Electroactivity of Nonconjugated Proteins and Peptides. Towards Electroanalysis of All Proteins

Present proteomics and biomedicine require sensitive analytical methods for all proteins. Recent progress in electrochemical analysis of peptides and proteins based on their intrinsic electroactivity is reviewed. Tyrosine and/or tryptophan‐containing proteins are oxidizable at carbon electrodes. At mercury electrodes all peptides and proteins (about 13 peptides and >25 proteins were tested) produce chronopotentiometric peak H at nanomolar concentrations. This peak is sensitive to changes in protein structure. Microliter sample volumes are sufficient for the analysis. Electrochemical methods can be used in studies of nucleic acid‐protein interactions and can be applied in biomedicine. Examples of such applications in neurogenerative diseases and cancer are presented.     

Evaluation of soybean seed protein extraction focusing on metalloprotein analysis

Two methods of protein extraction for soybean seeds were evaluated in terms of preservation of the metal ions bound to proteins after the extraction and separation procedures. The proteins were firstly separated according to their molar masses by polyacrylamide gel electrophoresis. Then, the protein bands were mapped by synchrotron radiation X-ray fluorescence in order to establish which metal ions were present in each one. Finally, some mapped protein bands were decomposed by microwave-assisted combustion and Ca, Cu, K, Mg, Mn, and Zn were quantified by inductively coupled plasma mass spectrometry or inductively coupled plasma optical emission spectrometry. The extraction methods studied were Method A (based on the treatment of ground soybean seeds with hexane and their extraction with Tris–HCl and β-mercaptoethanol) and Method B (based on the treatment of ground soybean seeds with petroleum ether and their extraction with Tris–HCl, dithiothreitol, phenylmethanesulfonyl fluoride, sodium dodecyl sulfate and potassium chloride). The best method was Method B, in which a 78% higher extraction efficiency was obtained when compared to Method A. Additionally, the metal-protein interactions were more appropriately preserved when Method B was applied, where the most affected ions were those that are bound weakly to proteins, such as Ca, K, and Mg.     

Protein dynamics tightly connected to the dynamics of surrounding and internal water molecules.

Proteins are key components of biological cells. For example, enzymes catalyze biochemical reactions, membrane transporters are responsible for uptake and release of critical and superfluous components from the cell environment, and structural proteins are responsible for the stability of the cell wall and cytoskeleton. Many of the diverse protein functions involve dynamic transitions ranging from small local atomic displacements up to large allosteric conformational changes. In any conformation, proteins are in contact with the universal solvent medium of cells, water. Water not only surrounds proteins but is often an integral part of proteins and also is involved in key mechanistic steps. This Minireview discusses recent experimental and theoretical results on the role of water for protein dynamics and function.     

Synthesis of 2′-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified oligonucleotides: hybridization affinity, resistance to nuclease, and protein binding characteristics

Synthesis of a series of 2′-O-[2-[(N,N-dialkylamino)oxy]ethyl]-modified 5-methyluridine nucleoside phosphoramidites and solid supports are described. Using these monomers, modified oligonucleotides containing phosphodiester linkages were synthesized in high yields. These modified oligonucleotides showed enhanced binding affinity to the complementary RNA (and not to DNA) and excellent nuclease stability with t_1/2>24 h. The human serum albumin binding properties of modified oligonucleotides have been evaluated to assess their transport and toxicity properties.     

Solvation of amino acid residues in water and urea-water mixtures: Volumes and heat capacities of 20 amino acids in water and in 8 molar urea at 25°C

The limiting partial molar volumes V ^o and heat capacities C _p ^o of 20 amino acids have been determined in water and in 8 molar urea at 25.0°C using flow calorimetry and flow densimetry. The side chain contributions to V ^o and C _p ^o were obtained as the difference between the properties of the various amino acids and those of glycine, both in water and in 8M urea. The solvent accessible surface area of the amino acid residues were obtained using a method developed by Hermann, and the total surface areas were separated into their hydrophobic A _Hb and hydrophilic components. In water, C _p ^o values for the various residues C _p ^o (R) were found well correlated with A _Hb , though much less so in the urea solution. Hence, C _p ^o (R) values, in water yield a good estimate of side chain hydrophobicity, but the (waterurea) transfer heat capacities appear strongly affected by specific solvation effects in the urea solution.Presented at the sixth Italian meeting on Calorimetry and Thermal Analysis (AICAT) held in Naples, December 4–7, 1984.     

蛋白芯片法同时检测食品中克伦特罗与莱克多巴胺的残留量

建立了食品中克伦特罗和莱克多巴胺残留含量同时检测的微阵列蛋白芯片法。结果显示:克伦特罗的线性范围为0.07～1.2 ng/g;莱克多巴胺的线性范围为0.05～0.8 ng/g。猪肉、猪肝中上述两种物质的加标回收率为74%～132%,相对标准偏差均小于10%。将所建立的方法与HPLC-MS方法进行对比,两者检测结果一致。该方法简单、快速、通量高,可用于实际样品中克伦特罗和莱克多巴胺残留量的检测。     

C反应蛋白免疫电极的研究

C反应蛋白(CRP)是临床医学中重要的检验项目之一。本文以醋酸纤维素-戊二醛-己二胺载体膜与自制的原电极研制成非标记CRP免疫电极。研究了载体膜的组成对电极灵敏度的影响,并采用均匀设计法对载体膜的活化条件、抗原抗体结合时间以及电极测试条件等进行了优化。该电极响应快、灵敏度高、重现性和稳定性良好。在最佳实验条件下,电极线性范围为1.8～60μg/ml,所得线性回归方程为E=0.2+0.091[CRP];相关系数0.9989。在用于血清样品的测定中,取得满意结果。     

纳米荧光小球标记在蛋白质微阵列检测中的应用研究

研究了不经分离、一次性制备氨基化联吡啶钌掺杂的双层二氧化硅纳米小球的方法。实验证明该纳米小球尺寸均匀,光稳定性、水溶性好,分散稳定。通过简单的偶联反应后,它能有效的和蛋白质结合,结合后的蛋白能保持其生物活性。以此纳米荧光小球为标记物,应用于蛋白质微阵列的定量检测,结果发现其效果明显优于相同条件下以异硫氰酸荧光素(FITC)为标记物的定量结果,检出限可以达到3.5 mg/L。