Protein retention in ion-exchange chromatography: effect of net charge and charge distribution

The charge regulated slab model is used to evaluate the salt dependence of the retention of Staphylococcal nuclease A and its mutants in cation-exchange chromatography. An important feature of this work is that the net charge of the proteins is varied in two different ways: (a) by changing the eluent pH so that the charges are created by protonation and (b) by point mutation at position 116. Since the structure of Staphylococcal nuclease and the mutants are known, the pH dependence of retention data of the different mutants gives detailed insights into the retention mechanism. Experimental results show that the salt dependence of retention is affected more strongly by changes of the eluent pH than by point mutations. This implies that the amino acid in position 116 has only a moderately strong interaction with the stationary phase surface and that a patch on one side of the protein surface is mainly responsible for the electrostatic interaction with the surface.     

Electrophoresis of cells and the biological relevance of surface charge

Recent developments in electrophoresis of cells are reviewed. Problems and progress in automation and miniaturization of analytical electrophoresis instruments as well as in the interpretation of experimentally determined electrophoretic mobility (EPM) data are summarized: In recent times, the EPM determination techniques not only became more reliable and faster, but also more knowledge could be gained about the cell surface electrical properties, the structure of the glycocalyx as well as its influence on the cell peripheral regions and microenvironment by applying cell electrophoresis. In addition, ways are shown to solve discrepancies between physical requirements of a preparative cell electrophoresis procedure and the quantities of ions, which have to be dissolved in cell suspension media. As the modern machines allow the purification of untagged cells suspended in more cell friendly and physiological media, they are likely to be valuable tools in several useful practical applications in clinical transplantation, gene therapy and treatment of disease states.     

Synthesis and anti-HIV-1 activity of the conjugates of gossypol with oligopeptides and D-glucosamine

A series of novel gossypol derivatives were synthesized and screened for their in vitro anti-HIV- 1I activity. The results showed that replacing the aldehyde groups of gossypol with certain oligopeptides and Dglucosamine not only reduced the cytotoxicity of gossypol derivatives but also enhanced their antiviral activity against HIV-1. Interestingly, D-glucosamine derivative of gossypol that lacked the COONa group also exhibited the same potent anti-HIV-1 activity as oligopeptide derivatives with the COONa group. These compounds blocked the entry of HIV-1ⅢB into target cell. which was similar to T20. Furthermore, the molecular docking analysis rationalized their anti-HIV-1 activity. The results also implied that certain oligopeptides and D-glucosamine were important moities to prepare gossypol derivatives as HIV- 1 entry inhibitors besides certain amino acids.     

Aqueous solution properties of polyampholytes: Effect of the net charge distribution

Viscometric and light scattering studies have been performed on aqueous solutions of polyampholyte terpolymers based on sodium-2-acrylamido-2-methylpropanesulfonate (Na-AMPS), 2-(methacryloyloxy)-ethyltrimethylammonium chloride (MADQUAT), and acrylamide (AM), prepared by an inverse microemulsion polymerization technique. The distribution of net charges among the chains was varied by adjusting the initial monomer composition and the degree of conversion. The effect of this distribution on the solubility of the samples and on the chain conformation was studied. It was found that samples with a narrow distribution of net charges were soluble in pure water even if the average net charge is small. Addition of salt induces a transition from an extended conformation to a more compact one, in qualitative agreement with theoretical predictions. A practically alternated NaAMPS-MADQUAT copolymer prepared by polymerization in homogeneous solution and with a small average net charge shows a behavior quite similar to that of the terpolymers. © 1996 John Wiley & Sons, Inc.     

Quantitative description of the relation between protein net charge and protein adsorption to air-water interfaces

In this study a set of chemically engineered variants of ovalbumin was produced to study the effects of electrostatic charge on the adsorption kinetics and resulting surface pressure at the air-water interface. The modification itself was based on the coupling of succinic anhydride to lysine residues on the protein surface. After purification of the modified proteins, five homogeneous batches were obtained with increasing degrees of modification and zeta-potentials ranging from -19 to -26 mV (-17 mV for native ovalbumin). These batches showed no changes in secondary, tertiary, or quaternary structure compared to the native protein. However, the rate of adsorption as measured with ellipsometry was found to decrease with increasing net charge, even at the initial stages of adsorption. This indicates an energy barrier to adsorption. With the use of a model based on the random sequential adsorption model, the energy barrier for adsorption was calculated and found to increase from 4.7 kT to 6.1 kT when the protein net charge was increased from -12 to -26. A second effect was that the increased electrostatic repulsion resulted in a larger apparent size of the adsorbed proteins, which went from 19 to 31 nm2 (native and highest modification, respectively), corresponding to similar interaction energies at saturation. The interaction energy was found to determine not only the saturation surface load but also the surface pressure as a function of the surface load. This work shows that, in order to describe the functionality of proteins at interfaces, they can be described as hard colloidal particles. Further, it is shown that the build-up of protein surface layers can be described by the coulombic interactions, exposed protein hydrophobicity, and size.     

Single isolated droplets with net charge as a source of ions

A levitation device for charged particles has been used to position a single isolated droplet at a time at atmospheric pressure near the sampling orifice of a vacuum chamber. Following a brief desolvation period (550 ms), a series of coluomb fission events were initiated. Several atmospheric pressure electrode designs were evaluated with respect to guiding the progeny droplets/ions, to the sampling orifice. The best design tested consisted of a series of four annular electrodes of decreasing radius positioned above the levitation ring electrodes, and, on average, 40 ions were counted per single isolated droplet. The ion utilization (charge detected versus charge in the original droplet) with this electrode design has been estimated to be 5 x 10(-6), a substantial improvement over the current utilization measured to be < or = 1 x 10(-9) with a conventional electrospray ion source using the same vacuum apparatus.     

Some biological transformations involving unsaturated linkages: the importance of charge separation and charge neutralization in enzyme catalysis

Current status of knowledge on the biological reduction of CC and CO is briefly reviewed. It is Argued that the crucial event in the reduction of CC is the addition of a proton to the more electron-rich terminal of the double bond to produce an electron-deficient species which is then neutralized through hydride transfer from NADPH. The activation for the reduction of a CO group may also be achieved by a related process in which the carbonyl oxygen is polarized by H-bonding to an acidic group on the enzyme, prior to hydride transfer from NAD(P)H.Thus with both these systems an early event in catalysis is the protonation of the substrate for which, normally, strong adds win be required. Since the groups available at the active-met of enzymes are weak acids, a mechanism through which powerful proton donating species could be transiently generated from them is proposed. The salient features of this mechanism may be enuinciatcd as follows: Let us consider the enzyme-substrate complex (A) in which an imidazolium group is about to play a role as a proton donating species. It is argued that rearrngement of initial complex(A)to the catalytic-complex(B), in which the negatively chared counter ion is removed away from the imidazolium cation, would transiently convert the latter group into a powerful proton danating species. the rearrangement (A)→(B) could occur through a protein conformational change or via a charge-relay system or a combination of both processes.     

Determination of the net electric charge of a polyacrylamide molecule by Donnan potential measurements

The net electric chargez _pof a polyacrylamide molecule (Praestol, Mol. Wt. 3.4×10⁶) in aqueous solution was determined for two pH values (7.2 and 10.0) by measuring the Donnan e.m.f. as function of the solutions NaCl contentc (2.7 mM<c<74 mM). Taking into account a non-ideal behavior of the counterions (Na⁺) mobility, the numerical values ofz _pturned out to be 3100 and 5500 electric charge units for pH=7.2 and 10.0, respectively. A measurable contribution of the assumed non-ideal model for the Na⁺ ions is found forc<15 mM.     

Effects of the net charge on abundance and stability of supramolecular surfactant aggregates in gas phase

Self-assembling of amphiphilic molecules under electrospray ionization (ESI) conditions is characterized by quite unexpected phenomenology. The noticeable differences with respect to the condensed phase are attributable to the absence of the surfactant-solvent interactions, the presence of net charge in the aggregates, and the strong deviation from equilibrium conditions. Aiming to investigate the effects of the net charge on abundance and stability of supramolecular surfactant aggregates, positively and negatively charged aggregates of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and sodium methane sulfonate (MetS), butane sulfonate (ButS) and octane sulfonate (OctS) have been studied by ESI mass spectrometry, energy resolved mass spectrometry and density functional theory calculations. The negatively charged aggregates are found to be less stable than their positive counterparts. The results are consistent with a self-assembling pattern dominated by electrostatic interactions involving the counterions and head groups of the investigated amphiphilic compounds while the alkyl chains point outwards, protecting the aggregates from unlimited growth processes.     

Tailoring the volatility and stability of oligopeptides

Amino acids are essential building blocks of life, and fluorinated derivatives have gained interest in chemistry and medicine. Modern mass spectrometry has enabled the study of oligo‐ and polypeptides as isolated entities in the gas phase, but predominantly as singly or even multiply charged species. While laser desorption of neutral peptides into adiabatically expanding supersonic noble gas jets is possible, UV–VIS spectroscopy, electric or magnetic deflectometry as well as quantum interferometry would profit from the possibility to prepare thermally slow molecular beams. This has typically been precluded by the fragility of the peptide bond and the fact that a peptide would rather ‘fry’, i.e. denature and fragment than ‘fly’. Here, we explore how tailored perfluoroalkyl functionalization can reduce the intermolecular binding and thus increase the volatility of peptides and compare it to previously explored methylation, acylation and amidation of peptides. We show that this strategy is essential and enables the formation of thermal beams of intact neutral tripeptides, whereas only fragments were observed for an extensively fluoroalkyl‐decorated nonapeptide. © 2017 The Authors. Journal of Mass Spectrometry Published by John Wiley & Sons Ltd.     

Docking of oligopeptides

Russian Chemical Bulletin - The paper presents the results concerning the use of the supercomputer docking program SOL-P, which performs a search for low-energy minima of protein—ligand...     

Catalytic activity correlation of Ni(II), Co(II) and Pd(II) complexes to metal atom net charge

The metal atom net charge correlation (MANCC) method was developed in prediction of catalyst activity of asymmetric late-transition metal complexes, 2-quinoxalinyl-6-iminopyridine Ni (II), 2-imino-1, 10-phenanthroline Co(II) and 2-methoxycarbonyl-6-iminopyridine Pd(II) complexes, from the net charge of the metal atom for ethylene polymerization. Dreiding force field was modified according to the X-ray diffraction data. We found that the asymmetric structure of the complexes resulted in a charge difference between two halogen atoms coordinated to the metal atom. In order to remove such contribution we introduced the effective charge Q _eff, which was obtained by the charge equilibration (QEq) approach. The results verified the successful introduction of Q _eff and showed that the catalytic activities of different complexes are related to central metal atom effective charge. Supported by the National Natural Science Foundation of China (Grant Nos. 90612015, 20674090, and 20474073), and 973 Project (Grant No. 2004CB720606)     

Absolute i.r. intensities,dipole derivatives and vibrational charge parameters in the perchlorate anion

The absolute intensities of the ν₃ and ν₄ fundamental bands of perchlorate anion have been measured in a state of matrix isolation in a potassium iodide host. Force constants were evaluated with the aid of the chlorine isotopic shift and used to define the transformation between normal coordinates and symmetry coordinates. The dipole derivatives with respect to symmetry coordinates were analyzed from the point of view of a charge and charge flux model, and the results were compared qualitatively with simple electronic bonding theory.     

Effect of background electrolyte on the estimation of protein hydrodynamic radius and net charge through capillary zone electrophoresis

Two physicochemical models are proposed for the estimation of both hydrodynamic radius and net charge of a protein when the capillary zone electrophoretic mobility at a given protocol, the set of pK of charged amino acids, and basic data from Protein Data Bank are available. These models also provide a rationale to interpret appropriately the effects of solvent properties on protein hydrodynamic radius and net charge. To illustrate the numerical predictions of these models, experimental data of electrophoretic mobility available in the literature for well-defined protocols are used. Five proteins are considered: lysozyme, staphylococcal nuclease, human carbonic anhydrase, bovine carbonic anhydrase, and human serum albumin. Numerical predictions of protein net charges through these models compare well with the results reported in the literature, including those found asymptotically through protein charge ladder techniques. Model calculations indicate that the hydrodynamic radius is sensitive to changes of the protein net charge and hence it cannot be assumed constant in general. Also, several limitations associated with models for estimating protein net charge and hydrodynamic radius from protein structure, amino acid sequence, and experimental electrophoretic mobility are provided and discussed. These conclusions also show clear requirements for further research.     

Absolute enantioselective separation: optical activity ex machina

The paper describes methodology of using three independent macroscopic factors affecting molecular orientation to accomplish separation of a racemic mixture without the presence of any other chiral compounds, i. e., absolute enantioselective separation (AES) which is an extension of a concept of applying these factors to absolute asymmetric synthesis. The three factors may be applied simultaneously or, if their effects can be retained, consecutively. The resulting three mutually orthogonal or near orthogonal directors constitute a true chiral influence and their scalar triple product is the measure of the chirality of the system. AES can be executed in a chromatography-like microfluidic process in the presence of an electric field. It may be carried out on a chemically modified flat surface, a monolithic polymer column made of a mesoporous material, each having imparted directional properties. Separation parameters were estimated for these media and possible implications for the natural homochirality are discussed.     

苯并异噻唑啉酮类化合物的合成与活性研究

通过烷基醇与固体三光气(BTC)反应后加入1,2-苯并异噻唑啉-2(3H)-酮(BIT)继续反应的“一锅法”制备了8种未见文献报道的2-(苯并异噻唑啉-3-酮-2-基)甲酸酯类化合物.化合物结构经IR、1HNMR和元素分析确认,并且对枯草芽孢杆菌、金黄色葡萄球菌、嗜水单细胞菌和大肠杆菌进行了初步抑菌活性实验,实验结果表...     

人参寡肽的合成

使用溶液和有机磷肽缩合试剂DEPBT合成了从人参中分离鉴定的三个寡肽,N,N'－双－(γ-谷氨酰甘氨酰)胱氨酸1,N,N'－双-γ－谷氨酰胱氨酰甘氨酸2,N-γ-谷氨酰胱氨酰-双-甘氨酸3,以及γ-谷氨酰甘氨酰半胱氨酸。4.产物经离子交换树脂柱或HPLC纯化,用质谱、核磁共振谱、氨基酸分析进行了验证。     

寡肽结构参数化及定量构效关系研究

从肽链的一级结构出发,基于分子中原子间距离及各原子的电负性,构建了能描述二肽与多肽分子的结构参数分子电负性边数矢量,简称分子电边矢量。据此,对58个血管紧张素转化酶抑制剂(二肽)及48个苦味二肽已知活性进行了定量结构活性相关研究,为考察分子电边矢量的实用性,还对血管舒缓激肽增效剂(五肽)已知活性进行了研究。结果表明：与其它表达方法相比,本研究的分子电边矢量具有计算简便,活性相关性好的特点,可望在生物分子的结构表征及活性预测方面有所作为。