Effects of peptide density and elution pH on affinity chromatographic purification of human immunoglobulins A and M

A family of linear hexamer peptide ligands HWRGWV, HYFKFD and HFRRHL, initially identified for their affinity to the Fc portion of human immunoglobulin G (hIgG), also have potential for use in the purification of human immunoglobulins A (hIgA) and M (hIgM). HWRGWV demonstrated the strongest binding affinity to hIgM, followed by hIgA and hIgG respectively. The effects of N-terminal acetylation of the peptide, as well as elution buffer pH, on the chromatographic elution of human IgG, IgA and IgM from HWRGWV resins at various peptide densities (0.04-0.55 meq/g) were investigated. Over 80% recovery and 90% purity were achieved for human IgG and IgA isolation from complete minimum essential medium (cMEM) using HWRGWV resin at optimum peptide densities. For human IgM, 75.7% recovery and 86.0% purity were achieved by using HWRGWV at a low peptide density of 0.04 meq/g. Although HYFKFD and HFRRHL exhibited their ability for isolation of human IgG, IgA and IgM from cMEM as well, HWRGWV is the best option among them for large-scale purification of human IgG, IgA and IgM based on conditions tested.     

聚乙烯亚胺对人血清白蛋白的构象和结合能力的影响

为了解基因载体材料聚乙烯亚胺（PEI）细胞毒性的分子作用机理,本文应用吸收光谱、荧光光谱、圆二色谱、动态光散射和zeta-电位测定分析平均相对分子量为1.8和25 kDa的PEI（记为PEI1.8k和PEI25k）对人血清白蛋白（HSA）构象的影响,同时以8-苯氨基萘-1-磺酸镁（ANS）和槲皮素为模型化合物,了解PEI对HSA结合能力的影响及机理。结果发现,PEI与HSA结合形成静态复合物,导致HSA流体动力学直径变小和分子内环境疏水性增强。PEI1.8k和低浓度的PEI25k引起HSA的a-螺旋结构增加,但是高浓度的PEI25k对HSA二级结构具有稳定作用。PEI对HSA结合能力的影响主要归因于PEI的竞争结合和PEI与HSA结合引起的蛋白质构象变化。PEI的竞争结合降低了HSA对ANS和槲皮素的结合效率,但是蛋白质的构象变化增强了HSA与ANS和槲皮素的结合能力。PEI与HSA的相互作用具有明显的分子尺寸效应,增加PEI的相对分子量可以增强对HSA构象和结合能力的影响。     

Voltammetric studies of the interaction of quinacrine with DNA

The binding interaction of the antimalarial drug quinacrine with herring sperm deoxyribonucleic acid (DNA) has been studied by square wave voltammetry. The binding parameters, the binding constant K and the binding site size s, were obtained simultaneously by the analysis of bound and free quinacrine concentration corresponding to the limits of slow and fast binding kinetics compared to the experimental timescale. The binding constant and the binding site size for the interaction of quinacrine with DNA were K=1.59 (±0.18)×10⁵ M^–1 and s=7.1 (±0.15) base pairs and K=7.35 (±0.83)×10⁵ M^–1, s=6.2 (±0.02) base pairs for the limiting conditions of static and mobile binding equilibrium respectively. The standard Gibbs free energy change (G⁰=–RT ln K) is approximately –29.67 kJ/mol at 25 °C, which highlights the spontaneity of the binding of quinacrine with DNA. The binding of quinacrine to herring sperm DNA results in peak potential shifts in voltammetric and a red shift in UV-absorption measurements. The ionic strength dependence of the binding constant is not large. Furthermore, the relative viscosity of DNA increases in the presence of quinacrine. These characteristics strongly support the intercalation of quinacrine into DNA. The results also show that the intercalation of quinacrine into DNA may occur at approximately every seventh base pair.     

The auxiliary determination of the binding site of berberine binding to human serum albumin by surface-enhanced Raman scattering

We report on the joint application of fluorescence, ultraviolet-visible (UV-Vis) and Raman spectroscopy to the study of berberine with human serum albumin (HSA). We propose the surface-enhanced Raman scattering (SERS) technique to improve the understanding of the quenching interaction caused by berberine which could be applied in recognition process of fluorescent drugs with large biomolecules. The fluorescence and UV-Vis spectroscopic results show that the fluorescence intensity of HSA is significantly decreased in the presence of berberine, and the quenching mechanism is static. The SERS technique demonstrates clear advantages over direct measurements in physiological conditions. By means of this method, we are able to deduce important information concerning the binding property of berberine when interacting with HSA. We show the nitrogen atom is free but the dioxolane is involved in the spontaneously electrostatic inducement and subsequently hydrophobic binding.     

Systematic investigation of sequence and structural motifs that recognize ATP

Interaction between ATP, a multifunctional and ubiquitous nucleotide, and proteins initializes phosphorylation, polypeptide synthesis and ATP hydrolysis which supplies energy for metabolism. However, current knowledge concerning the mechanisms through which ATP is recognized by proteins is incomplete, scattered, and inaccurate. We systemically investigate sequence and structural motifs of proteins that recognize ATP. We identified three novel motifs and refined the known p-loop and class II aminoacyl-tRNA synthetase motifs. The five motifs define five distinct ATP–protein interaction modes which concern over 5% of known protein structures. We demonstrate that although these motifs share a common GXG tripeptide they recognize ATP through different functional groups. The p-loop motif recognizes ATP through phosphates, class II aminoacyl-tRNA synthetase motif targets adenosine and the other three motifs recognize both phosphates and adenosine. We show that some motifs are shared by different enzyme types. Statistical tests demonstrate that the five sequence motifs are significantly associated with the nucleotide binding proteins. Large-scale test on PDB reveals that about 98% of proteins that include one of the structural motifs are confirmed to bind ATP.     

Photocurrent of purple membrane adsorbed onto a thin polymer film: action characteristics of the local anesthetics

The proton pumping activity of bacteriorhodopsin (bR) in the purple membrane adsorbed onto a thin polymer film as a solid support for electrical measurements has been examined in the presence of local anesthetics and 1-alcohols as an anesthetic model. This membrane adsorbed system provided high reproducibility of the photocurrents in bR due to the mechanical and the chemical stability and the electric properties of the thin polymer film. As the concentrations of the local anesthetics increased, the photocurrents generated by the proton pump of bR were cooperatively suppressed and the changes in the photocurrents were reversible. From the dose–response curves for the anesthetics, the concentration (EC₅₀) required for a 50% suppression showed a marked specificity in the order of lidocaine>bupivacaine>tetracaine>dibucaine. The suppression of the photocurrent in bR was more effective for the uncharged form of the local anesthetics than for the charged one. The absorption and fluorescence spectra suggested that the charged form of the anesthetics was bound to the purple membrane surface, while their uncharged form interacted with the hydrophobic portions of the purple membrane interior rather than with the membrane surface. From the dose–response curves for the 1-alcohols, an increase in hydrophobicity in their molecules effectively suppressed the photocurrent of bR. We found that the binding of hydrophobic organic cations such as tetracaine hydrochloride and bupivacaine hydrochloride to the blue membrane with loss of the proton pump, which was prepared by removal of the cations from the purple membrane, could regenerate the proton pumping activity. The photocurrent in bR in the purple membrane adsorbed onto a thin solid film sensitively responded to different local anesthetics.     

Review of binding methods and detection of Al(III) binding events in trypsin and DL-DPPC liposomes by a general thermodynamic model

In this paper are reviewed some of the most useful binding formalisms which have been developed in order to investigate the diversity of the commonly encountered receptor-ligand systems. Particularly, our attention is focused on the presentation and application of a general and rigorous thermodynamic treatment which explains in a simple and coherent way the non-linear profiles observed in the experimental binding, Scatchard, Hill and Adair plots. By applying this model, both qualitative and detailed quantitative investigations were carried out on the binding process of Al(III) to trypsin and dl-dipalmitoylphosphatidylcholine (DPPC) liposomes. Particularly, it has been demonstrated that: Al(III) interacts with liposomes in two binding sites with very different dissociation constants. The first one has been calculated to be 0.0168 μmol l⁻¹ and the second 2.833 μmol l⁻¹. The first is referred to the preferential interaction of Al³⁺ with the polar head of the phospholipid, while the second most likely regards the interaction of Al³⁺ with other peripheral sites. On the other hand, Al³⁺ interacts with trypsin in two types of binding sites. The first binding site with K_1,1=0.2531 μmol l⁻¹ and the second with K_2,1=1.424 μmol l⁻¹.     

重金属离子对猪胰α-淀粉酶活性影响的作用机理研究

通过在α-淀粉酶介质中加入Ce^3 ,Cd^2 和Pb^2 ,研究其对α-淀粉酶活性影响的作用机理。结果表明,低浓度重金属离子对酶活性有激活作用,高浓度则严重抑制酶活性。在高浓度下,Ce^3 ,Cd^2 和Pb^2 能完全竞争出α-淀粉酶中的Ca^2 。荧光滴定结果表明,Ce^3 ,Cd^2 和Pb^2 不仅可能完全占据Ca^2 的结合位点,而且还可能在Ca^2 的结合位点以外的氨基酸残基上结合,从而导致酶的构象改变。     

A Monte Carlo pharmacophore generation procedure: Application to the human PAF receptor

Summary A novel pharmacophore definition procedure is described, which uses a Monte Carlo method to superimpose molecules. Pharmacophore space is searched by a technique similar to high temperature annealing. Subsequent refinement of candidate pharmacophores by energy minimization produces low-energy conformations that may be involved in receptor binding. The method has been applied to compounds that bind to the human platelet-activating factor (PAF) receptor. Alternative binding site models for the PAF receptor are presented and discussed.A preliminary account of this work has been published elsewhere [1].