纤维素亲和膜用于内毒素的去除

报道了用于内毒素去除的３种新型纤维素亲和膜的制备及应用,并对它们的性能进行了比较。结果表明,这３种亲和膜均能有效去除盐溶液中的内毒素,去除率在９０％以上。对人血清白蛋白溶液,季铵盐和壳聚糖亲和膜对内毒素的去除率较高,仍然在９０％以上,金属螯合物亲和膜的去除效果不如前两种,去除率仅为８３．３％。实验选择不同的亲和膜,考察了它们对内毒素的吸附容量、样品处理量以及再生效果,结果表明吸附量在 ２．４×１０６ＥＵ／ｇ以上,样品处理量较大,适合用于小批量医药制剂的生产过程;再生效果较好,可重复使用。     

亲和膜用于去除医药及人血清白蛋白溶液中的内毒素(英文)

 以纤维素膜为基质材料 ,制备了 3种用于去除内毒素的亲和膜 ,分别为壳聚糖亲和膜 (KFCC5 17) ,疏水阳离子亲和膜 (KFCG316 )和金属螯合物亲和膜 (KFCG40 2 )。研究了亲和膜对内毒素的吸附容量及在内毒素去除方面的应用 ,结果表明这 3种亲和膜都可以用于多种溶液中内毒素的去除。考察了离子强度、pH值以及流速对去除效果的影响 ,并分析了原因。KFCC5 17,KFCG316 ,KFCG40 2都可用于人血清白蛋白溶液中内毒素的去除 ,KFCC5 17还可用于医药制剂如氢化可的松、葛根素、盐酸丁卡因和右旋糖苷 40葡萄糖注射液中内毒素的去除。     

新型亲和膜色谱用于去除胆红素的研究

 以纤维素膜为亲和基质 ,接枝后分别以聚赖氨酸和季铵盐为配基 ,制备了两种亲和膜介质 ,用于去除磷酸缓冲液 (pH 7 4)及人血白蛋白 (HSA)溶液中的胆红素。实验结果表明 ,该方法对磷酸缓冲液中胆红素的去除率能达到 70 %以上 ;对HSA溶液中的胆红素 ,其去除率稍低 ,但在较低浓度的HSA溶液中 ,也能获得高于 5 0 %的去除率。并对亲和膜与胆红素间的亲和作用进行了研究 ,考察了温度、流速、HSA浓度及胆红素初始浓度等相关条件对去除效果的影响。结果表明 ,在较高的温度条件下 ,去除效果较佳 ;在较高的初始浓度情况下 ,胆红素的吸附速率较大。     

高效亲和色谱法测定2种中药成分与人血清白蛋白的结合率

采用高效亲和色谱技术(HPAC)对中药成分与人血清白蛋白(HSA)的相互作用进行了研究。首先采用点击化学的方法制备了表面键合有HSA蛋白的硅胶固定相并装填成亲和色谱柱,根据药物在该色谱柱上与空白硅胶柱上的保留时间差计算得到药物与蛋白的结合率。利用该方法测得模型化合物华法令与HSA的结合率与文献中采用超滤法测得的结果基本一致,表明该方法可用于测定药物与HSA的结合率。在此基础上用该方法测定了葛根素和告依春两种中药成分与HSA的相对结合率分别为10.26%和10.20%。同时用超滤的方法测定了葛根素与HSA的结合率为14.25%。结果表明,HPAC可以作为研究药物与蛋白相互作用的一种简便可行的方法,其测定结果与超滤方法一致。     

Fractionation of human plasma proteins by hydrophobic interaction membrane chromatography

This paper discusses the fractionation of human plasma proteins HSA and HIgG by hydrophobic interaction membrane chromatography. A type of microporous polyvinylidine fluoride (PVDF) membrane having 0.1 μm pore size was identified as being suitable for carrying out this separation. This membrane bound HIgG at 1.5 M ammonium sulphate concentration, a condition at which HSA did not. Based on this selective binding resulting from the selective pressure induced by the high anti-chaotropic salt concentration, these human plasma proteins were fractionated. The HIgG binding capacity of the PVDF membrane examined in this study was 42.8 mg/ml at a feed concentration of 0.45 mg/ml. Separation of simulated HSA/HIgG mixtures were carried out in the pulse and step input modes and the HSA and HIgG fractions thus obtained were analysed for purity using affinity chromatography and SDS-PAGE. HSA and HIgG purities were typically in excess of 97–98%.     

Allosteric Modulation of Human Serum Albumin Induced by Peptide Ligand

Human serum albumin (HSA ) is an abundant protein in plasma that can bind and transport many small molecules, and the corresponding affinity‐controlled drug delivery shows great advantage in the biological system. Peptide SA06 is a reported ligand comprising 20 amino acids, and is known to non‐covalently bind with HSA to extend the lifetime and improve the pharmacokinetic performance. The structural information of the HSA ‐peptide complex is keen for obtaining molecular insight of the binding mechanism. We studied the secondary structural change and structure‐affinity relations of Peptide SA06 with HSA by using circular dichroism (CD ) spectroscopy in solution. Noticeable allosteric effect can be identified by compositional increase of α ‐helix structures when the peptide was co‐incubated with HSA . Furthermore, the equilibrium dissociation constant of Peptide SA06 with HSA can be determined by CD ‐based method. This work provides structural evidence on the allosteric interaction between peptide ligand and HSA , and sheds light on optimization of therapeutic properties in the affinity‐controlled delivery systems.     

Comprehensive two-dimensional HPLC to study the interaction of multiple components in Rheum palmatum L. with HSA by coupling a silica-bonded HSA column to a silica monolithic ODS column

A mode of comprehensive 2-D LC was developed by coupling a silica-bonded HSA column to a silica monolithic ODS column. This system combined the affinity property of the HSA column and the high-speed separation ability of the monolithic ODS column. The affinity chromatography with HSA-immobilized stationary phase was applied to study the interaction of multiple components in traditional Chinese medicines (TCMs) with HSA according to their affinity to protein in the first dimension. Then the unresolved components retained on the HSA column were further separated on the silica monolithic ODS column in the second dimension. By hyphenating the 2-D separation system to diode array detector and MS detectors, the UV and molecular weight information of the separated compounds can also be obtained. The developed separation system was applied to analysis of the extract of Rheum palmatum L., a number of low-abundant components can be separated on a single peak from the HSA column after normalization of peak heights. Six compounds were preliminarily identified according to their UV and MS spectra. It showed that this system was very useful for biological fingerprinting analysis of the components in TCMs and natural products.     

以赖氨酸为配基的内毒素吸附剂的研究

内毒素血症可出现于多种疾病中,如大面积烧伤、重症肝炎、肝硬化等疾病,使机体免疫功能严重受损,引起多器官功能衰竭等一系列严重的病理变化,最终导致不可逆休克和死亡,在美国每年都有超过10万人死于此病,临床上尚无有效的治疗方法,因此,及时、有效地清除或破坏患者体内的内毒素,     

以氨基酸为配体的血液灌流用内毒素吸附剂的制备及性能研究

以琼脂凝胶为载体,氨基酸为配体,合成了内毒素吸附剂,并对其吸附效果进行了比较.对吸附剂的各种性质进行了表征,选取琼脂-赖氨酸吸附剂对内毒素血症的兔子进行血液灌流,可将血浆中的内毒素降至接近正常值,最高清除率达73.6%,且吸附剂具有良好的血液相容性.     

亲和毛细管电泳法研究锌离子与人血清白蛋白的结合反应机制

建立了研究金属离子与人血清白蛋白(Human serum albumin,HSA)相互作用的亲和毛细管电泳(Affinity capillary electrophoresis,ACE)方法。生理条件下,构建配体(Zn2+)-受体(HSA)相互作用模型,以N,N-二甲基甲酰胺(N,N-Dimethylformamide,DMF)为内标物,基于Scatchard方程,依据有效淌度的变化,通过非线性模拟方程计算Zn2+-HSA结合反应的表观结合常数KB,定量表征了Zn2+-HSA相互作用的强度,并解析电泳谱图获得了Zn2+-HSA结合反应为一快平衡体系的结论。结果表明,建立的ACE方法简捷、有效,Zn2+-HSA相互作用的强度与Zn2+浓度之间存在明显的量效关系。     

化学改性纤维素亲和膜色谱用作人血清白蛋白中杂质的去除

用环氧法对纤维素微孔滤膜进行了化学改性，并在经改性的膜上共价结合了活性红染料配基Ｋ－２ＢＰ，设计并制作了特殊的膜分离器，利用所制备的亲和膜分离器成功地去除了人血清白蛋白中的杂质。     

Use of peak decay analysis and affinity microcolumns containing silica monoliths for rapid determination of drug-protein dissociation rates

This report examined the use of silica monoliths in affinity microcolumns containing human serum albumin (HSA) to measure the dissociation rates for various drugs from this protein. Immobilized HSA and control monolith columns with dimensions of 1 mm × 4.6 mm i.d. were prepared for this work and used with a noncompetitive peak decay method. Several drugs known to bind HSA were examined, such as warfarin, diazepam, imipramine, acetohexamide, and tolbutamide. Items that were studied and optimized in this method included the sample volume, sample concentration, and elution flow rate. It was found that flow rates up to 10 mL/min could be used in this approach. Work with HSA silica monoliths at these high flow rates made it possible to provide dissociation rate constants for drugs such as warfarin in less than 40s. The dissociation rate constants that were measured gave good agreement with values reported in the literature or that had been obtained with other solutes that had similar binding affinities for HSA. This approach is a general one that should be useful in examining the dissociation of other drugs from HSA and in providing a high-throughput method for screening drug-protein interactions.     

Exploring the interaction of sesamol as an antilung cancer compound with albumin through spectroscopic and bioinformatic analyses and the mechanism of anticancer effect

Sesamol has moved into biomedical research in recent years. However, its interactions with blood proteins and cancer cells have not been fully explored. Therefore, we aimed to investigate the interaction of sesamol with human serum albumin (HSA), A549 human nonsmall cell lung cancer (NSCLC) cell line, and Raw 264.7 macrophage. The interaction of HSA with sesamol was explored via application of fluorescence and circular dichroism (CD) spectroscopy studies as well as molecular docking analysis. Then, the cytotoxic effects of sesamol on A549 lung cancer cells and Raw 264.7 macrophages were evaluated by qPCR analysis. It was found that sesamol spontaneously (ΔG?=-45.89 kJ/mol) binds with HSA having a high affinity (log K_b = 8.05, n = 1.70, T = 298 K) and form a static complex trough contribution of hydrogen bonds and van der Waals interactions (ΔH?=-409.43 kJ/mol, TΔS?=-363.54 kJ/mol) which was supported by molecular docking study. Furthermore, by using CD and synchronous fluorescence spectroscopy analyses it was found that sesamol induced some minor secondary and tertiary structural changes, respectively in HSA structure. Cellular assays displayed that sesamol triggered selective cytotoxicity against A549 lung cancer cells through regulation of intrinsic apoptosis pathway mediated by mitigation of mitochondrial membrane potential, elevation of ROS generation, downregulation of Bax, and up regulation of caspase-9, ?3. In conclusion, it was found that sesamol could show high affinity with HSA and mediate intrinsic apoptosis pathway through ROS generation in the A549 lung cancer cell lines. These data indicate that the biochemical and anticancer mechanisms of sesamol can be further investigated in future studies to integrate it in the biomedical platforms.     

Protein adsorption and platelet adhesion of polysulfone membrane immobilized with chitosan and heparin conjugate

Polysulfone (PSF) membranes were treated with ozone to introduce peroxides, and then grafted with either acrylic acid or chitosan, followed by the immobilization of heparin. The effect of spacer arm on blood compatibility was investigated using three chitosans of different molecular weight [1170 (water soluble), 160 000, and 400 000] and similar degrees of deacetylation (75%). The hydrophilicity was evaluated by measuring the contact angle of water. Blood compatibility was evaluated using the activated partial thromboplastin time (APTT) as well as the adhesion of platelets. The protein affinity was determined by the absorption of human serum albumin (HSA) and human plasma fibrinogen (HPF). The results show that by the coupling of chitosan, the amount of heparin immobilized can be increased by four times. Water contact angle (from 78 ° to 41 °) decreased with the increase of the amount of heparin immobilized, showing increased wettability. The heparinized PSF membrane showed longer APTT and decreasing platelet adhesion, compared to that of unmodified PSF membrane. The adsorption of HSA and HPF were reduced to 17 and 6%, respectively. This suggests that longer spacer binding to heparin can increase the opportunity of anti‐coagulation on contacting blood. These results demonstrated that the hydrophilicity and blood compatibility of PSF membrane could be improved by chitosan and heparin conjugate. Copyright © 2003 John Wiley & Sons, Ltd.     

Zinc-human serum albumin association: testimony of two binding sites

The zinc cation (Zn²⁺) binding to human serum albumin (HSA) was studied using a non-equilibrium approach in order to prove two HSA binding sites. The effect of the bulk solvent pH and column temperature T on this binding and the corresponding thermodynamic data were also investigated. It appeared that the association process can be divided into two pH value ranges due to a predominant Zn²⁺ interaction with either HSA site I or site II. It was also demonstrated that the Zn²⁺ affinity for the site II was weakly affected by modifying the mobile phase pH whereas for the site I, the affinity constant increased strongly with increasing the pH of the bulk solvent.     

Purification and analysis of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography

We discuss the purification of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography. The hydrophobicity difference between the different fractionated species was induced by the addition of a lyotropic salt that caused phase transition of PEG (hydrophilic under normal condition) to a mildly hydrophobic form. The HSA PEGylation reaction mixture was mixed with lyotropic salt and passed through a stack of hydrophilized polyvinylidene fluoride membrane discs. Unmodified HSA was obtained in the flow through, while the PEGylated forms of the protein bound to the membrane and could be eluted by reducing the salt concentration. Among the three major PEGylated forms of HSA present in the feed (i.e. mono–, di–, and tri–), mono‐PEGylated HSA was eluted first and could be resolved from the others. The purified material was analyzed by SDS‐PAGE, dynamic light scattering, and SEC combined with multi‐angle light scattering. All these analytical techniques indicated the presence of species that has a molar mass consistent with mono‐PEGylated HSA. A scaled‐down version of the membrane chromatographic methods could be used for the rapid and sensitive analysis of PEGylated proteins.     

Investigation of non-covalent interactions between paramagnetic complexes and human serum albumin by electrospray mass spectrometry

Stable gadolinium(III) chelates are nowadays routinely used as contrast agents for magnetic resonance imaging (MRI). Their non-covalent binding to human serum albumin (HSA) has shown to improve their efficacy. Non-covalent interactions lead to complex formation that can be quantified by several techniques that are mostly tedious and time-consuming. In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate the interaction between HSA and several gadolinium(III) complexes. The results were compared with those obtained in the liquid phase. Four gadolinium complexes were investigated: Gd-DTPA 1, Gd-C(4)Me-DTPA 2, Gd-EOB-DTPA 3, and MP-2269 4. Relaxometry studies show that complexes 1 and 2 have no significant affinity for HSA, while complexes 3 and 4 have increasing affinities for the protein. 1:1 and 1:2 complexes between HSA and MP-2269 were detected by ESI-MS for a twofold excess of the contrast agent, whereas a ligand/protein molar ratio of 4:1 was necessary to observe a 1:1 stoichiometry for Gd-EOB-DTPA, an observation that is in good agreement with the known weaker affinity of the contrast agent for the protein. At a fourfold molar excess, no supramolecular complex was observed for Gd-DTPA 1 and Gd-C(4)Me-DTPA 2; a tenfold molar excess was necessary to detect a 1:1 complex, confirming the very weak affinity of these contrast agents for HSA.     

Electrophoretic immunodesorption of proteins according to molecular weight by use of a double-membrane system

Summary A simple method is described for electrophoretic desorption of proteins from antigen-antibody complexes, with more than 90% recovery and without denaturation, after immunosorbent affinity chromatography. Radiolabeled or unlabeled human serum albumin (HSA) and α-1-antitrypsin (AAT), conjugated to rabbit anti-HSA or anti-AAT polyclonal antisera, respectively, were electrophoretically desorbed from Sepharose 4B. In addition, purification and concentration of the major HSA protein band (monomer) of 68 kD from the other oligomeric protein bands were achieved by use of a two-membrane system in a simple electroelution apparatus. The system consisted of an upper cellulose acetate membrane, with pore size 20 nm and separation limit 70 kD, and a lower dialysis cellophane membrane with molecular weight cut-off from 1–50 kD that cnables separation according to size. Furthermore, purification of the monomer HSA or AAT from normal human serum was performed with 92% recovery. Homogeneity was implied by the presence of one band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, Western blot, and autoradiography.     

人血清白蛋白柱上药物的手性拆分

考察了４种酸性药物和１种中性药物对映体在人血清白蛋白手性固定相上的保留行为。这５种药物与人血清白蛋白结合的亲和力高，难于实现快速分离，作者提出在流动相中加入短链脂肪酸－正己酸，可快速手性拆分非诺洛芬、萘普生和布洛芬。酮基布洛芬对映体分离选择性随乙腈浓度升高而增大，流动相中加入适量异丙醇可使对映体选择性大大增加（α～１．２３），华法令同样可取得很好分离。     

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase.

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin-azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.