Altered enzymatic activity of lysozymes bound to variously sulfated chitosans

The purpose of this research is to investigate the effects of the variously sulfated chitosans on lysozyme activity and structure.It was shown that the specific enzymatic activity of lysozyme remained almost similar to the native protein after being bound to 6-O-sulfated chitosan(6S-chitosan) and 3,6-O-sulfated chitosan(3,6S-chitosan),but decreased greatly after being bound to 2-N-6-O-sulfated chitosan(2,6S-chitosan).Meanwhile,among these sulfated chitosans,2,6S-chitosan induced the greatest conformational change in lysozyme as indicated by the fluorescence spectra.These findings demonstrated that when sulfated chitosans of different structures bind to lysozyme,lysozyme undergoes conformational change of different magnitudes,which results in corresponding levels of lysozyme activity.Further study on the interaction of sulfated chitosans with lysozyme by surface plasmon resonance(SPR) suggested that their affinities might be determined by their molecular structures.     

Tris(hydroxymethyl)aminomethane-modified magnetic microspheres for rapid affinity purification of lysozyme

A novel affinity purification method for lysozyme (LZM) based on functionalized magnetic microspheres was developed. Tris(hydroxymethyl)aminomethane (Tris)-modified magnetic microspheres with specific affinity toward LZM were prepared using Tris as ligand and silica-coated magnetic microshperes as support. Transmission electron microscopy and magnetic property measurement results showed that the Tris-modified magnetic microspheres have a very good core-shell structure and high magnetization.The maximum binding capacity of LZM was about 108.6 mg/g magnetic microspheres. LZM purified from chicken egg white had high purity and well-maintained activity of 8140 U/mg. This magnetic-mediated LZM purification strategy has advantages of high efficiency, low cost and easy operation.     

Synthesis and chiral recognition of novel amylose derivatives containing regioselectively benzoate and phenylcarbamate groups

A new class of regioselectively substituted amylose derivatives bearing three different substituents at 2-, 3- and 6-positions, and two different substituents at 2-position and 3-, 6-positions were synthesized by a sequential process based on the esterification of 2-position of a glucose unit. Their chiral recognition abilities were evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). Each derivative had its own characteristic recognition ability depending on the arrangement of side chains at the three positions. Among the derivatives, amylose 2-(4-t-butylbenzoate) and amylose 2-(4-chlorobenzoate) series exhibited high chiral recognition. Some racemates can be efficiently separated on these derivatives as well as on the amylose tris-3,5-dimethylphenylcarbamate, which is commercially available as Chiralpak AD and one of the most powerful CSPs. The structures of the amylose derivatives were also investigated by circular dichroism spectroscopy.     

The interaction of anionic azo dyes with hen egg-white lysozyme: 2

In continuation of our preceding study, this report describes pH and ionic strength dependences of the binding constants of six anionic azo dyes to lysozyme, the competitive binding of the dyes with the substrate analogues of lysozyme and the change in the circular dichroism of lysozyme by the dye binding. The binding constants were obtained from the difference spectra of visible absorption. With an increase in pH from 5.0 to 7.0 the constants for the dyes (1st of the two modes of the binding of a dye named D3) are reduced. The increase in ionic strength from 0.1 to 0.2 also reduces the values of the constant. Competitive binding was found between D3 and the analogues, but not for the other dyes. The change in the circular dichroism due to the electronic perturbation of tryptophyl residues in lysozyme was found. From these evidences, Lys 33 in lysozyme is pinpointed as the most credible binding site for the dyes (1st mode binding of D3). An unspecified location near the subsiteB in lysozyme is addressed for 2nd mode binding of D3.     

Isomerization of light alkanes: preparation and characterization of platinum promoted sulfated zirconia catalysts

Summary Platinum-promoted sulfated zirconia catalysts active in n-hexane isomerization were prepared in a one-step synthesis and characterized. The crystallinity and sulfur contents depend mainly on the calcination temperature. The stability of the sulphate in the presence of hydrogen is reduced by the presence of Pt.     

Adsorption of lysozyme on a hemodialysis sulfonated polyacrylonitrile membrane, with and without preadsorbed poly(ethyleneimine) on the external faces

We present the influence of pH, from pH 4 to 10, with a focus on the neutral range, on the adsorption of lysozyme (isoelectric point pI=11) on a sulphonated membrane and the same membrane pre-treated with poly(ethyleneimine) (PEI). We found a steep increase of the adsorbed amount above pH 6 in phosphate buffer. The adsorbed amount was about twice as low in Tris buffer, around the neutral pH. The difference between the two types of buffer is attributed to their different ionic composition. High interfacial concentration in phosphate buffer is especially linked to the phosphate divalent anions. In the presence of divalent sulphate anions, we measured the same level of interfacial concentration than with phosphate buffer. With the PEI pre-treated membrane, we observed, on the time scale of our experiments (15–20 h), similar adsorbed amounts than on the raw membrane, showing that the PEI layer does not constitute a true barrier to the penetration of lysozyme into the membrane core. However, its presence leads to a slower adsorption rate in a system where convection does not occur through the membrane.     

Lysis of Escherichia coli cells by lysozyme: Discrimination between adsorption and enzyme action

The key factors of enzymatic lysis of cells are the interaction between the enzyme and the cell - catalytic and non-catalytic adsorption of enzyme on cell surface. Here, the studies of lysis of intact Escherichia coli cells by chicken egg white lysozyme were performed. It was found that the ionic strength has a dual effect onto the system. On the one hand, the desorption constant of the enzyme increases with the increase of the solution ionic strength, which results in a better enzyme performance. On the other hand, due to the higher osmosis, the cell lysis rate decreases with the increasing of ionic strength of the system. It was found that pH 8.6 and 30 mM NaCl are optimal conditions for lysis of E. coli cells by lysozyme.     

Binding of thermo-sensitive and pH-sensitive butylated poly(allylamine)s with lysozyme

Butyl modified poly(allylamine)s with butyl substitution degrees of 15%to 70%were prepared.The polymers show pH sensitive property and lower critical solution temperature(LCST)behavior.The LCST appears at lower temperature,lower pH and lower polymer concentration for the polymer with higher butylated degree.The binding of native lysozyme with the polymers depends on the hydrophobicity of the polymers at the pH range that the protein and the polymer carry the same positive charges.The increase of polymer hydrophobicity can increase the binding with lysozyme,but the self-aggregation of the polymer decreases the binding.The bound lysozyme molecules can recover their native activity completely after the dissociation of the complexes.Compared with native lysozyme,the denatured one which exposes the hydrophobic residues can increase the binding with the polymer and form stable complex nanoparticles.     

多糖类大分子与表面活性剂相互作用的研究进展

多糖类大分子具有天然、无毒、使用安全和可再生及来源丰富等优点. 将多糖类大分子与表面活性剂复配使用, 不仅可利用各自的优势和特性, 而且能发挥二者的协同作用, 大大改善二者的性能. 由于两者之间存在诸如静电作用、疏水作用、偶极相互作用、氢键作用、空间位阻效应等, 水体系中表面活性剂在多糖分子链上的缔合得到调控, 并引起表面活性剂的临界聚集浓度(cac)、临界胶束浓度(cmc)、结合量, 以及体系的表面吸附、界面流变性等呈现各种变化. 本文简要总结了近年来多糖类大分子与表面活性剂复配体系研究方面取得的一些进展, 述及复配体系研究中所采用的方法与手段, 主要讨论复配体系的物理化学性质以及多糖类大分子与表面活性剂相互作用的机制.     

Affinity switching for lysozyme and dual-responsive microgels by stopped-flow technique: Kinetic control and activity evaluation

The use of proteins as therapeutics in nanomedicine is an emerging research field and has developed rapidly.However,proteins are always vulnerable to renal excretion or digestion by the proteolytic system in vivo,which limits their usage to a large extent.Although biocompatible polymers have been covalently linked to proteins to protect them from recognition by the immune system and prolong their circulation time,the biological activity of them is sometimes decreased.To fill this gap,physical isolation,wrapping,or encapsulation techniques are employed.Up to now,various mature examples were reported,but the whole time scales for guest molecules loading and releasing,especially the initial rapid loading process,were rarely mentioned.Herein,a series of dual-responsive poly(N-isopropylacrylamide-co-methacrylic acid)(P(NIPAM-co-MAA)) microgels were synthesized and employed to investigate the kinetics of in situ complexation and release of lysozyme under external stimuli modulation upon a stopped-flow apparatus,which was suitable for rapid dynamic monitoring.Close inspection of the adsorption kinetics during the early stages( 50 s) revealed that the initial microgel collapse occurred within ~1 s,with more rapid transitions being observed when higher lysozyme concentrations were targeted.All the dynamic traces could be well fitted with a double exponential function,suggesting a fast(τ1) and a slow(τ2) relaxation time,respectively.Then,the kinetics of releasing bound lysozyme from microgels was carried on by utilizing the p H-responsive property,and the evaluation of the activity of released lysozyme was synchronously measured in a Micrococcus lysodeikticus(M.lysodeikticus) cell suspension.The corresponding relaxation time(τ) was also calculated by fitting the recorded dynamic traces.We speculate that this work can provide basic dynamics data and theoretical basis for microgels based nanocarriers to be used for protein delivery,controlled release,and possible chemical separation.     

The surface structure of sulfated zirconia: Studies of XPS and thermal analysis

Sulfated zirconias were prepared using two kinds of amorphous zirconia gels, XZO 631 and 632 supplied by MEL Chemicals, and their thermal gravimetrical analyses were carried out. DTG of the former sample showed two peaks based on decomposition of the sulfate species on the surface, the first peak at 680 °C and the second broad one centered at 850 °C. The latter sample indicated only broad peak at 850 °C in the range from 700 to >1000 °C. The first peak for the former sample was ascribed to the decomposition of Zr(SO₄)₂ remained on the surface, and the broad one at 700 to >1000 °C for the both samples was attributed to the catalytically active species. The acidic character of sulfated zirconia calcined at 1000 °C was examined in acid-catalyzed reactions of cumene, ethylbenzene, and butane together with the adsorption heat of Ar, showing a solid acid with acidity higher than that of silica-alumina. It was indicated from the XPS analysis that the S species are composed of SO₄²⁻. The results led to a structural model of the active surface to be polysulfate species containing mainly three or four S atoms with two ionic bonds of SOZr in addition to coordination bonds of SO with Zr, the active site being Lewis sites on the S atoms.     

Preparation of ion-exchange beads based on poly(methacrylic acid) brush grafted chitosan beads: Isolation of lysozyme from egg white in batch system

Poly(methacrylic acid) brush grafted crosslinked-chitosan (chitosan-g-poly(MAA)) beads were prepared in two sequential steps: in the first step, chitosan beads were prepared by phase-inversion technique and then were crosslinked with epichlorohydrin under alkaline condition; in the second step, the graft copolymerization of methacrylic acid onto the chitosan beads was initiated by ammonium persulfate (APS) under nitrogen atmosphere. The chitosan-g-poly(MAA) beads were first used as an ion exchange support for adsorption of lysozyme (LYZ) from aqueous solution. The influence of pH, equilibrium time, ionic strength and initial LYZ concentration on the adsorption capacity of the chitosan-g-poly(MAA) ion-exchange beads has been investigated in a batch system. Maximum LYZ adsorption onto chitosan-g-poly(MAA) beads was found to be 65.7 mg/g at pH 6.0. The experimental equilibrium data obtained LYZ adsorption onto chitosan-g-poly(MAA) ion-exchange beads fitted well to the Langmuir isotherm model. Kinetics parameters of this adsorption system were also analyzed by using the equilibrium experimental data. The result of kinetic analyzed for LYZ adsorption onto ion-exchange beads showed that the second order rate equation was favourable. Finally, the chitosan-g-poly(MAA) ion-exchange beads were used for the purification of LYZ from egg white in batch system and the purity of the eluted LYZ from ion-exchange chitosan-g-poly(MAA) beads was determined as 94% by HPLC from single step purification.     

L-半胱氨酸与牛血清白蛋白的相互作用

用差示扫描量热法(DSC)和荧光光谱法研究了L-半胱氨酸(L-Cys)与牛血清白蛋白(BSA)在一定离子强度的Tris-HCl缓冲溶液中的相互作用,发现随着L-Cys浓度的增大,BSA的DSC热转变曲线峰形变宽变平,变性温度(tm)和变性焓(△Hm)降低,说明L-Cys的加入使BSA分子的高级结构发生了变化,稳定性降低...     

量子点与蛋白质相互作用热力学

量子点(QDs)具有宽激发窄发射、量子产率高、发射波长可调和抗光漂白等优异的光学性质,因而在生物医学成像与示踪、生物传感等方面有着广泛的应用前景。量子点进入生命体系后,首先会遇到蛋白质,量子点与蛋白质之间发生相互作用后,蛋白质的结构和功能会因此发生变化,量子点的性能及应用也会发生改变。研究量子点与蛋白质的相互作用规律,可以为量子点的精细设计、高效应用以及生物安全性评价提供理论依据。本文在总结国内外相关文献和本课题组工作的基础上,介绍了量子点与蛋白质相互作用的热力学方法;重点从热力学角度揭示量子点与蛋白质相互作用的机制。     

Dendrimer与表面活性剂相互作用的研究进展

聚酰胺-胺(PAMAM)是目前最具应用前景的树枝状大分子(Dendrimer),它与表面活性剂相互作用后能够形成聚集体,这种聚集体能有效地改变体系的微观环境和物理化学性质。本文综述了小分子表面活性剂与Dendrimer相互作用研究方面所取得的进展,重点讨论了阴离子表面活性剂和阳离子表面活性剂与Dendrimer混合体系中溶液的疏水环境、浊度以及形成聚集体中值粒径等物理化学性质的变化,在此基础上,讨论了Dendrimer与小分子表面活性剂相互作用在机理方面所取得的研究进展,为进一步扩大Dendrimer的应用领域提供参考。     

NMR研究草酸双过氧钒配合物与精氨酸的相互作用

应用多核(^1H,^13c,^15N和^51V)NMR、二维扩散排序谱(2D DOSY)、变温NMR以及电喷雾质谱(ESI-MS)等谱学方法研究草酸双过氧钒配合物与精氨酸(Arg)的相互作用,发现该相互作用体系可生成以氨基配位的新过氧钒物种[OV(O2)2Arg]^-,研究结果表明综合利用上述谱学方法有助于揭示此类相互作用体系的反应过程和配位机制。     

NMR研究N-甲基咪唑与双过氧钒配合物的相互作用

为探讨过氧钒配合物上有机配体对反应平衡的影响, 在模拟生理条件下(0.15 mol/L NaCl溶液), 应用多核(¹H, ¹³C和⁵¹V)多维(COSY) NMR以及变温技术等谱学方法研究双过氧钒配合物[OV(O₂)₂LL']ⁿ− [n＝1～3, LL'＝oxalate (缩写为oxa)、picolinate(缩写为pic)、bipyridine(缩写为bipy)和1,10-phenanthroline(缩写为phen), 与它们配位的含钒物种分别缩写为bpV(oxa), bpV(pic), bpV(bipy)和bpV(phen)]与N-甲基咪唑(缩写为N-Me-Im)的相互作用, 实验结果表明N-Me-Im与4种双过氧钒配合物的反应活性从强到弱的顺序为: bpV(oxa)＞bpV(pic)＞bpV(bipy)＞bpV(phen). 研究表明金属中心上配体的配位能力和空间位阻都对反应平衡产生较大的影响, 同时竞争配位的结果导致新的过氧物种[OV(O₂)₂(N-Me-Im)]⁻的生成, 而利用上述谱学方法则有助于揭示此类相互作用体系的反应过程和配位方式.     

双过氧钒配合物与吡啶类配体相互作用的NMR研究

为探讨单齿/双齿吡啶类配体对反应平衡的影响,在模拟生理条件下(0.15mol·L-1NaCl溶液),应用多核(1H、13C和51V)多维(COSY,HSQC和HMBC)以及变温NMR技术研究双过氧钒配合物[OV(O2)2(D2O)]-/[OV(O2)2(HOD)]-(简写为bpV)与系列吡啶类配体的相互作用,研究结果表明bpV与有机配体的反应性从强到弱的顺序为:2,2′-联吡啶2,2′-联吡啶-4,4′-二甲酸根吡啶异烟酸根,这说明双齿吡啶类配体配位能力强于单齿配体,而不带羧基的吡啶类配体(单齿或双齿)配位能力强于所对应的带羧基的取代吡啶,竞争配位导致一系列新的6配位(配体为吡啶或异烟酸根)或7配位(配体为2,2′-联吡啶或2,2′-联吡啶-4,4′-二甲酸根)的过氧钒物种[OV(O2)2LL′]n-(LL′=吡啶类配体,n=1,2,3)生成。     

Interactions between gelatin and sodium dodecyl sulphate: binding isotherm and solution properties

The interaction between sodium dodecyl sulphate (SDS) and gelatin was studied at pH 4.5 and 6.5 where the gelatin is positively charged (i.e.p. 8). At pH 4.5 a SDS/gelatin concentration range was found where gelatin precipitates. At pH 6.5 the SDS-gelatin complex remains soluble although three SDS concentration domains were distinguished where the SDS-gelatin complex had very different affinities for the solvent. Below C₁ the complex was highly surface active but other measurements (viscosity, potentiometry, protons uptake) did not reveal any particular consequence of binding. Between C₁ and C₂ the molecular size decreased (viscosity lowering) upon charge neutralization and collapse about small SDS aggregates (17 SDS molecules per gelatin molecule). Above C₂ a cooperative binding mechanism lead to the formation of SDS aggregates; the complex stretched out and turned strongly hydrophilic (the viscosity increases, low surface activity). At saturation one gelatin molecule bound about 200 SDS molecules. Above the overlap concentration (about 3 wt%) SDS aggregates formed between several gelatin molecules, the viscosity increased continuously with SDS concentration and the binding ratio was lower than in dilute gelatin solutions. A very good correspondence was found between the different analytical data including turbidity, viscosity, surface tension, protons uptake and direct potentiometric SDS binding measurements.     

Protein-surfactant interaction: differences between fluorinated and hydrogenated surfactants

The interactions of proteins with fluorinated/hydrogenated surfactants were investigated by circular dichroism and turbidity measurement. Pairs of fluorinated and hydrogenated surfactants with similar critical micelle concentrations (cmc), including sodium perfluorooctanoate/sodium decylsulfate and lithium perfluorononanoate/sodium dodecylsulfate were compared in view of their interactions with proteins including BSA, lysozyme, β-lactoglobulin and ubiquitin. It was found that fluorinated surfactants exhibited stronger interactions with proteins than hydrogenated ones, which, however, depended on the structures of both proteins and surfactant molecules. If the proteins are very stable, or the surfactant–protein interactions are very strong, such differences between the two kinds of surfactants might be indistinguishable.