Probing Tertiary Structures of Proteins by Limited Proteolysis and HPLC-MS

It is generally accepted that one or more distinct, populated intermediates are involved in the folding of most proteins. Although the understanding of the tertiary structure of the intermediate is important to elucidate the hierarchic folding pathway, it is difficult to characterize the structure because it exists only transiently during the folding process. Recently, it has been shown that several globular proteins, when placed under certain denaturing conditions, can exist in equilibrium molten globule states.     

重氮法固定化糖化酶活力和稳定性

本文以苯氨基磺酸型多孔聚苯乙烯为载体，重氮化活化其苯氨基与糖化酶上的氨基反应。在固定化酶过程中，随反应时间的延长，不但固定化酶总活力增加，而且其比活和热稳定性也随之升高，最终可得活力为２００００Ｕ／ｇ干胶，比活为５００的高活力固定化酶，该固定化酶在ｐＨ４．５，６０℃无底物下保温，其半寿期是天然酶的５倍。以麦芽糖，糊精（分子量为１５００）和可溶性淀粉为底物测定该固定化酶活力表明：对某种孔度一定的载体     

Limited Proteolysis of Luciferase as a Reporter in Nanosystem Biology: A Comparative Study

Firefly luciferase is a 62 kDa protein that produces a flash of light on the oxidation of luciferin in the presence of ATP, Oxygen and Mg²⁺. Luciferase has a broad range of applications for analytical purposes and in vivo imaging for nanosystem biology studies. However, the enzyme is highly susceptible to proteolytic degradation that reduces its half-life. Rate of proteolytic digestion between two members of luciferase family ( Photinus pyralis and Lampyris turkestanicus ) is compared. Proteolytic sensitivity of L. turkestanicus luciferase was found to be more than P. pyralis luciferase, due to higher rate of hydrolysis under identical conditions. Both luciferases showed more sensitivity to chymotrypsin than trypsin with different digestion pattern. Digestion of P. pyralis by trypsin produced some fragments which were found to be more resistant to further degradation, whereas in L. turkestanicus initial fragments subdigested by trypsin, like chymotrypsin effect on both luciferases. Furthermore, both luciferases become increasingly labile to proteolysis as the temperature increases. The rate of inactivation and the rate of degradation between luciferases were different in a specific time of incubation. Appearance of similar bands for both luciferases confirmed exposure of specific regions, in spite of structural differences.     

Probing the 3-D Structure,Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS

Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.     

Stabilisation of polymers by catalytic antioxidants

Two types of catalytic antioxidant are now recognised. The first involves the catalytic destruction of hydroperoxides to non-radical products and the second consists of “stable” redox couples with the ability to remove both alkyl (CB-A) and alkylperoxyl (CB-D) radicals in autoxidising systems. The conditions under which both types of antioxidant can operate effectively are reviewed and the limitations to their activity are outlined.     

Functionalized Hyperbranched Aliphatic Polyester Polyols: Synthesis,Properties and Applications

Recently, hyperbranched polymers(HBPs), which differ significantly in structure and properties from linear, cross-linked and branched analogs, have become increasingly important. HBP have a spatial unloaded core and a shell of branched monomer units(dendrons), in which functional groups are predominantly located in the surface layer. The size of macromolecules ranges from 2 nm to 100 nm. Currently, there are a fairly large number of publications in the literature devoted to the modification of h...     

超嗜热酯酶APE1547中特殊位置氢键对酶活力和热稳定性的影响

探讨了APE1547蛋白的β-推进器结构中第3和第4“叶片”间的侧链氢键(Thr127-Gly154, Leu182-Arg145-Glu122)对蛋白质的作用.     

Thermostability testing and degradation profiles of doxycycline in bulk, tablets, and capsules by HPLC

A high-performance liquid chromatography (HPLC) method for the quantitation of doxycycline in bulk, tablets, and capsules after storage at -20, 5, 25, 40, 50, 60, and 70 degrees C, has been developed and validated. The samples are eluted from a micro-Bondapak C8 column (4.6x150-mm, i.d., 5-microm particle size) at 27 degrees C, with a mobile phase of acetonitrile-water-THF (29.5:70:0.5, v/v/v), adjusted to pH 2.5 with 1.0M HCl. The flow rate is 1.0 mL/min and detection by UV is at 350 nm. The stability of doxycycline in bulk and in pharmaceuticals is checked over 90 days. Doxycycline shows thermo-degradation after exposure to high temperature; tablets are more stable than capsules. The shelf lives (t90%) are determined to be 1.00, 2.84, and 5.26 years in bulk, capsules, and tablets, respectively, at 25 degrees C. Metacycline and 6-epidoxycyline are identified as degradation products at high temperatures. Amounts of doxycycline, metacycline, and 6-epidoxycycline in all samples are determined by HPLC, and the results compare with those from micellar electrokinetic capillary chromatography. After 90 days, metacycline and 6-epidoxycyline are almost equal in test samples from standard bulk form, tablets, and capsules. It is 27.8+/-0.3%, 13.7+/-0.1%, and 18.8+/-0.2%, respectively.     

Effect of Inoculated Lactic Acid Fermentation on the Fermentable Saccharides and Polyols,Polyphenols and Antioxidant Activity Changes in Wheat Sourdough

Inoculation of sourdough allows the fermentation medium to be dominated by desired microorganisms, which enables determining the kinetics of the conversion of chemical compounds by individual microorganisms. This knowledge may allow the design of functional food products with health features dedicated to consumers with special needs. The aim of the study was to assess the dynamics of transformations of fermentable oligosaccharide, disaccharide, monosaccharide and polyol (FODMAP) compounds from wheat flour as well as their antioxidant activity during inoculated and spontaneous sourdough fermentation. The FODMAP content in grain products was determined by the fructan content with negligible amounts of sugars and polyols. To produce a low-FODMAP cereal product, the fermentation time is essential. The 72 h fermentation time of L. plantarum-inoculated sourdough reduced the FODMAP content by 91%. The sourdough fermentation time of at least 72 h also positively influenced the content of polyphenols and antioxidant activity, regardless of the type of fermentation. The inoculation of both L. plantarum and L. casei contributed to a similar degree to the reduction in FODMAP in sourdough compared to spontaneous fermentation.     

Stabilisation of a heptamethine cyanine dye by rotaxane encapsulation

The crystal structure of a cyanine dye rotaxane shows that the cyclodextrin is tightly threaded round the polymethine bridge of the dye; encapsulation dramatically increases the kinetic chemical stability of the radicals formed on oxidation and reduction of the dye, making it possible to observe the rotaxane radical dication by ESR and UV-vis-NIR spectroscopy.     

Improved Separation of Polyols and Carbohydrates by High Performance Liquid Chromatography

Abstract

A reliable High Performance Liquid Chromatographic analysis of glucose-fructose-sucrose-sorbitol and mannitol is developed. A Sugar Pak I column at 85°C is used employing water as the mobile phase. The separation is completed within 20 minutes, and the resolution is very acceptable.     

Upregulation of an Artificial Zymogen by Proteolysis

Regulation of enzymatic activity is vital to living organisms. Here, we report the development and the genetic optimization of an artificial zymogen requiring the action of a natural protease to upregulate its latent asymmetric transfer hydrogenase activity.     

Improvement of Thermostability and Activity of Firefly Luciferase Through [TMG][Ac] Ionic Liquid Mediator

Firefly luciferase catalyzes production of light from luciferin in the presence of Mg²⁺?CATP and oxygen. This enzyme has wide range of applications in biotechnology and development of biosensors. The low thermal stability of wild-type firefly luciferase is a limiting factor in most applications. Improvements in activity and stability of few enzymes in the presence of ionic liquids were shown in many reports. In this study, kinetic and thermal stability of firefly luciferase from Photinus pyralis in the presence of three tetramethylguanidine-based ionic liquids was investigated. The enzyme has shown improved activity in the presence of [1, 1, 3, 3-tetramethylguanidine][acetate], but in the presence of [TMG][trichloroacetate] and [TMG][triflouroacetate] activity, it decreased or unchanged significantly. Among these ionic liquids, only [TMG][Ac] has increased the thermal stability of luciferase. Incubation of [TMG][Ac] with firefly luciferase brought about with decrease of K _m for ATP.     

环氧大豆油多元醇改性磺酸型水性聚氨酯合成与性能研究

用2-乙基己醇(异辛醇)对环氧大豆油进行开环,生成环氧大豆油多元醇,并利用其对由异佛尔酮二异氰酸酯(IPDI)、聚己内酯二元醇(PCL)和乙二胺基乙磺酸钠(AAS)为主要合成原料的磺酸型水性聚氨酯(SWPU)进行改性,考察了不同多元醇含量对磺酸型水性聚氨酯乳液以及胶膜性能的影响.首先,通过FTIR和1H-NMR证实了环氧大豆油多元醇的合成;然后经过粒径测试、耐水性测试、接触角测试、机械性能测试、TG测试等检测表明:磺酸型水性聚氨酯成功合成,随着多元醇含量的增加,水性聚氨酯乳液粒径逐渐增大;胶膜的吸水率逐渐减小,并趋于平缓;力学性能拉伸强度逐渐增强,而断裂伸长率下降.但当环氧大豆油多元醇添加量过大时,导致最后乳液无法分散,乳化失败.当多元醇含量与PCL质量比为0.2时,胶膜吸水率由50.14%减小至12.27%,接触角为103.3°,拉伸强度由8.16 MPa增至21.77 MPa.通过TG表明,分子结构中多元醇的引入,胶膜的耐热性有明显提高.     

Stabilisation of water-soluble platinum nanoparticles by phosphonic acid derivatives

Sodium 2-(diphenylphosphino)ethyl phosphonate (1) was investigated as a stabilising agent for platinum nanoparticles (Pt-NPs) in aqueous solution. This phosphino phosphonate is known to stabilise rhodium nanoparticles (NPs) in water. Here we report that in the case of Pt-NPs this ligand is indirectly involved in the stabilisation mechanism and the actual stabilisation agent is the platinum complex Na(2)[Pt(1)(2)] (2). The reduction of platinum(II) salts in the presence of the phosphonates 1, 2, sodium 2-(diphenylphosphoryl)ethyl phosphonate (3) and 3,3,3-triphenylpropyl phosphonate (4) leads to stable platinum NPs with a remarkably narrow particle size distribution. These platinum NPs show high catalytic activity in the hydrogenation of 1-hexene and 1-chloro-3-nitrobenzene under biphasic as well as heterogeneous (supported on charcoal) conditions. The activity of the supported NPs was 30 times higher than the commercially available catalyst Pt(0) EnCat?. Furthermore, the single-crystal X-ray structures of (1)(MeOH)(2)(H(2)O)(2), (3)(H(2)O)(4), and (4)(2)(H(2)O)(17) have been determined.     

醇热解法合成超顺磁性氧化铁纳米粒子及其性能

以甲氧基聚乙二醇同时作为溶剂、还原剂及修饰剂,在高温下分解乙酰丙酮铁,制备了纳米Fe3O4粒子,采用透射电子显微镜和X射线衍射分析表征材料的形貌和相组成,傅里叶变换红外光谱仪表征材料的表面修饰物,超导量子干涉仪测试合成的纳米粒子的磁性能,纳米粒度与zeta电势分析仪测试磁性纳米粒子在水中的zeta电势。 结果表明,纳米Fe3O4粒子的大小为（10.1±1.6） nm,粒度均一,单分散性好,在300 K下具有超顺磁性,饱和磁化强度为45 A·m2/kg。 红外结果表明,-COO-共价结合在粒子表面。 zeta电势为-25 mV。 其在水中的稳定性与以三甘醇为反应介质、高温分解法制备的纳米Fe3O4粒子作比较,表现出长时间（60 d以上）的良好分散性。 静电作用及空间位阻效应是其高稳定分散性的原因。     

Synthesis and Some Reactions of Polyols Formals

Synthesis of formals of some polyols such as dipentaerythritol, dietriol and diglycerin was proposed. Based on them, esters and carbamates were obtained.     

Improvement in the Thermostability of a Recombinant β-Glucosidase Immobilized in Zeolite under Different Conditions

β-Glucosidase is part of the cellulases and is responsible for degrading cellobiose into glucose, a compound that can be used to produce biofuels. However, the use of the free enzyme makes the process more expensive. Enzyme immobilization improves catalytic characteristics and supports, such as zeolites, which have physical-chemical characteristics and ion exchange capacity that have a promising application in the biotechnological industry. This research aimed to immobilize by adsorption a recombinant β-glucosidase from Trichoderma reesei, obtained in Escherichia coli BL21 (DE3), in a commercial zeolite. A Box Behnken statistical design was applied to find the optimal immobilization parameters, the stability against pH and temperature was determined, and the immobilized enzyme was characterized by SEM. The highest enzymatic activity was determined with 100 mg of zeolite at 35 °C and 175 min. Compared to the free enzyme, the immobilized recombinant β-glucosidase presented greater activity from pH 2 to 4 and greater thermostability. The kinetic parameters were calculated, and a lower K_M value was obtained for the immobilized enzyme compared to the free enzyme. The obtained immobilization parameters by a simple adsorption method and the significant operational stability indicate promising applications in different fields.