双水相萃取法从风干香肠中分离提取蛋白酶

双水相萃取(ATPS)是近年来发展起来的蛋白纯化方法。为了扩展该方法适应领域,同时为风干香肠形成过程酶系的研究提供具体方法。本实验研究了运用双水相技术分离提取风干香肠中蛋白酶,对构成双水相体系中的PEG分子量、浓度和类型以及盐浓度的影响进行了分析。确定了双水相组成体系为20%PEG1000(m/m)和25%MgSO4(m/m),在此体系中风干香肠的蛋白酶主要分布在上相,最高酶活12.37U/μg;纯化倍数为4.61;回收率为85%。通过分子筛层析对比,表明风干香肠经过双水相分离提取杂蛋白峰被除去,而蛋白酶峰几乎未受到影响,说明该双水相体系萃取香肠中蛋白酶具有良好的专一性。调解双水相pH值对蛋白酶的萃取没有影响,而添加电解质NaCl反而产生不利影响。     

Mechanism for the direct synthesis of tryptophan from indole and serine: a useful NMR technique for the detection of a reactive intermediate in the reaction mixture

The reaction mechanism for the biomimetic synthesis of tryptophan from indole and serine in the presence of Ac₂O in AcOH was investigated. Although the time‐course ¹H‐NMR spectra of the reaction of 5‐methoxyindole with N‐acetylserine were measured in the presence of (CD₃CO)₂O in CD₃CO₂D, the reactive intermediate could not be detected. This reaction was conducted without 5‐methoxyindole in order to elucidate the reactive intermediate, but the intermediate could not be isolated from the reaction mixture. Since the intermediate would be expected to have a very short life time, and therefore be very difficult to detect by conventional analytical methods, the structure of the intermediate was elucidated using a 2D‐NMR technique, diffusion‐ordered spectroscopy (DOSY). Two intermediates were detected and confirmed to be 2‐methyl‐4‐methyleneoxazol‐5(4H)‐one and 2‐methyl‐4‐hydroxymethyloxazol‐5(4H)‐one. The present results demonstrated that DOSY is a powerful tool for the detection of unstable intermediates. Copyright © 2010 John Wiley & Sons, Ltd.     

Two Tags in One Probe: Combining Fluorescence- and Biotin-based Detection of the Trypanosomal Cysteine Protease Rhodesain

Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a k_inac/K_i value of 37,000 M⁻¹s⁻¹. Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.     

New parameterization approaches of the LIE method to improve free energy calculations of PlmII‐Inhibitors complexes

The standard parameterization of the Linear Interaction Energy (LIE) method has been applied with quite good results to reproduce the experimental absolute binding free energies for several protein–ligand systems. However, we found that this parameterization failed to reproduce the experimental binding free energy of Plasmepsin II (PlmII) in complexes with inhibitors belonging to four dissimilar scaffolds. To overcome this fact, we developed three approaches of LIE, which combine systematic approaches to predict the inhibitor‐specific values of α, β, and γ parameters, to gauge their ability to calculate the absolute binding free energies for these PlmII‐Inhibitor complexes. Specifically: (i) we modified the linear relationship between the weighted nonpolar desolvation ratio (WNDR) and the α parameter, by introducing two models of the β parameter determined by the free energy perturbation (FEP) method in the absence of the constant term γ, and (ii) we developed a new parameterization model to investigate the linear correlation between WNDR and the correction term γ. Using these parameterizations, we were able to reproduce the experimental binding free energy from these systems with mean absolute errors lower than 1.5 kcal/mol. © 2010 Wiley Periodicals, Inc. J Comput Chem 2010     

含丝氨酸和组氨酸残基手性肽核酸单体的合成

设计合成了含组氨酸残基碱基为胸腺嘧啶的手性肽核酸单体, 咪唑氨基的最终保护基为2,4-二硝基苯基(Dnp). 对文献合成方法进行了适当改进, 制备了两种含丝氨酸和组氨酸残基碱基为腺嘌呤的手性肽核酸单体, 以上化合物均可作为制备手性肽核酸的基本构建单元.     

Quantum mechanical calculations on phosphate hydrolysis reactions

Multiple biological processes are regulated by kinases and phosphatases. This study aims to provide nonenzymatic models for phosphorylation and dephosphorylation of serine, threonine, and tyrosine phosphate using ab initio guantum mechanical calculations. We reduce the problem to methyl phosphate hydrolysis to model serine/threonine, and the hydrolysis of phenyl phosphate to model the tyrosine. HF, B3LYP, and MP2 calculations with a 6‐31+G(d) basis set were employed. The effect of water as a catalyst was also analyzed. As expected, the activation energy barrier is lowered. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 43–51, 2000     

Phosphorus-containing compounds regulate mineralization

Phosphorus is the main constitutive element of minerals and fat in the body. The process of mineral formation is defined as mineralization. The minerals in the body are mainly apatite, which is the inorganic phase that composes bones and teeth. It is worth noting that people with high fat content tend to cause excessive bone mineralization, which leads us to believe that different phosphorus-containing compounds in the body are mutually transformed and can regulate mineralization in different ways. The conversion and regulation of different phosphorus-containing compounds on the mineralization are essential for formation of a complex hierarchical structure and adaptation of the bone to various mechanical environments. Therefore, this review introduces the natural phosphorus-containing compounds in the body, introduces the hierarchical structure of the bone, and summarizes recent studies on different phosphorus-containing compounds (inorganic, organic, and phosphorus-containing proteases) involved in the biomineralization. We also discuss potential research directions of the biomineralization, offering the basis for future investigation of advanced bone substitute materials.     

脱氢丙氨酸合成方法研究

脱氢氨基酸(DHA)是许多天然抗生素肽和活性真菌代谢物如枯草菌素(subtilin)、乳酸链球菌(Nisin)等的重要组成部分及非天然氨基酸和肽合成中的一类重要中间体,同时也是研究催化氢化的极好的底物。脱氢丙氨酸的合成方法很多,但产率都不高(58％左右)。     

Simultaneous microchemical analysis of glycerol,fatty acid,and phosphorus in complex lipids

A single, small glycerol-containing lipid sample (50–200 nmoles) after a mild hydrolysis could be used for analysis of fatty acids, glycerol, and phosphorus. The range of the assays was from 10–500 nmoles for each lipid.