基于光波导分光光谱技术研究蛋白质与亚甲基蓝的竞争吸附行为

通过利用时间分辨光波导分光光谱技术原位测量从蛋白质-亚甲基蓝(MB)混合水溶液吸附到亲水玻璃光波导表面的MB可见光吸收谱,观测到在溶液pH值低于蛋白质等电点时MB与牛血清蛋白(BSA)以及MB与血红蛋白(Hb)存在竞争吸附行为,进一步测得这种竞争吸附行为对蛋白质浓度十分敏感,可以用于简单测定溶液中的蛋白质含量.基于Langmuir等温吸附理论推导出了两种分子竞争吸附的动力学方程,并利用该动力学方程对实验测得的吸光度随时间变化曲线进行了最佳拟合,揭示了玻璃表面吸附的MB分子个数在达到最大值后随时间呈指数衰减,同时得出拟合参数与蛋白质浓度呈准线性关系.     

Adsorption behavior of acidic and basic proteins onto citrate-coated Au surfaces correlated to their native fold, stability, and pI

The adsorption of eight different proteins (alpha-lactalbumin (types I and III), bovine serum albumin, hemoglobin, myoglobin, cytochrome c, alpha-casein, and lysozyme) onto a model anionic surface was performed at equivalent bulk (solvent, ionic strength, pH) and surface conditions. Adsorption was monitored on a quartz crystal microbalance with dissipation monitoring (QCM-D) with citrate-coated gold surfaces as adsorbents and has been correlated to native fold stability determined from near- and far-UV circular dichroism (CD) measurements. The proteins studied here were chosen based on their pI and documented knowledge about their structural stability and flexibility. Protein adsorption was found to be independent of global protein charge. Rather, binding occurs through oppositely charged patches on protein and surface. Moreover, data indicate that there is a correlation between secondary and tertiary structure stability and the adsorption characteristics at interfaces. Also, protein surface coverage, layer thickness, and flexibility can be tuned as a function of deposition method. This is discussed in terms of adsorption/spreading kinetics and intermolecular (protein-surface and protein-protein) interactions. Adsorption to surfaces can induce formation of supramolecular structures such as micelles (in the case of alpha-Cas) and multilayers (as for Hb). In the case of alpha-casein, this phenomenon depends on the deposition method and protein concentration. When ranking the surface coverage for proteins added in excess, the order is Lyz < Cyt c < Mb < BSA < alpha-La I < alpha-Cas < alpha-La III < Hb, which can be correlated to the proteins ability to form supramolecular structures (alpha-Cas, Hb), overall conformational flexibilities, and ability to form stable intermediates.     

In-vitro protein interactions with a bioactive gel-glass

Recent theories suggest that the local adsorption of biologically active peptide growth factors onto the surface of an implant may contribute to the unique osteogenic nature of silica-containing bioactive ceramics. A sol-gel derived glass is used as a model of the in-vivo reaction product of 45S5 bioactive glass at relatively short times (<48 hrs.) to investigate protein adsorption/desorption behavior. The adsorption kinetics of three heme-class proteins (cytochrome c, myoglobin, and hemoglobin) are measured spectroscopically. The rate of adsorption is shown to increase with average pore size, which is determined by the silica content of the gel. Adsorption rate decreases as protein size is increased and as solution pH is decreased. Biological function of an adsorbed peroxidase enzyme on pre-reacted Bioglass^® is shown to be retained. Desorption during physiologic conditions is shown to be linear with time and pH dependant, while independent of gel bioactivity.     

Experimental evidence of the reversibility of the first stage of protein adsorption at a hydrophobic quartz surface near the isoelectric point

The reversibility of the first stage of adsorption of zwitterionic cytochrome c on a hydrophobic quartz surface was investigated using time‐resolved slab optical waveguide (SOWG) absorption spectroscopy. Using a novel prism‐free broadband coupling approach, absorbance data were collected successfully at a 50 ms time interval during the first few seconds after solution–surface contact. Near the isoelectric point where the cytochrome c molecules possess a net charge of zero and hence cannot be influenced by an electric field, the speed at which adsorption proceeded was found to be dependent on cytochrome c concentration as well as on surface hydrophobicity. It was also observed that the degree of protein adsorption increased as the surface hydrophobicity was increased. Within 6 s the adsorption process appeared to be reversible, as revealed by extremely low chi‐squared values when the absorbance data were fitted into the reversible Langmuir‐type kinetic model. The standard Gibbs free energy of adsorption was also calculated from the absorbance data. Copyright © 2003 John Wiley & Sons, Ltd.     

Protein separation and enrichment by counter-current chromatography using reverse micelle solvent systems

A protein mixture consisting of myoglobin, cytochrome c, and lysozyme was separated by high-speed counter-current chromatography using a two-phase aqueous/reverse micelle-containing organic solvent system. About 50% stationary phase retention ratio was obtained in most chromatographic experiments. Separations were manipulated mainly by pH gradients that controlled the electrostatic interactions between the protein molecules and reverse micelles. Separations were further improved by incorporating an ionic strength gradient along with the pH gradient. Control of ionic strength in the aqueous solution helped fine-tune protein partitioning between the stationary and mobile phases. Although non-specific protein interactions affected baseline resolution, recovery of cytochrome c and lysozyme reached 90% and 82%. Furthermore, concentration or enrichment of these two proteins was achieved from a large-volume sample load. This technique can potentially be employed in the recovery and enrichment of proteins from large-volume aqueous solutions.     

Highly efficient enrichment and subsequent digestion of proteins in the mesoporous molecular sieve silicate SBA-15 for matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight/time-of-flight analyzer peptide mapping

Based on a previous study of protein digestion inside the nanoreactor channels of the mesoporous molecular sieve silicate SBA-15 (Chem. Eur. J. 2005, 11: 5391), we have developed a highly efficient enrichment and subsequent tryptic digestion of proteins in SBA-15 for matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) peptide mapping. The performance of the method is exemplified with myoglobin and cytochrome c. First, protein adsorption isotherms for two standard proteins with a range of initial concentration of proteins were investigated at room temperature. The results revealed that the kinetic adsorption rate of a protein within SBA-15 was independent of initial protein concentration, and a 15-min protein enrichment within SBA-15 could be enough for protein identification in biological samples. It was noticed that no washing steps were needed to avoid protein loss due to desorption from the mesochannels into solution. Second, protein digestion inside the channels of SBA-15 was also optimized. After adsorption of proteins into SBA-15 in 15 min, the trypsin solution (pH 8) was directly added to the SBA-15 beads with immobilized proteins by centrifugation, and then the digestion was performed for 15 min at 37 degrees C. It was observed that a higher peptide sequence covering of 98% for myoglobin was obtained by MALDI-TOF/TOF analysis, compared to in-solution digestion. So the protein digestion inside SBA-15 was proved to be significantly faster and yielded a better sequence coverage. The new procedure allows for rapid protein enrichment and digestion inside SBA-15, and has great potential for protein analysis.     

Protein adsorption in fused-silica and polyacrylamide-coated capillaries

The model proteins cytochrome c, myoglobin, ovalbumin, and beta-lactoglobulin were investigated with regard to their adsorption properties on capillaries for electrophoresis. The model compounds were selected to cover a wide range of properties. Cytochrome c is a basic protein (isoelectric point (pI): 9.6; M(r): 11.7 kDa), beta-lactoglobulin is rather acidic (pI: 5.4, M(r): 18.4 kDa), myoglobin was chosen as a neutral reference protein (pI: 6.8-7.4, M(r): 17.8 kDa), and ovalbumin (pI: 5.1, M(r): 45.0 kDa) was selected as a relatively larger analyte. First, the pH dependence of adsorption was investigated for the bare fused silica. A clear correlation to the respective pIs was noted. For myoglobin and ovalbumin, none or negligible adsorption was found above the pI, whereas strong adsorption was noted just below this parameter. Cytochrome c and beta-lactoglobulin already showed distinct adsorption above their pIs. However, none of the proteins showed any significant adsorption more than one pH unit above the pIs. For linear polyacrylamide-coated capillaries, a decreased but not a complete lack of adsorption was observed. Here, pH-dependent adsorption was noted as well. Regeneration of the capillaries by rinsing with buffers containing 200 mM SDS was also investigated. This method was completely successful for myoglobin, but that too for only freshly-adsorbed protein. After a storage time of 24 h and due to the aging of the adsorbate, a sufficient regeneration was no longer possible.     

Investigations on the Q and CT Bands of Cytochrome c Submonolayer Adsorbed on an Alumina Surface Using Broadband Spectroscopy with Single-Mode Integrated Optical Waveguides

In this work, we report experimental results on the molar absorptivity of cytochrome c adsorbed at different submonolayer levels onto an aluminum oxide waveguide surface; our data show a clear dependence of the protein optical properties on its surface density. The measurements were performed using the broadband, single-mode, integrated optical waveguide spectroscopic technique, which is an extremely sensitive tool able to reach submonolayer levels of detection required for this type of studies. This investigation focuses on the molar absorptivity at the Q-band (centered at 525 nm) and, for the first time to our knowledge, the weak charge transfer (CT) band (centered at 695 nm) of surface-adsorbed cyt c. Polarized light in the spectral region from 450 to 775 nm was all-coupled into an alumina thin film, which functioned as a single-mode planar optical waveguide. The alumina thin-film waveguide used for this work had a thickness of 180 nm and was deposited on a glass substrate by the atomic layer deposition process. The protein submonolayer was formed on the alumina waveguide surface through electrostatic adsorption from an aqueous buffer solution at neutral pH. The optical properties of the surface-adsorbed cyt c were investigated for bulk protein concentrations ranging from 5 nM to 8200 nM in the aqueous buffer solution. For a protein surface density of 2.3 pmol/cm(2), the molar absorptivity measured at the charge transfer band was 335 M(-1) cm(-1), and for a surface density of 15 pmol/cm(2) was 720 M(-1) cm(-1), which is much closer to the value of cyt c dissolved in an aqueous neutral buffer (830 M(-1) cm(-1)). The modification of the protein molar absorptivity and its dependence on the surface density can most likely be attributed to conformational changes of the surface-adsorbed species.     

A cationic β‐cyclodextrin as a dynamic coating for the separation of proteins in capillary electrophoresis

A cationic cyclodextrin was used as dynamic coating for the capillary electrophoresis of a model mixture of proteins (i.e., ubiquitin, α‐lactoglobulin, cytochrome‐c, and myoglobin) as positively charged species in a fused silica capillary. An interesting feature of the coating is that by simple adjustment of the concentration of cyclodextrin added into the background electrolyte, a neutral or positively charged surface, which was beneficial in preventing protein adsorption at the inner capillary wall surface, was obtained. This is the first demonstration of a dynamic coating that yielded a neutral surface for protein separations in capillary electrophoresis. Based on electro‐osmotic flow measurements, addition of 0.05 to 0.10 mg/mL quaternary β‐cyclodextrin in a low pH electrolyte resulted in a neutral or positive surface (undetectable to very slow anodic electro‐osmotic flow). The coating approach afforded the electrophoretic separation of the mixture of proteins at positive polarity with good repeatability and separation performance.     

Micropreparative isoelectric focusing protein separation in a suspended drop

IEF protein binary separations were performed in a 12-μL drop suspended between two palladium electrodes, using pH gradients created by electrolysis of simple buffers at low voltages (1.5-5 V). The dynamics of pH gradient formation and protein separation were investigated by computer simulation and experimentally via digital video microscope imaging in the presence and absence of pH indicator solution. Albumin, ferritin, myoglobin, and cytochrome c were used as model proteins. A drop containing 2.4 μg of each protein was applied, electrophoresed, and allowed to evaporate until it splits to produce two fractions that were recovered by rinsing the electrodes with a few microliters of buffer. Analysis by gel electrophoresis revealed that anode and cathode fractions were depleted from high pI and low pI proteins, respectively, whereas proteins with intermediate pI values were recovered in both fractions. Comparable data were obtained with diluted bovine serum that was fortified with myoglobin and cytochrome c.     

Trypsin entrapped in poly(diallyldimethylammonium chloride) silica sol-gel microreactor coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An enzyme-immobilized capillary microreactor for rapid protein digestion and proteomics analysis is reported. The inner surface of the fused-silica capillary was coated with poly(diallyldimethylammonium chloride) (PDDA)-entrapped silica sol-gel matrix, followed by assembly of trypsin onto the PDDA-modified surface via electrostatic adsorption. The immobilization parameters such as PDDA content in the sol-gel matrix, trypsin concentration and pH were investigated in detail. Protein samples including beta-casein, myoglobin and cytochrome c could be effectively digested and electrophoretically separated simultaneously in such a modified capillary. Just 2.26 ng (corresponding to 0.10-0.14 picomole) of sample was sufficient for on-line capillary electrophoresis peptide mapping. The efficiency of the digestion was further demonstrated by digestion of a human liver cytoplasm sample and 253 proteins were identified in one unique run.     

Radiochemische und elektrochemische untersuchung der adsorption von cytochrom c an der phasengrenze elektrolyt/quecksilber

The surface area of an adsorbed cytochrome-c molecule was calculated both from the radiochemically determined surface concentration and from the adsorption kinetics. The value thus obtained is 2000±200 Ų. This accordance shows that the Koryta equation holds for the adsorption kinetics of proteins. The comparison with the cross-section of the molecule in crystalline state allows us to conclude that cytochrome c is unfolded at the electrode. While at low concentrations the molecules are strongly adsorbed, at high concentrations an exchange of adsorbed molecules with molecules in the bulk occurs. The mechanism of charge transfer is proposed to be a superposition of electron transport through adsorbed molecules and exchange of already reduced molecules.     

Loose ultrafiltration of proteins using hydrolyzed polyacrylonitrile hollow fiber

Ultrafiltration of proteins using hydrolyzed polyacrylonitrile hollow fiber was investigated in this work. Polyacrylonigrile hollow fiber was spun and hydrolyzed with NaOH aqueous solution under various conditions. A thin layer of polyacrylic acid was formed on the inner surface of the hollow fiber, and the ionic density on the hydrolyzed surface was determined through titration. The hydrolyzed hollow fiber was characterized with the hydraulic permeability and the retention of dextran. A sharp decrease in the hydraulic permeability was observed between pH 5 and 6, possibly due to the swelling of the hydrolyzed layer. The retention of dextran slightly increased with the ionic density of the hydrolyzed hollow fiber. The retentions of two proteins, myoglobin and cytochrome c, were measured over a range of pH values (4∼10). The results show that the retention of protein changes with pH, and is the lowest at the isoelectric point. Separation of mixtures of cytochrome c and myoglobin was performed at pH 5, 7 and 9 using hydrolyzed PAN hollow fiber. The selectivity of the cytochrome c over myoglobin was about 40 at pH 5, about 1 at pH 7, and about 0.5 at pH 9.     

The adsorption and unfolding kinetics determines the folding state of proteins at the air-water interface and thereby the equation of state

Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends on the rate of adsorption to the interface, which can be controlled by bulk concentration. Therefore, the adsorption of proteins with varying structural stabilities at several protein concentrations was studied using ellipsometry and surface tensiometry. For beta-lactoglobulin the adsorbed amount (Gamma) needed to reach a certain surface pressure (Pi) decreased with decreasing bulk concentration. Ovalbumin showed no such dependence. To verify whether this difference in behavior is caused by the difference in structural stability, similar experiments were performed with cytochrome c and a destabilized variant of this protein. Both proteins showed identical Pi-Gamma, and no dependence on bulk concentration. From this work it was concluded that unfolding will only take place if the kinetics of adsorption is similar or slower than the kinetics of unfolding. The latter depends on the activation energy of unfolding (which is in the order of 100-300 kJ/mol), rather than the free energy of unfolding (typically 10-50 kJ/mol).     

A new model of protein adsorption kinetics derived from simultaneous measurement of mass loading and changes in surface energy

We describe a novel technology based on changes in the resonant frequency of an acoustically actuated surface and use it to measure temporal changes in the surface energy gamma (N m(-1)) of an elastomeric polymer membrane due to the adsorption of macromolecules from aqueous solution. The resonant elastomeric surface-tension (REST) sensor permits simultaneous determination of mass loading kinetics and gamma(t) for a given adsorption process, thereby providing a multivariable data set from which to build and test models of the kinetics of adsorption at solid-liquid interfaces. The technique is used to measure gamma(t) during the adsorption of either sodium dodecyl sulfate (SDS) or hen egg-white lysozyme (HEWL) onto an acrylic polymer membrane. The adsorption of SDS is reversible and is characterized by a decrease in gamma over a time period that coincides with that required for the mass loading of the membrane. For the adsorption of HEWL labeled with Alexa Fluor 532 dye, gamma continues to change long after the surface concentration of labeled HEWL, measured by using the elastomeric polymer membrane as an optical waveguide, reaches steady state. Gradual but significant changes in gamma(t) are observed as long as the concentration of protein in the bulk solution, c(b), remains nonzero. HEWL remains adsorbed to the membrane when c(b) = 0, but changes in gamma(t) are not observed under this condition, indicating that the interaction of bound protein molecules with those free in solution contribute to the prolonged change in the surface energy. This observation has been used to define a new model for the kinetics of globular protein adsorption to a solid-liquid interface that includes a mechanism by which the molecules in the bulk can facilitate the desorption of a sorbate molecule or change the energetic states of adsorbed molecules and, thus, the overall surface energy. The model is shown to capture the unique features of protein adsorption kinetics, including the relatively fast mass loading, the much more gradual change in surface energy that does not cease until the protein is removed from the bulk, the rapid desorption of an incubation-time-dependent fraction of bound protein when the protein is removed from the bulk, and the fixing of the residual surface concentration and surface energy at constant values once the removal of reversibly bound protein and free protein is complete.     

Iceberg Model of the Structure of Globular Protein Adsorption Layers at the Air–Water Interface. Study by the Tritium Planigraphy Technique

The organization of the adsorption layers of insulin, pancreatic inhibitor of trypsin, ribonuclease S, lysozyme, myoglobin, carboxypeptidase A, subtilisin, and thermolysin at the air–water interface was studied by tritium planigraphy technique. The location of globular protein molecules relative to the interface was determined for solution concentrations corresponding to the formation of saturated adsorption layers (10^–4 M, pH 6). It is established that the fraction of solution surface occupied by protein component is equal to 7–62%. Only a small part of globules with the height from 2 to 19% of their effective diameter is above the solution surface, whereas the main part of globules is submerged into water (the iceberg model).     

Preparation of Keggin‐type phosphomolybdate by a one‐step solid‐state reaction at room temperature and its application in protein adsorption

Keggin‐type phosphomolybdate ((C₁₉H₄₂N)₃PMo₁₂O₄₀) is prepared by a one‐step solid‐state reaction at room temperature and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X‐ray diffraction, thermogravimetric analysis, and elemental analysis. The as‐prepared phosphomolybdate is demonstrated to be an efficient adsorbent for proteins. In this particular case, the selective adsorption of neutral protein hemoglobin is achieved. While under the same conditions virtually no adsorption of acidic and basic proteins, represented by bovine serum albumin and cytochrome c, are observed. A solid‐phase extraction procedure is developed for the selective isolation of hemoglobin. At pH 6, a sorption efficiency of 91.4% is achieved for 100 μg/mL hemoglobin in 1.0 mL solution by using 5.0 mg of the phosphomolybdate. The adsorption behavior of hemoglobin fits well with a Langmuir adsorption model, corresponding to a theoretical adsorption capacity of 55.86 mg/g. The retained hemoglobin could be readily recovered by using a 60 mmol/L imidazole solution at pH 7, giving rise to a recovery of 64.7%. The practical application of phosphomolybdate for protein adsorption is demonstrated by the selective isolation of hemoglobin from human whole blood followed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis assay.     

The isolation of basic proteins by solid-phase extraction with multiwalled carbon nanotubes

Multiwalled carbon nanotubes (MWCNTs) have been employed for the first time as sorbents for the isolation of basic proteins from other protein species in biological sample matrices by solid-phase extraction (SPE). A microcolumn packed with MWCNTs was incorporated after appropriate pretreatment into a sequential injection system, which facilitates online selective sorption of basic protein species (hemoglobin and cytochrome c in this particular case). The retained protein species were afterwards separated from each other by sequential elution from the microcolumn through the employment of appropriate eluents. A 0.025 mol L(-1) phosphate buffer solution of pH 8.0 facilitated the efficient collection of hemoglobin, while a 0.5 mol L(-1) NaCl solution ensured the quantitative recovery of the retained cytochrome c. With a sample loading volume of 2.0 mL, enrichment factors of 11 and 15 were derived for hemoglobin and cytochrome c, along with retention efficiencies of 100% for both species and recovery rates of 98 and 90%, respectively. A sampling frequency of 8 h(-1) was achieved, and the precisions were 3.0% and 0.8% (RSD) for hemoglobin and cytochrome c at a concentration of 5.0 microg mL(-1). The practical applicability of this system was demonstrated by processing of human whole blood for isolation of hemoglobin, and satisfactory results were obtained by assay with SDS-PAGE.     

基于ITO电极的细胞色素c吸附层的直接电化学

采用未经修饰的铟锡氧化物(ITO)工作电极直接探测到了细胞色素c(Cytc)吸附层的氧化还原峰,并得出了Cytc的表面浓度,随着溶液浓度从2μmo·lL-1增大到10μmo·lL-1,Cytc的表面浓度相应地从0.35×10-12mo·lcm-2增大到1.53×10-12mo·lcm-2.实验获得的表面浓度倒数与溶液浓度倒数的准线性关系说明Cytc在ITO表面的吸附基本满足Langmuir等温吸附理论.对Cytc溶液的循环伏安测试结果表明参与电极反应的Cytc包括游离分子和吸附分子,前者的贡献大于后者,电极反应主要受扩散控制并呈准可逆过程.根据Nicholson方法估算得到反应物的标准异相速率常数的平均值为1.65×10-3cm·s-1.实验结果显示在室温下放置1h后Cytc吸附层电化学活性部分丧失,在80℃下放置1h后吸附层完全失活.失活的Cytc吸附层对铁氰化钾溶液在Au电极上的电极反应具有明显的阻碍作用.     

Adsorption of Water-Soluble Proteins onto Bubbles in Continuous Foam Separation

The mechanism of water-soluble protein enrichment in continuous foam separation was studied. The liquid flow rate and the protein concentration in the foam phase were measured at various heights from the interface between the bulk liquid and foam layer, and the intrinsic values at the interface were estimated by the extrapolation method to determine the accurate adsorption density on the bubble surface. Ovalbumin (OA) and hemoglobin (HB) were used as the soluble proteins. The solution pH values were varied from 3.5 to 6.0 for OA and from 6.0 to 8.0 for HB. The experimental isotherms for OA and HB were compared to the Langmuir isotherm, and the two adsorption parameters of the equilibrium constant, K, and the saturated density, gamma, at each pH were determined. Both gamma values obtained for OA and HB showed maxima at their isoelectric point (pH 4.6 for OA and pH 6.8 for HB). Assuming that OA and HB molecules are spherical in shape and are adsorbed on the bubble surface in a close-packed structure at saturation, the calculated diameters for OA and HB molecules were quite similar to the literature values. The variation in gamma for both OA and HB is discussed qualitatively in relation to the net charge of the protein molecule. Copyright 2000 Academic Press.