type of hardener. An autocatalytic kinetic reaction occurs in epoxy resin systems with phenol novolac hardener, regardless of the kinds of epoxy resin and the epoxy resin systems using Xylok and DCPDP (dicyclopentadiene‐type phenol resin) curing agents following an nth‐order kinetic mechanism. The kinetic parameters of all epoxy resin systems were reported in terms of a generalized kinetic equation that considered the diffusion term. The fastest reaction conversion rate among the epoxy resin systems with a phenol novolac curing agent was obtained in the EOCN‐C epoxy resin system, and for systems with Xylok and DCPDP hardeners, the highest reaction rate values were obtained in NC‐3000P and EOCN‐C epoxy resin systems, respectively. The system constants in DiBenedetto's equation of each epoxy resin system with different curing agents were obtained, and their curing characteristics can be interpreted by the curing model using a curing agent as a spacer. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1942–1952, 2002     

Investigation of the reaction mechanism of different epoxy resins using a phosphorus‐based hardener

In this work, the fundamental kinetic and structure/property information for a novel phosphorus‐based hardener, bis(4‐aminophenoxy) phosphonate is cured with a range of common epoxy resins such as diglycidyl ether of bisphenol A, tri glycidyl p‐amino phenol and tetra glycidyl diamino diphenyl methane (TGDDM) at various cure temperatures. The rate coefficients k₁ and k₂ for the primary and secondary amine epoxide addition reactions, respectively, were determined and were found to exhibit a positive substitution effect for the TGAP and TGDDM epoxy resins. Etherification or internal cyclization were shown to be important at higher levels of cure conversion, with these reactions being more significant for the TGAP/BAPP system. Some basic structure/property relationships were established between the glass transition temperature (T_g) and epoxide conversion. The master curve obtained for the superimposition of the various cure temperatures for each epoxy demonstrated the independence of the cure mechanism with temperature. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 99:3288–3299, 2006