Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability

The influence of protein stability on the adsorption and desorption behavior to surfaces with fundamentally different properties (negatively charged, positively charged, hydrophilic, and hydrophobic) was examined by surface plasmon resonance measurements. Three engineered variants of human carbonic anhydrase II were used that have unchanged surface properties but large differences in stability. The orientation and conformational state of the adsorbed protein could be elucidated by taking all of the following properties of the protein variants into account: stability, unfolding, adsorption, and desorption behavior. Regardless of the nature of the surface, there were correlation between (i) the protein stability and kinetics of adsorption, with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability. All of the above correlations could be explained by the rate of denaturation and the conformational state of the adsorbed protein. In conclusion, protein engineering for increased stability can be used as a strategy to decrease irreversible adsorption on surfaces at a liquid-solid interface.     

Quantitation of Group-specificaAntigen in Hepatitis B Vaccines by Anti-HBs/aMonoclonal Antibody

Balb/c mice were immunized with aluminium hydroxide [alum, Al(OH)₃]-adjuvanted hepatitis B (HB) vaccines of subtypesadr,ayworadw. Spleen cells from the immune animals were fused with SP2/O cells. Eight hybridoma clones producing antibodies specific or HB surface antigen (HBsAg) were selected. Monoclonal antibodies (mAbs) of four clones were specific for group-specific antigen/a, and the other of four clones were specific for subtype antigen/d,y,r, orw. The anti-HBs/amAbs were classified into three non-competitive groups.Quantitation of group-specific determinantaof HBsAg (HBsAg/a) was performed by sandwich enzyme-linked immunosorbent assay (ELISA), in which a solid phase of anti-HBs guinea-pig polyclonal antibodies (pAb), the HBsAg for testing, anti-HBs/amouse mAb and horseradish peroxidase (HRP)-conjugated anti-mouse IgG were used.The unadsorbed HBsAg was used to establish the standard curve HBsAg/a. The lower detection limits were 0·5 to 1 ng/ml of HBsAg. Methods of solubilization of alum were investigated to quantify HBsAg/ain adsorbed HB vaccines. The recovery rate was more than 60% if vaccines were prediluted. The recovery of HBsAg/ain HB vaccines produced by the same manufacturer showed the similar recovery rate, and the contents of HBsAg/ain adsorbed HB vaccines could be estimated by the recovery rate determined for adsorbed HB vaccines.     

Optimization of adsorption conditions of papain on dye affinity membrane using response surface methodology

The adsorption of papain on Reactive Blue 4 dye–ligand affinity membrane was investigated in a batch system. The combined effects of operating parameters such as initial pH, temperature, and initial papain concentration on the adsorption were analyzed using response surface methodology. The optimum adsorption conditions were determined as initial pH 7.05, temperature 39 °C, and initial papain concentration 11.0 mg/ml. At optimum conditions, the adsorption capacity of dye–ligand affinity membrane for papain was found to be 27.85 mg/g after 120 min adsorption. The papain was purified 34.6-fold in a single step determined by fast protein liquid chromatography. More than 85% of the adsorbed papain was desorbed using 1.0 M NaCl at pH 9.0 as the elution agent. The purification process showed that the dye–ligand immobilized composite membrane gave good separation of papain from aqueous solution.     

Trypsin immobilization by direct adsorption on metal ion chelated macroporous chitosan-silica gel beads

Silica gel bead coated with macroporous chitosan layer (CTS-SiO₂) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu²⁺, Zn²⁺, Ni²⁺ ions, respectively on CTS-SiO₂, and trypsin could be adsorbed on the IMAC adsorbent through metal–protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu²⁺-CTS-SiO₂ with the value of 4980 ± 125 IU g⁻¹ of the adsorbent, while the maximal adsorption capacity for trypsin on Zn²⁺ and Ni²⁺ loaded adsorbent was 3762 ± 68 IU g⁻¹ and 2636 ± 53 IU g⁻¹, respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5–10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO₂ beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 °C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO₂ could effectively digest BSA revealed by HPLC peptide mapping.     

Salt-independent adsorption of human serum proteins on cyanocarbon gels

Electron donor acceptor gels based on cyanocarbons have been tested for human serum protein adsorption in the absence of salt-promotion by water-structuring salt. This phenomenon was compared with a normal adsorption process in the presence of salt. The tricyanoaminopropene–divinyl sulfone–agarose displayed unusual protein adsorption properties as binding could occur both independently or dependently of the salt-promotion. The absence of hydrophobic or ionic character of the salt-independent interaction suggests an electron donor acceptor adsorption mechanism which is shown, for the first time, to occur independently of salt-promotion in aqueous solution. Study of the protein adsorption specificity showed similar protein selectivity for the fractions adsorbed in both conditions.     

Selective adsorption/desorption of nucleic acids on submicron-sized polymeric particles

DNA can be removed or separated by the selective adsorption/desorption on positively charged submicronsized polymeric particles (SSPP). The selective adsorption of DNA, in the presence of protein, on positively charged SSPP was accomplished by increasing the concentration of potassium phosphate or sodium phosphate. The adsorption of DNA was not affected by the concentration of potassium phosphate or sodium phosphate up to 1.2M. On the other hand, the adsoprtion of a protein (bovine serum albumin) was completely impeded by 170mM potassium phosphate. DNA adsorbed on SSPP could be desorbed by increasing the concentration of NaCl or KCl, thus it can be recovered. DNA desorbed from SSPP when the concentration of NaCl or KC was higher than 0.6M. A complete desorption of DNA was achieved at the concentration of NaCl or KCl above 1.2M.     

Cell adsorption and selective desorption for separation of microbial cells by using chitosan-immobilized silica

Cell adsorption and selective desorption for separation of microbial cells were conducted by using chitosan-immobilized silica (CIS). When chitosan was immobilized onto silica surfaces with glutaraldehyde, bacterial cells adsorbed well and retained viability. Testing of the adsorption and desorption ability of CIS using various microbes such as Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus casei, Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Saccharomyces cerevisiae, Saccharomyces ludwigii, and Schizosaccharomyces pombe revealed that most microbes could be adsorbed and selectively desorbed under different conditions. In particular, recovery was improved when L-cysteine was added. A mixture of two bacterial strains adsorbed onto CIS could also be successfully separated by use of specific solutions for each strain. Most of the desorbed cells were alive. Thus, quantitative and selective fractionation of cells is readily achievable by employing chitosan, a known antibacterial material.     

Adsorption, desorption, and activity of glucose oxidase on selected clay species.

The adsorption of the enzyme glucose oxidase (EC 1.1.3.4) to clays followed the pattern described for other proteins as being pH dependent. Maximum adsorption occurred at or below the isoelectric point of the enzyme. The amount of enzyme adsorbed to clay was influenced by the type of clay used, and also the saturating cations. Initially adsorbed enzyme showed low specific activities, and as amounts of enzyme adsorbed approached maximum stauration of clay, specific activities increased approaching that determined for free enzyme. The adsorption of glucose oxidase involved a temperature-independent cation-exchange mechanism, and enzyme adsorbed to surfaces of clay could be desorbed in active form by elevation of pH of suspending solution. This was followed by a slower temperature-dependent fixation, probably by hydrogen bonding, which resulted in protein being irreversibly adsorbed to clay surfaces. It is proposed that on adsorption of glucose oxidase to clay surfaces unravelling of the protein structure occurred, which allowed penetration of protein into the interlamellar spaces of montmorillonite. This proposal was based on the observed expansion of montmorillonite to 23 A, and the decreases in amount of a second-protein lysozyme adsorbed with extended incubation times of glucose oxidase - clay complexes at pH 4.5.     

Protein adsorption at solid-liquid interfaces: Part II--Adsorption from binary protein mixture

Extent of adsorption of proteins at alumina-water interface from solutions containing binary mixture of beta-lactoglobulin and bovine serum albumin (BSA), beta-lactoglobulin and gelatin, and gelatin and bovine serum albumin has been estimated as functions of protein concentrations at varying pH, ionic strength, temperature and weight fraction ratios of protein mixture. The extent of adsorption (gamma lacw) of lactoglobulin in the presence of BSA increases with increase of protein concentration (Clac) until it reaches a maximum but a fixed value gamma lacw(m). Extent of adsorption gamma serw also initially increases with increase of protein concentrations until it reaches maximum value gamma serw(m). Beyond these protein concentrations, adsorbed BSA is gradually desorbed due to the preferential adsorption of lactoglobulin from the protein mixture. In many systems, gamma serw at high protein concentrations even becomes negative due to the strong competition of BSA and water for binding to the surface sites in the presence of lactoglobulin. For lactoglobulin-gelatin mixtures, adsorption of both proteins is enhanced as protein concentration is increased until limiting values for adsorption are reached. Beyond the limiting value, lactoglobulin is further accumulated at the interface without limit when protein concentration is high. For gelatin-albumin mixtures, extent of gelatin adsorption increases with increase in the adsorption of BSA. The limit for saturation of adsorption for gelatin is not reached for many systems. At acid pH, adsorbed BSA appears to be desorbed from the surface in the presence of gelatin. From the results thus obtained the role of electrostatic and hydrophobic effects in controlling the adsorption process has been analysed.     

Cadmium removal from human plasma by Cibacron Blue F3GA and thionein incorporated into polymeric microspheres

Poly(2-hydroxyethylmethacrylate–ethyleneglycoldimethacrylate) [poly(HEMA–EGDMA)] microspheres carrying Cibacron Blue F3GA and/or thionein were prepared and used for the removal of cadmium ions Cd(II) from human plasma. The poly(HEMA–EGDMA) microspheres, in the size range of 150–200 μm in diameter, were produced by a modified suspension copolymerization of HEMA and EGDMA. The reactive triazinyl dye-ligand Cibacron Blue F3GA was then covalently incorporated into the microspheres. The maximum dye incorporation was 16.5 μmol/g. Then, thionein was bound onto the Cibacron Blue F3GA-incorporated microspheres under different conditions. The maximum amount of thionein bound was 14.3 mg/g. The maximum amounts of Cd(II) ions removed from human plasma by poly(HEMA–EGDMA)–Cibacron Blue F3GA and poly(HEMA–EGDMA)–Cibacron Blue F3GA–thionein were of 17.5 mg/g and 38.0 mg/g, respectively. Cd(II) ions could be repeatedly adsorbed and desorbed with both types of microspheres without significant loss in their adsorption capacity.     

Fractionation of lignin-carbohydrate complexes by hydrophobic-interaction chromatography

Hydrophobic properties of lignin-carbohydrate complexes (LCC) isolated from Pinus densiflora Sieb. et Zucc. have been analysed by hydrophobic-interaction chromatography on Phenyl- and Octyl-Sepharose CL-4B gels. The ability of LCC to be adsorbed by these hydrophobic gels was exclusively dependent on their lignin content. Materials adsorbed on Octyl-Sepharose were desorbed with a lower concentration of 2-ethoxyethanol than those adsorbed on Phenyl-Sepharose. In the adsorption of LCC by Phenyl-Sepharose, ππ interactions between the aromatic ligands and the benzene skeletons of lignin play an important role, whereas hydrophobic interaction is the exclusive driving-force for adsorption in the case of Octyl-Sepharose.     

Binding of the protoxin and toxin proteins ofBacillus thuringiensis subsp.kurstaki on clay minerals

The equilibrium adsorption and binding of the delta-endotoxin proteins, i.e., the protoxins (M_r=132 kDa) and toxins (M_r=66 kDa), fromBacillus thuringiensis subsp.kurstaki were greater on montmorillonite than on kaolinite (five-fold more protoxin and three-fold more toxin were adsorbed on montmorillonite). Approximately two- to three-fold more toxin than protoxin was adsorbed on these clay minerals. Maximum adsorption occurred within 30 min (the shortest interval measured), and adsorption was not significantly affected by temperatures between 7° and 50°C. The proteins were more easily desorbed from kaolinite than from montmorillonite; they could not be desorbed from montmorillonite with water or 0.2% Na₂CO₃, but they could be removed with Tris-SDS (sodium dodecyl sulfate) buffer. Adsorption was higher at low pH and decreased as the pH increased. Adsorption on kaolinite was also dependent on the ionic nature of the buffers. The molecular mass of the proteins was unaltered after adsorption on montmorillonite, as shown by SDS-PAGE (polyacrylamide gel electrophoresis) of the desorbed proteins; no significant modifications occurred in their structure as the result of binding on the clay, as indicated by infrared analysis; and there was no significant expansion of the clay by the proteins, as shown by x-ray diffraction analysis. The bound proteins appeared to retain their insecticidal activity against the third instar larvae ofTrichoplusia ni.     

磷酸盐在土壤中的竞争吸附与解吸机制

本文概述了近年来国内外有关磷酸盐的竞争吸附与解吸的研究成果。土壤中许多阴离子都能与磷竞争吸附点位,使得磷的吸附下降。有机质可促进或抑制磷的吸附,pH是影响竞争吸附的主要因子。磷被吸附后大多固持在表面而难于解吸,往往呈现明显的滞后现象。通常只有拟物理吸附的磷能被解吸,化学吸附的磷因与表面金属离子作用形成双齿配位而极难被淋洗下来。解吸受多种因素的影响,其中解吸剂的类型是主要因子之一。     

Adsorption-desorption of recombinant hepatitis B surface antigen (r-HBsAg) from P. pastoris on a diatomaceous earth matrix: Optimization of parameters for purification

Recombinant hepatitis B surface antigen (r-HBsAg) produced in yeast is adsorbed on a diatomaceous earth matrix for purification purposes. A pH dependence in the adsorption-elution behavior was found. The capacity of celite (Hyflo Super Cei) for adsorbing r-HBsAg increased with decreasing pH. Nonspecific proteins were also adsorbed, but a low pH dependence was found. Elution from the matrix was performed using a basic pH buffer, in which r-HBsAg is more specifically adsorbed/desorbed than contaminant proteins, permitting the purification of the r-HBsAg. A pH of 4.0 was used for adsorption and pH 8.2 was used for desorption. The described protocol allows a purification factor between three- and fivefold with respect to contaminant proteins and sixfold with respect to contaminant DNA. Finally, the adsorption step was successfully scaled-up for production purposes. (c) 1993 John Wiley & Sons, Inc.     

Fast separation of bromelain by polyacrylic acid-bound iron oxide magnetic nanoparticles

The adsorption of bromelain from an aqueous solution by polyacrylic acid (PAA)-bound iron oxide magnetic nanoparticles was studied. The magnetic composite nanoparticles were shown to be efficient for the separation of bromelain. Except at pH <3, the adsorption of bromelain increased with the decrease in solution pH and reached almost 100% at pH 3–5. The adsorbed bromelain could be desorbed by the addition of KCl and complete desorption was achieved at pH 7 when [KCl]>0.6 M. The adsorption behaviour followed the Langmuir isotherm with a maximum adsorption amount of 0.476 mg/mg and a Langmuir adsorption equilibrium constant of 58.4 ml/mg at pH 4 and 0.1 M phosphate. In addition, it was notable that both the adsorption and desorption of bromelain were quite fast and could be completed in about 1 min due to the absence of internal diffusion resistance. Bromelain retained 87.4% activity after adsorption/desorption.     

Bt毒素在三种矿物表面的吸附与解吸

研究了Bt库斯塔克亚种(kurstaki）毒素（65 kDa）在高岭土、针铁矿和氧化硅表面的吸附和解吸特性.结果表明：在磷酸盐缓冲体系（pH 8）中,3种矿物的等温吸附曲线均符合Langmuir方程（R²>0.9661）,它们对Bt毒素的吸附顺序为：针铁矿﹥高岭土﹥二氧化硅.矿物对Bt毒素的吸附1 h就基本达到了吸附平衡.在pH 6～8范围内,针铁矿、高岭土和二氧化硅对Bt毒素的吸附量随pH值的升高而降低.10 ℃～50 ℃范围内,针铁矿和氧化硅对Bt毒素吸附量随温度升高有所下降（8.39％和47.06％）,高岭土对Bt毒素吸附则略有升高（5.91％）.红外光谱分析显示,Bt毒素被矿物吸附后结构仅有微小变化.被矿物吸附的Bt毒素不易被去离子水解吸,水洗3次总解吸率为28.48%～42.04%.     

Thermoresponsive protein adsorption of poly(N-isopropylacrylamide)-modified streptavidin on polydimethylsiloxane microchannel surfaces

The control of protein adsorption on microchannel surfaces is important for biosensors. In this study, we demonstrated protein adsorption method that is controlled through temperature change, i.e., thermoresponsive protein adsorption, on polydimethylsiloxane (PDMS) microchannel surfaces using a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). To provide general protein adsorption control method, we adopted biotin-streptavidin chemistry and synthesized streptavidin covalently modified with PNIPAAm (PNIPAAm-StAv). Modification of streptavidin, a hydrophilic protein, with PNIPAAm induced successful thermoresponsive adsorption on a PDMS microchannel surfaces: PNIPAAm-StAv adsorbed at 37 degrees C and desorbed at 10 degrees C on the surfaces. We also demonstrated the thermoresponsive adsorption of biotinylated immunoglobulin G (IgG-b) using PNIPAAm-StAv. Conjugation of IgG-b with PNIPAAm-StAv induced successful thermoresponsive IgG-b adsorption on PDMS. Modification of PDMS surfaces with PNIPAAm reduced physical adsorption of the partially hydrophobic IgG-b on the surface and contributed to the high-contrast thermoresponsive adsorption of IgG-b: less than 1% of the IgG-b adsorbed at 37 degrees C was detected after the PNIPAAm-PDMS surface was washed at 10 degrees C. The controllable adsorption of this system is expected to be applied to the regeneration of biosensor chips and to on-chip protein manipulation.     

Effect of Solute Adsorption Properties on Its Separation from Supercritical Carbon Dioxide with a Thin Porous Silica Membrane

A thin porous silica membrane (average pore size of 3.3 mm) was prepared by the sol–gel method and used to separate the solute from supercritical carbon dioxide. The characteristics of solute permeation were investigated in respect of the adsorption properties of the solute, the desorption rate of the solute from the membrane being measured and the potential energy of solute near the silica surface being calculated by the molecular modeling technique. It was found that caffeine was strongly adsorbed to the surface and then slowly desorbed to form an adsorption layer, making the pores narrower and causing a molecular-sieving effect. Therefore, the rejection value was positive. On the other hand, the rejection value of n-octanoic acid, which was well adsorbed and rapidly desorbed, was negative. It is presumed that the molecules filled the pores due to their potential energy and were then forced to flow through the pores by the transmembrane pressure.     

Adsorption of Ni on amine-modified mycelium of Penicillium chrysogenum

An effective heavy metal ion adsorbent was prepared by a simple amine modification on mycelium from biomass of penicillin fermentation. The maximum adsorption capacity for Ni²⁺ could reach 260 mg/g after modification. The adsorbed Ni²⁺ could be easily desorbed by 0.01% EDTA or 0.2–0.5% HCl solution. The mechanical strength of the adsorbent was also improved after modification and the new adsorbent could be reused for at least 10 cycles without losing its uptake. The low cost adsorbent has a potential in large scale heavy metal ion wastewater treatment.     

Dye-ligand poly(GMA–TAIC–DVB) affinity adsorbent for protein adsorption

Macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) was prepared by a radical suspension copolymerization. Reaction of the copolymer with 2-hydroxyethyl amine was employed to obtain a hydrophilic matrix. An affinity dye, Cibacron blue 3GA, was then coupled covalently to prepare a novel macroporous affinity adsorbent. The surface and pore structure of the affinity adsorbent were examined by scanning electron micrography (SEM). SEM observations showed that the affinity adsorbent abounded in macropores. Bovine serum albumin (BSA) and lysozyme (Lys) were used as samples to examine the adsorption properties of the adsorbent. Under appropriate conditions, the affinity adsorbent had a capacity of 15.5 mg BSA/g and 22.3 mg Lys/g (wet adsorbent weight). The adsorbed proteins could be desorbed by increasing liquid phase ionic strength or by using a NaOH solution, and the adsorbent could be recycled for protein adsorption.