氧气和一氧化碳在人血红蛋白迁移过程研究

为了揭示CO和O₂竞争性结合人血红蛋白血红素位点的机制及其与人血红蛋白结构转换之间的关系,本文采用全原子分子动力学模拟（MD）结合马尔科夫状态模型（MSMs）研究氧气（O₂）和一氧化碳（CO）分子从水溶液迁移进入人血红蛋白四聚体α链和β链的全过程。分子动力学模拟揭示了O₂和CO结合α链和β链的稳态结合位点和瞬态结合位点、迁移通道以及α链的结构变化。结果显示,分子模拟不仅仅能够再现全部实验中所观察到的离散Xe结合位点和分子扩散通道,而且揭示了实验中无法观测的瞬态结合位点和多重气体迁移途径。上述结果表明人血红蛋白因其结构柔性所形成的瞬态通道对于气体分子迁移过程的重要性。除此之外,利用MSM和过渡路径理论（TPT）构建了人血红蛋白α链结构变化与气体分子迁移之间的关系,阐释了血红蛋白中影响气体迁移的关键结构及其微观机制。     

体外孵化模型中乳酸盐对肌红蛋白结构影响的机理

为了研究乳酸盐与肌红蛋白(Myogolobin,Mb)血红素辅基的相互作用机制。本试验以牦牛背最长肌为原材料,对牦牛背最长肌中的肌红蛋白进行分离纯化,纯化后加入乳酸钙,在4 ℃条件下贮藏0、12、24、36、72 h,建立体外模型系统,采用紫外-可见吸收光谱、荧光光谱和圆二色光谱测定其在特征峰的吸光度,研究肌红蛋白的结构变化。结果表明紫外-可见光谱在冷藏期间4个时间点409 nm的特征峰处吸收强度都略有增加,但峰位和峰形几乎没变化;荧光光谱在597 nm处发射峰强度减弱,肌红蛋白的铁卟啉环的发射峰位置也未发生位移,但荧光强度随着贮藏时间的延长而减弱;圆二色谱表明对照组和处理组在远紫外区192、208和222 nm 3个特征峰的形状和肩峰的位置未发生明显改变,在190~240 nm处的图谱在乳酸钙处理组中和对照组一样曲线平滑。说明了乳酸盐和Mb的共价加合作用并未发生在血红素辅基上,而是通过非血红素配体结合的方式对肌红蛋白进行调节。

     

恶性骨肿瘤患者红细胞的拉曼光谱研究

通过对正常人和恶性骨肿瘤患者红细胞的拉曼光谱进行了研究.发现对于恶性骨肿瘤患者,部分谱线如1001.2,1228.8,1398.7,1372.8cm-1红移了2～4cm-1,说明这些谱线对应的基团内部构象发生微小变化.以1586cm-1为标记峰得到各峰的相对峰强比,发现恶性骨肿瘤患者红细胞的拉曼峰相对于正常人的相对强度的比值在1505,1228,1087,1001和747cm-1处显著减小,说明亚铁血红素、苯丙氨酸和脱氧核糖在红细胞内的含量减少.由统计分析可知Ⅰ1586/1228,Ⅰ1586/1001和Ⅰ1586/1169的峰-峰比的变化可为恶性骨肿瘤的早期诊断提供一定的依据.     

酶解鹅血制备高氨基酸血红素

利用Box-Behnken中心组合实验设计对碱性蛋白酶水解鹅血提取高氨基酸鹅血血红素的工艺条件进行了优化。结果表明:对水解度的影响次序为酶浓度>水解温度>水解时间>溶液p H。鹅血提取高氨基酸血红素的最佳酶解工艺参数:酶浓度5%,温度50℃,p H8.80,水解时间5.15 h,水解度为14.36%±0.18%。水解后,水解液中血红素含量为1.210 mg/m L,铁离子的含量为90.42μg/m L。      

Use of “filtered smoke” and carbon monoxide with fish

This review deals with the importance of flesh colour for quality assessment of muscle food and possibilities of influencing the colour via muscle colour pigments. Carbon monoxide as reagent to stabilise muscle colour and processes producing CO for this purpose are highlighted concentrating on those using filtered smoke (FS) to treat fish muscle. The scientific background is discussed and an overview is given on the situation concerning food law in this respect in different countries. Received: September 5, 2007     

Enhancing the stability of microsomal cytochrome b5: a rational approach informed by comparative studies with the outer mitochondrial membrane isoform

The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is much less prone to lose heme than the microsomal isoform (Mc b5), with a conserved difference at position 71 (leucine versus serine) playing a major role. We replaced Ser71 in Mc b5 with Leu, with the prediction that it would retard heme loss by diminishing polypeptide expansion accompanying rupture of the histidine to iron bonds. The strategy was partially successful in that it slowed dissociation of heme from its less stable orientation in bMc b5 (B). Heme dissociation from orientation A was accelerated to a similar extent, however, apparently owing to increased binding pocket dynamic mobility related to steric strain. A second mutation (L32I) guided by results of previous comparative studies of Mc and OM b5s diminished the steric strain, but much greater relief was achieved by replacing heme with iron deuteroporphyrin IX (FeDPIX). Indeed, the stability of the Mc(S71L) b5 FeDPIX complex is similar to that of the FeDPIX complex of OM b5. The results suggest that maximizing heme binding pocket compactness in the apo state is a useful general strategy for increasing the stability of engineered or designed proteins.     

LICRED: A Versatile Drop‐In Vector for Rapid Generation of Redox‐Self‐Sufficient Cytochrome P450s

Cytochromes P450 (P450s) are a family of haem‐containing oxidases with considerable potential as tools for industrial biocatalysis. Organismal genomes are revealing thousands of gene sequences that encode P450s of as yet unknown function, the exploitation of which will require high‐throughput tools for their isolation and characterisation. In this report, a ligationindependent cloning vector “LICRED” is described that enables the high‐throughput generation of libraries of redox‐self‐sufficient P450s by fusing a range of P450 haem domains to the reductase of P450RhF (RhF‐Red) in a robust and generically applicable way. Cloning and expression of fusions of RhF‐Red with the haem domains of P450cam and P450‐XplA resulted in soluble, active, redox‐self‐sufficient, chimeric enzymes. In vitro studies also revealed that electron transfer from NADPH to haem was primarily intramolecular. The general applicability of the LICRED platform was then demonstrated through the creation of a library of RhF‐Red fusion constructs by using the diverse complement of P450 haem domains identified in the genome of Nocardia farcinica. The resultant fusion‐protein library was then screened against a panel of substrates; this revealed chimeric enzymes competent for the hydroxylation of testosterone and methyltestosterone, and the dealkylation of 7‐ethoxycoumarin.     

Insertion of heme b into the structure of the Cys34-carbamidomethylated human lipocalin α(1)-microglobulin: formation of a [(heme)(2) (α(1)-Microglobulin)](3) complex

α(1)-Microglobulin (α(1)m) is a 26 kDa plasma and tissue protein belonging to the lipocalin protein family. Previous investigations indicate that the protein interacts with heme and suggest that it has a function in heme metabolism. However, detailed characterizations of the α(1)m-heme interactions are lacking. Here, we report for the first time the preparation and analysis of a stable α(1)m-heme complex upon carbamidomethylation of the reactive Cys34 by using recombinantly expressed human α(1)m. Analytical size-exclusion chromatography coupled with a diode-array absorbance spectrophotometry demonstrates that at first an α(1)m-heme monomer is formed. Subsequently, a second heme triggers oligomerization that leads to trimerization. The resulting (α(1)m[heme](2))(3) complex was characterized by resonance Raman and EPR spectroscopy, which support the presence of two ferrihemes, thus indicating an unusual spin-state admixed ground state with S=(3)/(2), (5)/(2).     

Single-Step Reconstitution of Apo-Hemoproteins at the Disruption Stage of Escherichia coli Cells

Hemoproteins on their metal: We report a novel strategy for the reconstitution of hemoproteins with non-natural metal complexes; simple addition of manganese and ruthenium porphyrin to E. coli cells immediately prior to homogenization yields the reconstituted proteins. We believe that this simple approach could become a standard reconstitution method for hemoproteins.