Optimization of Functional Monomer Content in Protein‐Imprinted Polymers

Abstract

We have prepared synthetic materials having binding specificity toward lysozyme using molecular imprinting. The lysozyme‐imprinted polymers were prepared on silica beads using acrylic acid, acrylamide, and N,N′‐methylenebisacrylamide. The molar ratio of acrylic acid to the template affected the selectivity and the maximum binding specificity was yielded with the molar ratio of 5 to 1 (acrylic acid/template). No binding specificity toward lysozyme was observed when there was no acrylic acid, and the binding specificity decreased when the amount of acrylic acid was high. The functional monomer‐template ratio indicated in this study could be useful for improving the binding specificity in molecular imprinting of proteins.     

Simultaneous acquisition of all four forms of circular polarization Raman optical activity: results for α‐pinene and lysozyme

A new circularly polarized (CP) Raman spectrometer is described that demonstrates simultaneous acquisition of all four forms of circular polarization Raman optical activity (ROA). The instrument is a design extension of a commercially available back scattering circular polarization (SCP) ROA spectrometer. Circular polarization of the incident beam is introduced with a quarter‐wave plate, and a half‐wave plate alternately positioned in and out of the beam controls the modulation between right circular polarization (RCP) or left circular polarization (LCP) states. Combining this modulation with the simultaneous detection of RCP and LCP scattered Raman radiation allows the measurement of incident circular polarization (ICP), SCP, in‐phase dual circular polarization(DCP_I) and out‐of‐phase DCP_II‐ROA. In addition, three different forms of backscattered Raman spectra, namely unpolarized, highly polarized, and depolarized Raman spectra, as well as a degree of circularity spectrum are obtained. The performance of the new all‐CP ROA spectrometer is evaluated with neat α‐pinene and aqueous hen lysozyme solution. Copyright © 2011 John Wiley & Sons, Ltd.     

The dissolution phenomenon of lysozyme crystals

Dissolution studies on lysozyme crystals were carried out since the observed dissolution pattern look different from non‐protein dissolved crystals. The Tetragonal, High Temperature and Low Temperature Orthorhombic morphologies, crystallized using sodium chloride, were chosen and the influence of different pH, salt and protein concentration on their dissolution was investigated. An increase in pH and/or salt concentration can modify the dissolution behaviour. The pattern of the crystals during the dissolution process will, therefore, develop differently. Frequently a skeleton like crystal pattern followed by a falling apart of the crystals is observed. The multi‐component character of the lysozyme crystal (protein, water, buffer, salt) as well as “solvatomorphism” gives first insights in the phenomena happening in the dissolution process. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A thermodynamic study of protein-induced lipid lateral phase separation. Effect of lysozyme on mixed lipid vesicles

In this work we study by differential scanning calorimetry (DSC) the lateral phase separation induced by a globular protein (lysozyme) on vesicles built-up by charged (phosphatidic acid) and neutral (phosphatidylcholine) lipids.The adsorption of the positively charged protein onto the negative vesicle surface induces the formation of micro-domains richer in the charged lipid component. This phenomenon is revealed as a splitting of the excess heat capacity peak associated to the melting of the lipid hydrocarbon chains.Also, the peak associated to the protein denaturation is shifted, suggesting the presence of adsorbed proteins onto the vesicle surface. The surface electrostatic potentials, both of proteins and vesicles, have been modulated by pH and ionic strength variations, showing a deep influence of the electric charges in modifying protein adsorption, rate of denaturation (related to unfolding enthalpy variation), and lipid micro-domain formation.Some of the present results have been rationalized on the basis of a theoretical model recently developed by the authors.     

Refolding of Denatured／Reduced Lysozyme Using Weak－Cation Exchange Chromatography

Oxidative refolding of the denatured/reduced lysozyme was investigated by using weak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming intermolecular aggregates. At the same time urea and ammonium sulfate were added to the mobile phase to increase the elution strength for lysozyme. Ammonium sulfate can more stabilize the native protein than a common eluting agent,sodium chloride. Refolding of lysozyme by using this WCX is successfully. It was simply carried out to obtain a completely and correctly refolding of the denatured lysozyme at high concentration of 20.0 mg/mL.     

Volumetric Properties of Amino Acids and Hen-Egg White Lysozyme in Aqueous Triton X-100 at 298.15 K

The apparent molar volumes, V_,2, of glycine, alanine, -amino-n-butyric acid, valine, leucine, and lysine monohydrochloride have been determined in aqueous solutions of 0.05, 0.1, and 0.4 mol-kg^–1 Triton X-100 (TX-100), and the partial specific volume, v⁰, of hen-egg-white lysozyme in 0.4 mol-kg^–1 TX-100 by density measurements at 298.15 K. These data have been used to calculate the infinite dilution apparent molar volumes, V_2,m⁰, for the amino acids in aqueous TX-100 solutions and the standard partial molar volumes of transfer, _tr V_2,m⁰, of the amino acids from water to the aqueous surfactant solutions. The linear correlation of V_2,m⁰ for a homologous series of amino acids has been utilized to calculate the contribution of the charged end groups (NH₃⁺, COO^–), CH₂ group and other alkyl chains of the amino acids to V_2,m⁰. The results on _tr V_2,m⁰, of amino acids from water to aqueous TX-100 solutions have been interpreted in terms of ion–ion, ion–polar, hydrophilic–hydrophilic and hydrophobic–hydrophobic group interactions. For all the six amino acids studied, the values of _tr V_2,m⁰ from water to all the studied concentrations of aqueous TX-100 are small in spite of their different hydrophobic content, indicating an overall balance in interactions of zwitterionic/hydrophilic groups of amino acids with the hydrophilic groups of TX-100, and of hydrophobic and ionic/hydrophilic groups of the amino acids with hydrophobic groups of TX-100. Comparison of the interactions of the amino acids with nonionic, anionic and cationic surfactants has also been made and discussed. The partial specific volume of transfer of lysozyme from water to aqueous TX-100 solutions also indicates a balance of the hydrophobic and hydrophilic interactions in the protein–nonionic surfactant system.     

基于异硫氰酸荧光素标记溶菌酶和荷正电纳米金构建荧光共振能量转移平台检测广谱细菌

通过异硫氰酸荧光素标记的溶菌酶（FITC－Lys）和聚乙烯亚胺修饰的荷正电纳米金粒子（PEI－AuNPs）与细菌结合,构建了一个基于荧光共振能量转移（FRET）的平台用于广谱细菌检测。待检样本中存在细菌时,荷正电的FITC－Lys（能量供体）通过与细胞壁肽聚糖的识别作用和静电作用与细菌结合,荷正电的PEI－AuNPs（能量受体）也通过静电作用与细菌结合,使能量供体和受体之间的距离缩短,荧光猝灭,显示出低荧光强度的FRET“开”的信号读出模式。采用水热法合成了PEI－AuNPs,通过紫外光谱、透射电镜、激光粒度仪等对PEI－AuNPs进行了表征。结果表明,PEI－AuNPs在526.5 nm处有最大紫外吸收,直径约为9.6 nm,荷正电。该方法可对10种细菌（如金黄色葡萄球菌、肺炎链球菌、粪肠球菌、表皮葡萄球菌、李斯特菌、大肠杆菌、鼠伤寒沙门氏菌、甲型副伤寒沙门氏菌、副溶血弧菌和铜绿假单胞菌）进行检测。考察了实际样本（以牛奶和果汁为例）中可能的干扰组分对检测的干扰情况。结果表明,将样本稀释10倍后,样本中蛋白、离子等不会对分析结果产生干扰。该研究构建的广谱细菌检测方法,对于致病菌的防控具有积极意义。     

Structural Compactness in Hen Egg White Lysozyme Induced by Bisphenol S: A Spectroscopic and Molecular Dynamics Simulation Approach

The endocrine disrupting compound Bisphenol and its analogues are widely used in food packaging products and can cause serious health hazards. The protein, Lysozyme (Lyz), showing anti-microbial properties, is used as a “natural” food and dairy preservative. Herein, we explored the interaction between Lyz and Bisphenol S (BPS) by multi-spectroscopic and theoretical approaches. Lyz interacts with BPS through static quenching, where hydrophobic force governed the underlying interaction. Molecular docking results reveal that tryptophan plays a vital role in binding, corroborated well with near UV-CD studies. A decrease in the radius of gyration (from 1.43 nm to 1.35 nm) of Lyz substantiates the compactness of the protein conformation owing to such an interaction. This structural alteration experienced by Lyz may alter its functional properties as a food preservative. Consequently, this can degrade the quality of the food products and thereby lead to severe health issues.     

Changes in the characteristics of acid‐treated clay after the inclusion of proteins

The aim of this work is to study the difference in the characteristics of commercial clay after the inclusion of two proteins. Bovine serum albumin and egg white lysozyme were immobilized on the structure of commercial montmorillonite, which was previously treated with a hydrochloric acid solution. Studies were carried out at two different concentrations, fixing the pH to ensure the charge of biomolecules and clay surface. Acid‐treated clay containing proteins was characterized by X‐ray diffraction, Fourier transformed infrared spectroscopy, thermogravimetric analysis, and nitrogen adsorption at 77 K. The powders showed similar thermal behavior after the inclusion of proteins but with variations in the amount of mass loss in each sample. Moreover, changes in the surface characteristics of the final solid were observed, depending on both the concentration and the nature of the incorporated protein. The differences observed in the acid‐treated clay characteristics after the inclusion of each type of protein are discussed. The characterization of materials after the inclusion of protein molecules can be useful to understand the adsorption mechanism of biomolecules on solid surfaces. Copyright © 2014 John Wiley & Sons, Ltd.     

The small‐angle and wide‐angle X‐ray scattering set‐up at beamline BL9 of DELTA

The multi‐purpose experimental endstation of beamline BL9 at the Dortmund Electron Accelerator (DELTA) is dedicated to diffraction experiments in grazing‐incidence geometry, reflectivity and powder diffraction measurements. Moreover, fluorescence analysis and inelastic X‐ray scattering experiments can be performed. Recently, a new set‐up for small‐angle and wide‐angle X‐ray scattering utilizing detection by means of an image‐plate scanner was installed and is described in detail here. First small‐angle X‐ray scattering experiments on aqueous solutions of lysozyme with different cosolvents and of staphylococcal nuclease are discussed. The application of the set‐up for texture analysis is emphasized and a study of the crystallographic texture of natural bio‐nanocomposites, using lobster and crab cuticles as model materials, is presented.