Effects of forced solution flow on lysozyme crystal growth

In order to study quantitatively the effects of forced solution on crystal growth, we designed a new set of experimental equipment, in particular, a microchannel mixer was used as crystallization container so that the consumption of protein samples was much reduced and thus an exact syringe pump could be used for precise control of the flow rates. Since the mixer's section was designed to be rectangular, the solution velocity in its center was steady and constant, and thus repeatable experiments were facilitated. Experimental results showed that the effects of forced solution on protein crystal growth were different under different levels of supersaturation, and new results were obtained for cases of high supersaturation. When the supersaturation is σ = 2.3, with increasing flow rates the growth rates of the lysozyme crystal's (110) face hardly change when the flow rates are lower than 1300 μm/s, and decrease quickly afterwards. When the flow rate reaches 2000 μm/s, the crystal nearly ceases to grow. When the supersaturation is σ = 2.7, with increasing flow rates the (110) face growth rates increase at the beginning then reach the maximum values at 1700 μm/s – 1900 μm/s and decrease afterwards, approaching zero or so when the flow rate reaches 12000 μm/s. The higher the supersaturation, the larger the flow rate at which the crystal ceases to grow. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

等转化率法分析溶菌酶在添加剂影响下的热变性行为

利用差示扫描量热法(DSC)研究了溶菌酶在不同浓度的添加剂(蔗糖、葡萄糖、果糖、甘露醇)和不同浓度的磷酸盐缓冲液影响下的热变性过程,并用等转化率法对该过程进行了分析.变性温度Tm随扫描速率和添加剂浓度的增加而提高,随磷酸盐缓冲液浓度的增加而降低.磷酸盐加速了溶菌酶的变性过程,降低了溶菌酶的热稳定性,而添加剂则增强了溶菌酶的热稳定性.在添加剂存在的条件下,溶菌酶变性过程部分为聚集不可逆过程,部分则可逆.等转化率法表明溶菌酶在所有条件下,其表观活化能在不同的转化率下并未保持不变,而是随转化率增大而减小,说明了一个简单的反应机理并不能用来描述溶菌酶的变性过程,其过程并不是标准两态可逆过程,而是一个涉及多种蛋白质状态的复杂过程.     

LC of sulfonamide residues in poultry muscle and eggs extracts using fluorescence pre‐column derivatization and monolithic silica column

A new, rapid, sensitive and selective HPLC method with fluorescence detection is described for the simultaneous determination of 12 sulfonamides, in the presence of putrescine as internal standard, after pre‐column derivatization with fluorescamine. The drugs were separated on a Chromolith Performance RP‐18 column (100×4.6 mm), using a gradient elution with a binary mobile phase of methanol/0.05 M acetate buffer (pH 3.4). Linearity of derivatization was obtained for concentrations from 3.0 to 300 μg/L in standard solutions. The whole procedure was evaluated and fully validated, according to the European Union Decision 2002/657/EC, for the determination of sulfonamides in turkey muscle and hen eggs following SPE. The LODs varied from 2 to 17 μg/kg in turkey and 2 to 15 μg/kg in egg samples. The average recoveries ranged between 96.9–108.6% in turkey muscle and 96.0–108.4% in egg samples, respectively.     

A synthetic camel anti-lysozyme peptide antibody (peptibody) with flexible loop structure identified by high-resolution affinity mass spectrometry

We describe the synthesis and characterisation of the fully functional molecular recognition structure of a 26-amino acid residue peptide antibody, referred to as peptibody, designed from a monoclonal single-domain antibody fragment derived from a camel heavy-chain antibody. The CDR3 region (CDR = complementarity determining region) of the cAbLys3 camel antibody fragment, which binds to the active site of hen eggwhite lysozyme (HEL) and acts as a potent enzyme inhibitor by mimicking an oligosaccharide substrate, was prepared by solid-phase peptide synthesis. To obtain a closed loop-like structure resembling that in the crystal structure, N- and C-terminal cysteine residues were added to the linear peptide and oxidised to a cyclic disulfide-bridged peptide by using dimethylsulfoxide. A further, internal cysteine-12 residue was acetamidomethyl-protected to prevent possible oxidative byproducts. Affinity separation on a lysozyme microcolumn combined with MALDI-TOF mass spectrometry revealed that the peptide resumed high affinity to lysozyme only after deprotection of Cys-12, suggesting the importance of this paratope sequence for epitope recognition. The complex of lysozyme and active peptibody was characterised directly by conducting high-resolution ESI-FTICR mass spectrometry, which provided a molecular comparison of affinities for linear and cyclic peptibodies.     

The Effect of pH Difference Between Two Phases on the Partition of Lysozyme in Aqueous Two-Phase System

Aqueoustwo-phasesystems(ATPS)areformedwhentwoaqueoussolutionsofhydrophilicpolymersorapolymerandasaltaremixedathighconcentration.Forthechargedproteins,AlbertssonhasdevelopedtheclassicalmodelfortheelectrochemicalpartitioninginATPSfInK,=lnK, (Z,F/RT)Arp(l)HereK,denotesthepartitioncoefficientofproteins,InK,includesalltheotherfactorsaffectingproteinpartitioning,and(Z,F/RT)A(PistheelectrostatictermasaproductofproteinsurfacechargeZPandAam,theelectrostaticpotentialdifferencebetweenthetwopha…     

Hen ovomucoid-agarose: A new conjugate for the isolation by affinity chromatography of UDP-galactose: Glycoprotein galactosyl-transferase

A new conjugate for the affinity chromatography of UDP-galactose:glycoprotein galactosyltransferase has been synthesized by coupling hen ovomucoid, a ligand similar to the acceptor substrate, to agarose. The hen ovomucoid-Sepharose conjugate binds galactosyl transferase more tightly that other acceptor-Sepharose conjugates. The new adsorbent gives comparable yields and purifications with those obtained by ligands similar to the nucleotide moiety of the substrate and to the “specifier” protein, α-lactalbumin. The soluble galactosyltransferase from rat ventral prostate is effectively removed from the high speed supernatant by an ovomucoid-Sepharose column. The enzyme can be eluted with buffer containing EDTA andN-acetylglucosamine in a high yield (75–80%) and in a purified form (4000-fold purification). The stability of ovomucoid to heat and to high concentrations of urea and its inhibition of some proteases makes the conjugate easy to operate with an quite useful even with rather crude preparations.     

脲和盐酸胍诱导溶菌酶去折叠的荧光相图法研究

用荧光相图法分别研究了脲和盐酸胍诱导卵清溶菌酶去抓叠的过程。当变性体 系中无还原剂2－巯基乙醇存在、脲浓度从0变化至4.0 mol/L（或盐酸胍浓度从0变 化至3.0 mol/L）时,溶菌酶从天然态转变为部分折叠中间态,当脲浓度从4.0 mol/L变化至8.0 mol/L（或盐酸胍浓度从3.0 mol/L变化至6.0 mol/L）时,溶菌 酶从中间态转变为去折叠态,此时该蛋白的变性过程符合“三态模型”。而当变性 体系中有该还原剂存在时,溶菌酶则由天然态直接转变为去折叠态,此时脲诱导该 蛋白去折叠的过程符合曲型的“二态模型”。实难结果表明荧光相图法可以检测蛋 白南去抓叠的中间态。     

疏水表面上吸附态溶菌酶的DSC和FTIR研究

联合差示扫描量热 (DSC)和傅里叶变换红外吸收光谱(FTIR)分别研究了鸡蛋白溶菌酶(Lyz)在适度疏水吸附剂(PEG-600)表面上吸附和折叠时,不同盐(硫酸铵)浓度、表面覆盖度和变性剂(盐酸胍)浓度对无水环境的吸附态天然和变性溶菌酶构象变化及热稳定性的影响。研究发现：随着硫酸铵浓度和溶菌酶表面覆盖度的增加,吸附态天然和变性溶菌酶的吸热峰温度都逐渐降低,同时在较高温度下的微扰也增多。吸附发生后,α-螺旋结构减少,β-折叠和β-转角结构增多。在FTIR图谱中,吸附态变性溶菌酶在1 400～1 425 cm-1处的C—C拉伸振动峰和1 650～1 670 cm-1处的酰胺Ⅰ带特征峰都能明显观测到。但是,与之相比,相同条件下吸附态天然溶菌酶在1 650～1 670 cm-1处特征峰却几乎看不到。吸附态天然溶菌酶发生了结构丢失,表现得更加不稳定。     

聚乙烯醇与溶菌酶的相互作用及其对溶菌酶构象的影响

在生理条件下, 使用凝胶过滤色谱、荧光光谱法、差示扫描量热分析和傅里叶变换红外光谱法(FT-IR)研究了溶菌酶与聚乙烯醇(PVA)的相互作用. 结果表明PVA与溶菌酶结合形成复合物, 在它们的相互作用过程中, 溶菌酶酪氨酸的发射荧光部分被猝灭, 但是, 相互作用并没有改变酪氨酸的微环境; 差示扫描量热分析结果表明, 溶菌酶与PVA之间的相互作用没有破坏溶菌酶的高级结构; 进一步使用红外光谱法结合可增强分辨率的傅里叶去卷积技术和高斯曲线拟合技术共同用于对溶菌酶与PVA复合物冻干粉中溶菌酶酰胺I带的定量分析, 发现冻干粉溶菌酶分子中与分子间相互作用相关的β-折叠组分含量减少了, 但是, 用于衡量冻干状态蛋白质结构完整性的α-螺旋组分含量没有降低. 活性分析结果进一步确认, PVA与溶菌酶的相互作用没有破坏溶菌酶的三级结构.