Protein adsorption at solid-liquid interfaces: Part IV--Effects of different solid-liquid systems and various neutral salts.

Adsorption isotherms of BSA at the solid-water interfaces have been studied as a function of protein concentration, ionic strength of the medium, pH and temperature using silica, barium sulphate, carbon, alumina, chromium, ion-exchange resins and sephadex as solid interfaces. In most cases, isotherms for adsorption of BSA attained the state of adsorption saturation. In the presence of barium sulphate, carbon and alumina, two types in the isotherms are observed. Adsorption of BSA is affected by change in pH, ionic strength and temperature of the medium. In the presence of metallic chromium, adsorbed BSA molecules are either denatured or negatively adsorbed at the metallic interface. Due to the presence of pores in ion-exchange resins, adsorption of BSA is followed by preferential hydration on resin surfaces in some cases. Sometimes two steps of isotherms are also observed during adsorption of BSA on the solid resins in chloride form. Adsorption of BSA, beta-lactoglobulin, gelatin, myosin and lysozyme is negative on Sephadex surface due to the excess adsorption of water by Sephadex. The negative adsorption is significantly affected in the presence of CaCl2, KSCN, LiCl, Na2SO4, NaI, KCl and urea. The values of absolute amounts of water and protein, simultaneously adsorbed on the surface of different solids, have been evaluated in some cases on critical thermodynamic analysis. The standard free energies (delta G0) of excess positive and negative adsorption of the protein per square meter at the state of monolayer saturation have been calculated using proposed universal scale of thermodynamics. The free energy of adsorption with reference to this state is shown to be strictly comparable to each other. The magnitude of standard free energy of transfer (delta G0B) of one mole of protein or a protein mixture at any type of physiochemical condition and at any type of surface is observed to be 38.5 kJ/mole.     

Binding of long-chain fatty acids to bovine serum albumin

We have studied the binding of long-chain free fatty acids (FFA) to crystalline bovine serum albumin (BSA) that had been extracted with charcoal to remove endogenous fatty acids. The data were analyzed in terms of a model consisting of six high-energy binding sites and a large number of weak binding sites. The high-energy sites were resolved into two distinct classes, each containing three sites. At 37 degrees C and pH 7.4, k'(1) (the apparent association constant of a class of binding sites) was about 10(6) m(-1) for binding to the three primary sites, and k'(2) was about 10(5) m(-1) for binding to the three secondary sites. The number of weak (tertiary) sites was estimated to be 63 with a k'(3) of 10(3) m(-1). In general, palmitate and palmitoleate were bound more tightly than oleate, linoleate, stearate, or myristate, and much more tightly than laurate. The association of palmitate with human and rabbit albumin also was analyzed in terms of this model. Palmitate was bound less firmly by human or rabbit albumin than by BSA. Palmitate binding to BSA was dependent upon the pH and temperature of the incubation medium. Long-chain hydrocarbons that did not contain a free carboxyl group (methyl palmitate, cetyl alcohol, and hexadecane) were bound to a limited extent and weakly. The presence of positively charged protein sites and native protein tertiary structure were required for maximal binding of palmitate to BSA. Of nine other proteins tested, only -lactoglobulin exhibited a significant capacity to bind palmitate.     

Protein adsorption on core-shell resins for flow-through purifications: Effect of protein molecular size,shape, and salt concentration

This work addresses the functional properties of the core-shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose-based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.     

Modification of cellulose films by adsorption of CMC and chitosan for controlled attachment of biomolecules

The adsorption of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) on cellulose supports were investigated. The dynamics and extent of related adsorption processes were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Amine groups were installed on the cellulose substrate by adsorption of chitosan from aqueous solution, which allowed for hIgG to physisorb from acid media and produced a functionalized substrate with high surface density (10 mg/m(2)). hIgG adsorption from neutral and alkaline conditions was found to yield lower adsorbed amounts. The installation of the carboxyl groups on cellulose substrate via carboxymethylated cellulose (CMC) adsorption from aqueous solution enhanced the physisorption of hIgG at acidic (adsorbed amount of 5.6 mg/m(2)) and neutral conditions. hIgG adsorption from alkaline conditions reduced the surface density. BSA was used to examine protein attachment on cellulose after modification with chitosan or carboxymethyl cellulose. At the isoelectric point of BSA (pI 5), both of the surface modifications enhanced the adsorption of this protein when compared to that on unmodified cellulose (a 2-fold increase from 1.7 to 3.5 mg/m(2)). At pH 4, the electrostatic interactions favored the adsorption of BSA on the CMC-modified cellulose, revealing the affinity of the system and the possibility of tailoring biomolecule binding by choice of the surface modifier and pH of the medium.     

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.     

Preferential interaction of albumin-binding proteins, gp30 and gp18, with conformationally modified albumins. Presence in many cells and tissues with a possible role in catabolism.

Albumin binding to the endothelial surface apparently initiates its transcytosis via plasmalemmal vesicles and also increases capillary permselectivity. Several albumin-binding proteins, which, we call gp60, gp30, and gp18, have been identified; however, their functional relationship to each other is unclear. In this study, we show that gp30 and gp18 are both variably expressed by cultured rat fibroblasts, smooth muscle cells, and endothelial cells and are present in all tissues examined (heart, lung, skeletal muscle, diaphragm, duodenum, kidney, fat, brain, adrenal, pancreas, and liver). The binding of albumin-gold complexes (A-Au) to gp30 and gp18 was compared with that of native and modified albumins. Monomeric native bovine serum albumin (BSA) interacted much less avidly than A-Au and BSA that was chemically modified by formaldehyde (Fm-BSA) or maleic anhydride (Mal-BSA). Mal-BSA and A-Au have similar affinity constants for gp30 and gp18 (KD approximately 3-7 micrograms/ml (50-100 nM)), which is 1000-fold greater than BSA. These interactions were Ca(2+)-independent but sensitive to pH (< 6.0) and high salt concentrations (> or = 1.0 M). Comparative biochemical characterization provided evidence of conformational changes for Mal-BSA, Fm-BSA, and A-Au. Anti-native BSA serum recognizes BSA much more avidly than modified BSA. Mal-BSA, Fm-BSA, and A-Au are much more sensitive to trypsin digestion than BSA. Cellular processing was also examined. A-Au and Mal-BSA bound at the endothelial cell surface were degraded, whereas BSA was not. Our results indicate that: (i) gp30 and gp18, unlike gp60, are expressed in all tissues tested regardless of the type of endothelia lining the microvasculature and the local mechanism of transendothelial albumin transport; (ii) BSA conformationally modified by either surface adsorption or chemical means not only interacts more avidly with gp30 and gp18 than native albumin but also is preferentially degraded by the cells; (iii) A-Au and native albumin are not equivalent probes for detecting albumin interaction sites; and (iv) gp30 and gp18 exhibit binding behavior resembling scavenger receptors. The possible roles of gp30 and gp18 in albumin binding, transcytosis, endocytosis, and even protein catabolism are discussed.     

The influence of radioiodination on the adsorption of IgG and serum albumin to polystyrene

The adsorption of radioiodinated rabbit IgG and bovine serum albumin (BSA) to polystyrene tubes was investigated. Adsorption isotherms where the proportion of the protein bound was relatively constant over a range of intermediate protein concentrations, and where the proportion bound was protein dependent, were obtained. To investigate the effects of radioiodination, proteins labeled to give a wide range of substitution ratios (0.03 to 3.7 125I/protein molecule) were employed. While labeling did not appear to affect BSA adsorption, the kinetics of IgG binding were altered in a number of ways. The proportion bound in the concentration independent region was decreased even at substitution ratios less than or equal to 0.2. In addition, while all preparations of iodinated BSA, and IgG preparations with less than or equal to 1.6 125I/IgG, gave bimodal adsorption isotherms, with more heavily labeled IgG (greater than or equal to 2.5 125I/IgG) the apparent high affinity binding to the plastic surface was abolished. These results indicate that radioiodination substantially alters the kinetics of the binding of IgG to polystyrene. In addition, the results obtained are discussed with respect to previous relevant and often apparently contradictory findings.     

Lipid lateral organization in fluid interfaces controls the rate of colipase association.

Colipase, a cofactor of pancreatic triacylglycerol lipase, binds to surfaces of lipolysis reactants, like fatty acid and diacylglycerol, but not to the nonsubstrate phosphatidylcholine. The initial rate of colipase binding to fluid, single-phase lipid monolayers was used to characterize the interfacial requirements for its adsorption. Colipase adsorption rates to phosphatidylcholine/reactant mixed monolayers depended strongly on lipid composition and packing. Paradoxically, reactants lowered colipase adsorption rates only if phosphatidylcholine was present. This suggests that interactions between phosphatidylcholine and reactants create dynamic complexes that impede colipase adsorption. Complex formation was independently verified by physical measurements. Colipase binding rate depends nonlinearly on the two-dimensional concentration of phosphatidylcholine. This suggests that binding is initiated by a cluster of nonexcluded surface sites smaller than the area occupied by a bound colipase. Binding rates are mathematically consistent with this mechanism. Moreover, for each phosphatidylcholine-reactant pair, the complex area obtained from the analysis of binding rates agrees well with the independently measured collapse area of the complex. The dynamic complexes between phosphatidylcholine and lipids, like diacylglycerols, exist independently of the presence of colipase. Thus, our results suggest that lipid complexes may regulate the fluxes of other proteins to membranes during, for example, lipid-mediated signaling events in cells.     

Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography

Weak partitioning chromatography (WPC) has been proposed for the purification of monoclonal antibodies using an anion exchange (AEX) resin to simultaneously remove both acidic and basic protein impurities. Despite potential advantages, the relationship between resin structure and WPC performance has not been evaluated systematically. In this work, we determine the structure of representative AEX resins (Fractogel® EMD TMAE HiCap, Q Sepharose FF, and POROS 50 HQ) using transmission electron microscopy and inverse size exclusion chromatography and characterize protein interactions while operating these resins under WPC conditions using two mAb monomers, a mAb dimer, mAb multimers, and BSA as model products and impurities. We determine the isocratic elution behavior of the weakly bound monomer and dimer species and the adsorptive and mass transfer properties of the strongly bound multimers and BSA by confocal laser scanning microscopy. The results show that for each resin, using the product K_p value as guidance, salt, and pH conditions can be found where mAb multimers and BSA are simultaneously removed. Isocratic elution and adsorption mechanisms are, however, different for each resin and for the different components. Under WPC conditions, the Fractogel resin exhibited very slow diffusion of both mAb monomer and dimer species but fast adsorption for both mAb multimers and BSA with high capacity for BSA, while the Sepharose resin, because of its small pore size, was unable to effectively remove mAb multimers. The POROS resin was instead able to bind both multimers and BSA effectively, while exhibiting a greater resolution of mAb monomer and dimer species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:425–434, 2017     

Restricted diffusion effect on the binding of proteins to porous polymer resins as studied by repetitive injection method

A solution of bovine serum albumin (BSA) is repeatedly injected into a column packed with highly porous and hydrophobic polymer resins at appropriate intervals. The injected BSA is thoroughly retained in the column for 10 injections and, afterwards, starts to be eluted from the column gradually. Taking into consideration the restricting effect of already bound BSA upon the diffusion of newly injected BSA into the pores of the polymer resins, we can interpret the BSA elution profile from columns packed with polymer resin of various pore sizes and porosities. The effects of the binding rate constant and BSA concentration upon the elution profiles of BSA are also analyzed. Formyl groups are introduced into the polymers as a binding site with proteins, and the elution profiles of BSA from the column packed with the formylated resin are also analyzed.     

Characterization of covalent protein conjugates using solid-state 13C NMR spectroscopy.

Cross-polarization magic-angle spinning (CPMAS) 13C NMR spectroscopy has been used to characterize covalent conjugates of alachlor, an alpha-chloroacetamide hapten, with glutathione (GSH) and bovine serum albumin (BSA). The solid-state NMR method demonstrates definitively the covalent nature of these conjugates and can also be used to characterize the sites of hapten attachment to proteins. Three different sites of alachlor binding are observed in the BSA system. Accurate quantitation of the amount of hapten covalently bound to GSH and BSA is reported. The solid-state 13C NMR technique can easily be generalized to study other small molecule/protein conjugates and can be used to assist the development and refinement of synthetic methods needed for the successful formation of such protein alkylation products.     

Effect of histidine residues in antigenic sites on pH dependence of immuno-adsorption equilibrium

Summary Polyclonal anti-myoglobin antibodies were fractionated into five subpopulations directed against five specific antigenic sites, respectively. The equilibrium characteristics of each subpopulation and orginal anti-myoglobin immobilized to CNBr-activated Sepharose 4B were compared. The four subpopulations of antibodies lost their binding abilities at around pH 4.5 because of the conformational changes of myoglobin. However, the subpopulation directed against the region containing three histidine residues dissociated with the antigenic site at higher pH, and such equilibrium characteristics were considered to be caused by the dissociation characteristics of histidine residues. Therefore, the effects of histidine modification in BSA on the adsorption capacities of original anti-BSA antibody and a pH sensitive fraction of it were compared. The adsorption capacity of the pH sensitive fraction showed greater decrease than that of original antibody by histidine modification in BSA. These results imply that the antigenic sites in which histidine residues play an important role for the binding to antibodies show equilibrium characteristics sensitive to pH.     

Identical independent sites for dye ligand on bovine serum albumin demonstrated by multivariate analysis

The most fundamental parameters concerning an interaction between a ligand and a protein are equilibrium constants and the number of binding sites. The Scatchard plot has for a long time been widely used to obtain those parameters. However, controversy in 1982-1983 over the reliability of this plot (the graphical estimation of the number of identical independent sites from the x-intercept) indicated that some methodologies other than the Scatchard plot are expected. Over the past decade, we have developed a method for applying multivariate analysis to the problem of determining spectral features of a ligand associated with a protein molecule. In principle, this method is based mainly on the computer-assisted adjustment of dissociation constants to an assumed reaction model. We discovered in this process that an n-parameter, introduced into an equation for calculating the amount of dye ligand bound to a protein, coincided with the number of identical independent sites, under a certain condition in principal factor analysis calculation. In this study, we established a new methodology for determining the number of identical independent sites using synthesized spectral series, and we then applied this method to a simple reaction system composed of bovine serum albumin (BSA) and bromocresol purple (BCP) anions. BSA was found to have two identical independent sites for BCP anions at pH 8.8.     

Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling

The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern-Volmer quenching constants of (1.1+/-0.1)x10(4) M(-1), (3.2+/-0.1)x10(3) M(-1) and (2.1+/-0.1)x10(3) M(-1) for SDS, HPS and CTAC, respectively, which are associated to the 'effective' association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA-myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W131, corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.     

原位椭圆偏振术研究牛血清清蛋白在固/液界面的吸附

用原位椭圆偏振术系统研究了硅片表面因素及缓冲液环境因素对牛血清清蛋白在固/液界面吸附的影响。在生理条件下,疏水表面与亲水表面相比BSA吸附量较大。随着硅片表面电荷密度增加,BSA吸附量增加。BSA吸附量当体溶液pH值等于BSA等电点时达到最大。而随着体溶液离子强度增加,BSA吸附量亦上升。实验结果提示:除了熵驱动作用之外,硅片表面与BSA分子及BSA分子之间的静电作用在BSA吸附中起着十分重要的作用。     

The prevention of adsorption of interferents to radiolabelled protein by Tween 20

Radiolabels are often used to quantitatively determine the amount of protein immobilized on chromatographic supports, immunochemical plates and biosensor surfaces. Bovine serum albumin (BSA) was chosen as a model protein for quantitative deposition studies. BSA was radioiodinated (125I-) or fluorescently labelled (fluorescein), then incubated with the following surfaces: quartz, quartz derivatized by 3-aminopropyltriethoxysilane (Qz-APTES), and Qz-APTES reacted with glutaraldehyde or tresyl chloride. The amounts of BSA immobilized to the different surfaces were compared using data from radioactivity and fluorescence assays. Irreproducible results were obtained with radioiodinated BSA due to adsorption/desorption behaviour of an unidentified radioactive species. When the non-ionic detergent Tween 20 was added to the protein/surface incubation mixture, radiolabelled BSA gave reproducible protein binding results which agreed with fluorescent protein binding patterns. The effect of Tween 20 was due to either the binding to BSA displacing the interferent and/or the solubilization of the interferent.     

Binding of pentagalloyl glucose to two globular proteins occurs via multiple surface sites

The interaction between pentagalloyl glucose (PGG) and two globular proteins, bovine serum albumin (BSA) and ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco), was investigated by isothermal titration calorimetry (ITC). ITC data fit to a binding model consisting of two sets of multiple binding sites, which reveal similarities in the mode of binding of PGG to BSA and rubisco. In both cases, the interaction is characterized by a high number of binding sites, which suggests that binding occurs by a surface adsorption mechanism that leads to coating of the protein surface, which promotes aggregation and precipitation of the PGG-protein complex. This model was confirmed by turbidimetry analysis of the PGG-BSA interaction. Analysis of tryptophan fluorescence quenching during the interaction of PGG with BSA suggests that binding of PGG leads to some conformational changes that are energetically closer to the unfolded state of the BSA structure, because small red shifts in the resulting emission spectra were observed.     

The binding of pyridoxal 5'-phosphate to human serum albumin

Most of the pyridoxal 5'-phosphate (PLP) in plasma is bound to protein, primarily albumin. Binding to protein is probably important in transporting PLP in the circulation and in regulating its metabolism. The binding of PLP to human serum albumin (HSA) was studied using absorption spectral analysis, equilibrium dialysis, and inhibition studies. The kinetics of the changes in the spectrum of PLP when mixed with an equimolar concentration of HSA at pH 7.4 followed a model for two-step consecutive binding with rate constants of 7.72 mM-1 min-1 and 0.088 min-1. The resulting PLP-HSA complex had absorption peaks at 338 and 414 nm and was reduced by potassium borohydride. The 414-nm peak is probably due to a protonated aldimine formed between PLP and HSA. The binding of PLP to bovine serum albumin (BSA) at equimolar concentrations at pH 7.4 occurred at about 10% the rate of its binding to HSA. The final PLP-BSA complex absorbed maximally at 334 nm and did not appear to be reduced with borohydride. Equilibrium dialysis of PLP and HSA indicated that there were more than one class of binding sites of HSA for PLP. There was one high affinity site with a dissociation constant of 8.7 microM and two or more other sites with dissociation constants of 90 microM or greater. PLP binding to HSA was inhibited by pyridoxal and 4-pyridoxic acid. It was not inhibited appreciably by inorganic phosphate or phosphorylated compounds. The binding of PLP to BSA was inhibited more than its binding to HSA by several compounds containing anionic groups. It is concluded that PLP binds differently to HSA than it does to BSA.     

Phage as a molecular recognition element in biosensors immobilized by physical adsorption

Biosensors based on landscape phages immobilized by physical adsorption on the surface of a quartz crystal microbalance was used for detection of beta-galactosidase from Escherichia coli. The sensor had a detection limit of a few nanomoles and a response time of a approximately 100 s over the range of 0.003-210 nM. The binding dose-response curve had a typical sigmoid shape and the signal was saturated at the beta-galactosidase concentration of about 200 nM. A marked selectivity for beta-galactosidase over BSA was observed in mixed solutions even when the concentration of BSA exceeded the concentration of beta-galactosidase by a factor of approximately 2000. The apparent value of the dissociation constant (K(d)) of the interaction of free phage with beta-galactosidase (9.1+/-0.9 pM) was smaller compared with the one calculated for the bound phage (1.7+/-0.5 nM). The binding was specific with three binding sites needed to bind a single molecule of beta-galactosidase. The K(d) obtained from the enzyme-linked immunosorbent assay (ELISA) for the phage and the monoclonal anti-beta-galactosidase antibodies were 21+/-2 and 26+/-2 nM, respectively. Although the method of physical adsorption is simpler and more economical in comparison with Langmuir-Blodgett and molecular assembling methods the performances of the sensors made by these technologies compare well. This work provides evidence that phage can be used as a recognition element in biosensors using physical adsorption method for immobilization of phage on the sensor surface.     

Evidence for the binding states of copper(II)–serum albumin complexes as revealed by transient electric dichroism measurements

Transient electric dichroism has been measured for a Cu(II)–bovine serum albumin (BSA)–2-(2-pyridylazo)-1-naphthol (αPAN) complex at pH 5.5–12. From the magnitude of the reduced linear dichroism and the disorientation rate of the oriented chromophore, at least three kinds of binding states of Cu(αPAN)⁺ complex exist. They are present predominantly at pH 5.5–10, 7.5–10, and 10–12, with the αPAN plane approximately parallel, vertical, and parallel with respect to the oriented axis of a BSA molecule.