Z及logI对离子交换色谱中脲变溶菌酶分子构象变化的表征

以溶菌酶(Lys)为目标蛋白,用计量置换保留理论(SDT-R)中的参数Z和logI对弱阳离子交换色谱(WCX)中“准天然态”和脲变还原与非还原的两种变性状态的Lys的分子构象变化进行了表征。发现在流动相中含有脲时,蛋白的保留仍服从SDT理论,可以准确测定在特定脲浓度条件下Lys的Z及logI值。结果表明,3种分子构象状态的Lys的Z值均随脲浓度改变呈现不连续变化;“准天然态”Lys在同一脲浓度条件下的Z值比变性状态的大,logI比变性状态的小,而非还原变性态和“准天然态”Lys的Z和logI值比较接近。还     

Slow dynamics of supercooled hydration water in contact with lysozyme: examining the cage effect at different length scales

ABSTRACT

We study by computer simulation the dynamics of hydration water in solution with lysozyme upon approaching the glassy state of water. We calculate the self-density correlation function at different wavelengths to test the Mode Coupling Theory (MCT) of glassy dynamics at different length scales. The results show a strict and clear relation of the behaviour of the structural relaxation with the cage effect. We find a good agreement with the predictions of the MCT in the short and medium scale range, while at increasing length scales the interaction of water molecules with the protein's substrate induces deviations from the MCT behaviour, as found in previous studies. Besides at low temperatures the slow dynamics deviates from MCT due to hopping processes, similar to the bulk, as witnessed by a crossover from a fragile behaviour to a strong behaviour. We show that this deviation is evident at all length scales. Interestingly, we find that in the fragile region the confining cage decreases in radius with temperature while in the strong region it appears stable.     

溶菌酶响应纳米钻石载药体系制备及其与HepG2细胞作用

通过缩水甘油(GLY)与阿霉素(DOX)发生开环反应,赋予阿霉素活性羟基基团,以氨基己酸(ACA)为桥梁,通过共价键合方法将缩水甘油化阿霉素(GLY-DOX,GDOX)偶联于氨基己酸化纳米钻石(ND-ACA)载体上,获得具有溶菌酶响应特性的ND-ACA-GLY-DOX(NAGD)药物输送体系。 采用荧光光谱法测定了ND表面偶联ACA和DOX的量分别为(185±10.0) μg/mg和(115±5.2) μg/mg。 体外释药发现NAGD在生理环境(pH=7.4)中药物释放量很低,而在肿瘤细胞溶酶体环境中(溶菌酶存在下),酯键水解断裂,大量释放DOX。 以肝癌细胞HepG2为模型,采用细胞形态测试法表明NAGD可有效杀死肿瘤细胞。 上述研究结果表明,NAGD可作为一种良好的药物输送体系,并为共价偶联药物开拓新思维。     

Complex of lysozyme and Myramistin: formation and adsorption at the water–xylene interface

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Enhancing the Sensitivity of Aptameric Detection of Lysozyme with a “Feed‐Forward” Network of DNA‐Related Reaction Cycles

In this study, a network of DNA‐related reaction cycles was established to enhance the sensitivity of lysozyme detection with dual signal amplification, and aptamer‐based reactions were integrated into this system to provide high specificity. The network was organized in a feed‐forward manner: the “upstream cycles” recognized the lysozyme (the target) and released the “messenger strands” from probe A (a DNA construct); the “downstream cycles” received them and then released the “signal strands” from another DNA construct, probe B, in multiplied quantities to that of the original inputted lysozyme. The upstream cycles centered on “target‐displacement polymerization”, which circulates the lysozyme to provide primary amplification; the downstream cycles centered on “strand‐displacement polymerization”, which circulates the messenger strand to provide further amplification. There were also several “nicking–polymerization” cycles in both reaction groups that provide extra signal amplification. In total, the network enclosed eight interconnected and autonomic reaction cycles, with only two probes, two primers, and two enzymes needed as raw feeds, and the network can be operated simply in one‐pot mode. With this network, lysozyme could be quantified at lysozyme concentrations as low as 2.0×10^?14 M , with a detection limit of 3.6×10^?15 M (3σ rule), which was seven orders of magnitude lower than that obtained without any amplification(1.8×10^?8 M ). Detection of lysozyme in real serum samples confirmed the reliability and practicality of the assay based on this reported reaction network.     

光谱法比较喜树碱与溶菌酶、过氧化氢酶的相互作用

采用紫外可见吸收、红外、荧光等多种光谱学方法研究了抗癌药物喜树碱和溶菌酶及过氧化氢酶两种蛋白之间的相互作用,结果表明,喜树碱与之均形成基态复合物并引起蛋白内源荧光猝灭。通过计算确定了喜树碱和蛋白相互作用的结合常数、结合位点数、主要作用力类型以及与蛋白中色氨酸的结合距离。喜树碱对过氧化氢酶具有较高的结合能力,这主要是由于喜树碱不仅与过氧化氢酶中的色氨酸存在相互作用,也与酶中的酪氨酸及血红素产生相互作用。喜树碱的存在可引起蛋白构象发生变化,使α-螺旋二级结构减少。     

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.     

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)     

Development of high-performance and rapid immunoassay for model food allergen lysozyme using antibody-conjugated bacterial magenetic particles and fully automated system

A high-performance and rapid chemiluminescence immunoassay for model food allergen lysozyme, one of the major allergenic components in egg white, using antibody-conjugated bacterial magnetic particles and a fully automated system was developed. This system contians a reaction station, tip rack, and an eight-tip pipettor that is alble to attach and detach a strong magnet to the tip surface. The immunoreaction time was shortened to 5 min, and the assay was completed within 20 min. The lower detection limit for lysozyme was 10 ng/mL. This system can be used to perform 24 samples in 60 min within 10% coefficient of variation.