Multilayer adsorption of lysozyme on a hydrophobic substrate.

Macromolecular adsorption is known to occur as a complex process, often in a series of steps. Several models are discussed in the literature which describe the microscopic structure of the adsorbate. In the present study we investigated the adsorption of hen egg white lysozyme on alkylated silicon oxide surfaces. A combination of fluorescence excitation in the evanescent field and fluorescence recovery after photobleaching allowed us to measure the amount of adsorbed fluorescent lysozyme and the equilibrium exchange kinetics with molecules in solution. We found that a model with at least three classes of adsorbed molecules is necessary to describe the experimental results. A first layer is formed by the molecules which adsorb within a short time after the beginning of the incubation. These molecules make up approximately 65% of the final coverage. They are quasi-irreversibly adsorbed and do not measurably exchange with bulk molecules within one day even at temperatures up to 55 degrees C. A second layer, which reaches equilibrium only after several hours of incubation, shows a pronounced exchange with bulk molecules. The on-off kinetics show a distinct temperature dependence from which an activation barrier of delta E approximately 22 kcal/mol is derived. A third layer of molecules that exchange rapidly with the bulk can be seen to comprise approximately 10% of the total coverage. The exchange rate is on the order of fractions of a second. The binding of the latter two classes of adsorbed molecules is exothermic. From the temperature dependence of the coverage, the binding enthalpy of the slowly exchanging layer was estimated to be delta Hads approximately 3.8 kcal/mol. The second and third class of molecules remain enzymatically active as a muramidase, which was tested by the lysis of the cell walls of Micrococcus lysodeiktikus. The molecules in the first layer, on the other hand, showed no enzymatic activity.     

Effects of high temperature on Escherichia coli F pili.

The effects of high temperatures (46 to 50 degrees C) on the production of F pili by Escherichia coli were studied by electron microscopy. Attached F pili rapidly disappeared at 48 and 50 degrees C but not at 46 degrees C. Free pili were not denatured at these temperatures. The pili that disappeared from the cells at 50 degrees C did not appear as free pili in the culture supernatant fluid, indicating that the pili had retracted to the cell surface or into the cell. The adsorption of either R17 phage or F pili antibody to the sides of pili prevented retraction. The disappearance of pili was accompanied by a loss in the ability to adsorb R17 phage but not M13 phage, suggesting that the tip of a pilus remains exposed after retraction.     

Potential and structure controlled interfacial behavior of uracil derivatives

The adsorption and related interfacial behavior of uracil, various methylated uracil derivatives, uridine, uridine-5'-monophosphate and uridine-3'5'-cyclic monophosphate has been studied by surface electrochemical measurements at a mercury electrode. All uracil derivatives exhibit an initial "dilute" adsorption region where the virtually flat uracil residue is absorbed flat on the electrode surface. In the case of uracil and its methylated derivatives the area occupied by one molecule is about 60-70 A2. Uracil, thymine and 1,5-dimethyluracil exhibit a second adsorption region where they rearrange on the surface and adopt a perpendicular orientation and occupy about 40 A2 per molecule. In this perpendicular orientation the uracils are bound to the electrode through the N(3)-H or perhaps N(1)-H functions in a manner similar to their Watson-Crick bonding in nucleic acids. When in the perpendicular orientation the adsorbed molecules undergo extensive stacking (association) interactions, again similar to those observed between adjacent bases in nucleic acids. The ability of a uracil derivative to undergo a surface reorientation is critically dependent on electrode potential, bulk-solution concentration and molecular structure.     

Adsorption of human beta 2-microglobulin at a water/mercury interface

Adsorption of human beta 2-microglobulin from a neutral solution of 0.15 M NaCl on a mercury surface was studied at 25 degrees C by measurement of the differential capacity of the electrical double layer. From the diffusion-controlled adsorption kinetics, the surface concentration and hence the area occupied by the adsorbed beta 2-microglobulin molecule were determined at various potentials of the mercury surface. The results indicate unfolding or flattening of beta 2-microglobulin molecules adsorbed in particular on the electrically uncharged surface. The extent of this interfacial conformational rearrangement was reduced with growing positive or negative surface charge density.     

Adsorption properties of polar/apolar inducers at a charged interface and their relevance to leukemia cell differentiation.

The interfacial adsorption properties of polar/apolar inducers of cell differentiation (PAIs) were studied on a mercury electrode. This study, on a clean and reproducible charged surface, unraveled the purely physical interactions among these compounds and the surface, apart from the complexity of the biological membrane. The interfacial behavior of two classical inducers, hexamethylenebisacetamide (HMBA) and dimethylsulfoxide, was compared with that of a typical apolar aliphatic compound, 1-octanol, that has a similar hydrophobic moiety as HMBA but a much smaller dipolar moment. Both HMBA and Octanol adsorb flat in contact with the surface because of hydrophobic forces, with a very similar free energy of adsorption. However, the ratio of polar to apolar moieties in PAIs turned out to be crucial to drive the adsorption maximum toward physiological values of surface charge density, where octanol is desorbed. The electrostatic effects in the interfacial region reflected the adsorption properties: the changes in the potential drop across the interfacial region as a function of the surface charge density, in the physiological range, were opposite in PAIs as compared with apolar aliphatic compounds, as exemplified by octanol. This peculiar electrostatic effect of PAIs has far-reaching relevance for the design of inducers with an adequate therapeutic index to be used in clinical trials.     

Adsorption of beta-galactosidase of Alicyclobacillus acidocaldarius on wild type and mutants spores of Bacillus subtilis

ABSTRACT: BACKGROUND: The Bacillus subtilis spore has long been used as a surface display system with potential applications in a variety of fields ranging from mucosal vaccine delivery, bioremediation and biocatalyst development. More recently, a non-recombinant approach of spore display has been proposed and heterologous proteins adsorbed on the spore surface. We used the well-characterized beta-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius as a model to study enzyme adsorption, to analyze whether and how spore-adsorption affects the properties of the enzyme and to improve the efficiency of the process. RESULTS: We report that purified beta-galactosidase molecules were adsorbed to purified spores of a wild type strain of B. subtilis retaining ca. 50% of their enzymatic activity. Optimal pH and temperature of the enzyme were not altered by the presence of the spore, that protected the adsorbed beta-galactosidase from exposure to acidic pH conditions. A collection of mutant strains of B. subtilis lacking a single or several spore coat proteins was compared to the isogenic parental strain for the adsorption efficiency. Mutants with an altered outermost spore layer (crust) were able to adsorb 60-80% of the enzyme, while mutants with a severely altered or totally lacking outer coat adsorbed 100% of the beta-galactosidase molecules present in the adsorption reaction. CONCLUSION: Our results indicate that the spore surface structures, the crust and the outer coat layer, have an negative effect on the adhesion of the beta-galactosidase. Electrostatic forces, previously suggested as main determinants of spore adsorption, do not seem to play an essential role in the spore-beta-galactosidase interaction. The analysis of mutants with altered spore surface has shown that the process of spore adsorption can be improved and has suggested that such improvement has to be based on a better understanding of the spore surface structure. Although the molecular details of spore adsorption have not been fully elucidated, the efficiency of the process and the pH-stability of the adsorbed molecules, together with the well documented robustness and safety of spores of B. subtilis, propose the spore as a novel, non-recombinant system for enzyme display.     

Removal of dissolved metals by plant tissue

Various types of microbial biomass have been shown to adsorb metals dissolved in aqueous media. It has now been demonstrated that certain plant tissues are also effective for this type of adsorption process. In particular, tomato and tobacco roots harvested from field-grown plants were shown to adsorb Sr from an aqueous solution of SrCl(2). Distribution coefficients in excess of 550 were measured and the adsorption isotherms at 25 degrees C could be fitted to Langmuir-type expressions. The bioadsorbent could be regenerated and metals recovered by either a reduction in the pH to less than 2.0 or by use of a concentrated chloride salt solution.     

Repair of impurity-poisoned protein crystal surfaces

The surface morphology of Bence-Jones protein (BJP) crystals was investigated during growth and dissolution by using in situ atomic force microscopy (AFM). It was shown that over a wide supersaturation range, impurities adsorb on the crystalline surface and ultimately form an impurity adsorption layer that prevents further growth of the crystal. At low undersaturations, this impurity adsorption layer prevents dissolution. At greater undersaturation, dissolution takes place around large particles incorporated into the crystal, leading to etch pits with impurity-free bottoms. On restoration of supersaturation conditions, two-dimensional nucleation takes place on the impurity-free bottoms of these etch pits. After new growth layers fill in the etch pits, they cover the impurity-poisoned top layer of the crystal face. This leads to the resumption of its growth. Formation of an impurity-adsorption layer can explain the termination of growth of macromolecular crystals that has been widely noted. Growth-dissolution-growth cycles could be used to produce larger crystals that otherwise would have stopped growing because of impurity poisoning.     

Fullerene modified supported lipid membrane as sensitive element of sensor for odorants

A supported lipid bilayer membrane (s-BLMs) formed on a freshly cleaved metallic surface by the Tien method was applied for the design of an electrochemical sensor for detection of neutral odorant molecules. The lipid bilayer was modified by saturation with fullerene C₆₀, which possesses electron mediator properties and facilitates a redox reaction occurring at the border of the lipid membrane and metal surface. I₂/I⁻ and ferrocenyl trimethyl bromide were used as electroactive marker ions. The smell compounds adsorb on the surface of the lipid layer and change its structure. As a consequence the ratio of marker ion penetration to the lipid membrane is altered. The magnitude of these changes depends on the amount and chemical structure of adsorbed molecules. The research presented was carried out by cyclic voltammetry. The magnitude of the electrochemical signal generated by smell compounds was correlated with other parameters describing their molecular properties such as: octanol/water partition coefficients and dipole moments.     

Protein adsorption at solid-liquid interfaces: Part I--Affinities of proteins for alumina surface

Extent of adsorption (gamma pw) of bovine serum albumin, beta-lactoglobulin, gelatin and myosin at the alumina-water interface has been measured as function of protein concentration (Cp) at several temperatures, pH, and ionic strengths of the medium. gamma pw for proteins in most cases increases with increase of protein concentration but it attains maximum value gamma pw(m) when Cp is high. Values of maximum adsorption have been examined in terms of molecular orientation, molecular size and shape and unfolding of the packed proteins at the interface. In few cases, gamma pw increases with increase of Cp without reaching a real state of saturation as a result of aggregation of molecules or extensive unfolding of the protein at the interface. In the case of beta-lactoglobulin at pH 5.2 and ionic strength 0.05, gamma pw in high concentration region decreases to zero value when Cp increases. For myosin at 45 degrees C and pH 6.4, and also at 27 degrees and pH 7.8, the values of gamma pw are all negative and these negative values increase with increase of Cp. All these results have been explained in terms of significant competitions of water and protein for binding to the surface sites of the powdered alumina. Adsorption of myosin has also been found to be affected in the presence of NaCl, KCl, CaCl2, KI, Na2SO4, LiCl and urea. The relative affinities of the adsorption of various proteins for the surface of alumina at different physical conditions of the system have been compared in terms of maximum values of adsorption attained when gamma pw is varied with Cp. The affinities are shown to be compared more precisely in terms of the standard free energy decrease for the saturation of the surface by protein as a result of the change in its concentration from zero to unity in the mole fraction scale.     

Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions.     

Adsorption of monomers on microspherical structures of thermal heterocomplex molecules from amino ACIDS

The surface of a microspherical structure formed in the aqueous suspension of thermal heterocomplex molecules made by heating aspartic acid and proline can adsorb basic amino acids such as histidine, lysine and arginine. It can also adsorb adenine, cytosine, adenosine and cytidine. Electrostatic interactions acting between those monomers to be adsorbed and the adsorbing surface are responsible for the adsorption.Presented at the 1993 ISSOL Meeting in Barcelona, Spain.     

Theoretical study of adsorption of nitrogen-containing environmental contaminants on kaolinite surfaces

The adsorption of nitrogen-containing compounds (NCCs) including 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), 2,4-dinitroanisole (DNAN), and 3-nitro-1,2,4-triazol-5-one (NTO) on kaolinite surfaces was investigated. The M06-2X and M06-2X-D3 density functionals were applied with the cluster approximation. Several different positions of NCCs relative to the adsorption sites of kaolinite were examined, including NCCs in perpendicular and parallel orientation toward both surface models of kaolinite. The binding between the target molecules and kaolinite surfaces was analyzed and bond energies were calculated applying the atoms in molecules (AIM) method. All NCCs were found to prefer a parallel orientation toward both kaolinite surfaces, and were bound more strongly to the octahedral than to the tetrahedral site. TNT exhibited the strongest interaction with the octahedral surface and DNAN with the tetrahedral surface of kaolinite. Hydrogen bonding was shown to be the dominant non-covalent interaction for NCCs interacting with the octahedral surface of kaolinite with a small stabilizing effect of dispersion interactions. In the case of adsorption on the tetrahedral surface, kaolonite–NCC binding was shown to be governed by the balance between hydrogen bonds and dispersion forces. The presence of water as a solvent leads to a significant decrease in the adsorption strength for all studied NCCs interacting with both kaolinite surfaces.     

Adsorption of monorhamnolipid and dirhamnolipid on two <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strains and the effect on cell surface hydrophobicity

Previously, adsorption feature of a dirhamnolipid biosurfactant on diverse microbial cells was studied and the effect of the adsorption on cell surface hydrophobicity was compared. In this paper, the adsorption behavior of a monorhamnolipid and a dirhamnolipid on cells of two Pseudomonas aeruginosa strains was investigated in order to further reveal the influence of biosurfactant structure and cell property on the adsorption and the relation between the adsorption and cell surface hydrophobicity. Experimental results showed that the adsorption capacity of all the cells to monorhamnolipid was much stronger than to dirhamnolipid, and the rhamnolipid-sourced P. aeruginosa cells, no matter grown on glucose or hexadecane, released extra dirhamnolipid when aqueous concentration of dirhamnolipid was too high. Length of surfactant alkyl chain as well as the type of carbon source used to cultivate the cell adsorbents had only minor influence on the adsorption. The adsorption was assumed to be driven by polar interaction between the rhamnolipid molecules and the cell surface chemical groups. The directional orientation of the rhamnolipid molecules with hydrophobic moiety extending to the environment may account for the rapid increase of cell surface hydrophobicity at low aqueous concentrations of the surfactant, while the stable or decreased cell hydrophobicity was probably the consequence of multiple surfactant layer formation or hemimicelle accumulation.     

Application of molecular dynamics DL_POLY codes to interfaces of inorganic materials

Three recent applications of the DL_POLY molecular dynamics code are described, which demonstrate the flexibility and viability of the code for extending our understanding of the structure, stability and reactivity of ceramics and minerals at the atomic level. The first is an investigation into differences in oxygen atom mobility in bulk and at the most stable {111} surface of ceria. The results show enhanced surface transport but that it is via subsurface oxygen. Secondly, we investigate how polychloro-dibenzo-pdioxins (PCDDs) molecules might adsorb on clay surfaces. The resulting adsorption energies show a clear relationship with chlorine content of the molecule. Finally, we apply DL_POLY to comparing the aggregation of magnesium oxide and calcium carbonate nanoparticles. We find that very small calcium carbonate nanoparticles are amorphous and their aggregation shows no preferred orientation in contrast to magnesium oxide, which remain highly crystalline and combine in a highly structural specific way.     

Interaction of fibrinogen with magnetite nanoparticles

The interaction between fibrinogen and magnetite nanoparticles in solution has been studied by the methods of spin labeling, ferromagnetic resonance, dynamic and Rayleigh light scattering. It is shown that protein molecules adsorb on the surface of nanoparticles to form multilayer protein covers. The number of molecules adsorbed on one nanoparticle amounts to ∼65 and the thickness of the adsorption layer amounts to ∼27 nm. Separate nanoparticles with fibrinogen covers (clusters) form aggregates due to interactions of the end D domains of fibrinogen. Under the influence of direct magnetic field, nanoparticles with adsorbed proteins form linear aggregates parallel to the force lines. It is shown that the rate of protein coagulation during the formation of fibrin gel under the action of thrombin on fibrinogen decreases ∼2 times in the presence of magnetite nanoparticles, and the magnitude of the average fiber mass/length ratio grows.     

Kinetics of protein adsorption and desorption on surfaces with grafted polymers

The kinetics of protein adsorption are studied using a generalized diffusion approach which shows that the time-determining step in the adsorption is the crossing of the kinetic barrier presented by the polymers and already adsorbed proteins. The potential of mean-force between the adsorbing protein and the polymer-protein surface changes as a function of time due to the deformation of the polymer layers as the proteins adsorb. Furthermore, the range and strength of the repulsive interaction felt by the approaching proteins increases with grafted polymer molecular weight and surface coverage. The effect of molecular weight on the kinetics is very complex and different than its role on the equilibrium adsorption isotherms. The very large kinetic barriers make the timescale for the adsorption process very long and the computational effort increases with time, thus, an approximate kinetic approach is developed. The kinetic theory is based on the knowledge that the time-determining step is crossing the potential-of-mean-force barrier. Kinetic equations for two states (adsorbed and bulk) are written where the kinetic coefficients are the product of the Boltzmann factor for the free energy of adsorption (desorption) multiplied by a preexponential factor determined from a Kramers-like theory. The predictions from the kinetic approach are in excellent quantitative agreement with the full diffusion equation solutions demonstrating that the two most important physical processes are the crossing of the barrier and the changes in the barrier with time due to the deformation of the polymer layer as the proteins adsorb/desorb. The kinetic coefficients can be calculated a priori allowing for systematic calculations over very long timescales. It is found that, in many cases where the equilibrium adsorption shows a finite value, the kinetics of the process is so slow that the experimental system will show no adsorption. This effect is particularly important at high grafted polymer surface coverage. The construction of guidelines for molecular weight/surface coverage necessary for kinetic prevention of protein adsorption in a desired timescale is shown. The time-dependent desorption is also studied by modeling how adsorbed proteins leave the surface when in contact with a pure water solution. It is found that the kinetics of desorption are very slow and depend in a nonmonotonic way in the polymer chain length. When the polymer layer thickness is shorter than the size of the protein, increasing polymer chain length, at fixed surface coverage, makes the desorption process faster. For polymer layers with thickness larger than the protein size, increases in molecular weight results in a longer time for desorption. This is due to the grafted polymers trapping the adsorbed proteins and slowing down the desorption process. These results offer a possible explanation to some experimental data on adsorption. Limitations and extension of the developed approaches for practical applications are discussed.     

Kinetics of adsorption of globular proteins at an air-water interface.

Adsorption of globular proteins at an air-water interface from an infinite stagnant medium was modeled as one-dimensional diffusion in a potential field. The interaction potential experienced by an adsorbing molecule consisted of contributions from electrostatic interactions, work done against the surface pressure to clear area at the interface in order to anchor the adsorbed segments, and the change in the free energy due to exposure of penetrated surface hydrophobic functional groups to air. The assumption of irreversible adsorption is employed in the present analysis. The energy barrier to adsorption, present at sufficiently large surface pressures, was found to be higher for smaller surface hydrophobicities, larger surface pressures, larger size molecules, and oblate orientation of an ellipsoidal molecule. Consequently, more adsorption occurred at larger surface hydrophobicities, smaller size molecules, and for prolate orientation of ellipsoidal molecules. The subphase concentration has been shown to be zero at short times, increasing with time at larger times, and eventually becoming close to the bulk concentration as a result of increasing energy barrier to adsorption. The predicted evolution of surface concentration with time for adsorption of lysozyme at an air-water interface agreed well with the experimental data of Graham and Phillips (1979a).     

Surface charging by large multivalent molecules. Extending the standard Gouy-Chapman treatment.

Traditionally, Gouy-Chapman theory has been used to calculate the distribution of ions in the diffuse layer next to a charged surface. In recent years, the same theory has found application to adsorption (incorporation, partitioning) of charged peptides, hormones, or drugs at the membrane-water interface. Empirically it has been found that an effective charge, smaller than the physical charge, must often be used in the Gouy-Chapman formula. In addition, the large size of these molecules can be expected to influence their adsorption isotherms. To improve evaluation techniques for such experiments, comparatively simple extensions of the standard Gouy-Chapman formalism have been studied which are based on a discrete charge virial expansion. The model allows for the mobility of charged groups at the interface. It accounts for finite size of the adsorbed macromolecules and for discrete charge effects arising from pair interactions in the interface plane. In contrast to previous discrete charge treatments this model nearly coincides with the Gouy-Chapman formalism in the case where the adsorbing molecules are univalent. Large discrepancies are found for multivalent molecules. This could explain the reduced effective charges needed in the standard Gouy-Chapman treatment. The reduction factor can be predicted. The model is mainly limited to low surface coverage, typical for the adsorption studies in question.     

Rapid chromatography for evaluating adsorption characteristics of cellulase binding domain mimetics

The cost of cellulolytic enzymes is one barrier to the economic production of fermentable sugars from lignocellulosic biomass for the production of fuels and chemicals. One functional characteristic of cellulolytic enzymes that improves reaction kinetics over mineral acids is a cellulose binding domain that concentrates the catalytic domain to the substrate surface. We have identified maleic acid as an attractive catalytic domain with pK(a) and dicarboxylic acid structure properties that hydrolyze cellulose while producing minimal degradation of the glucose formed. In this study we report results of a rapid chromatographic method to assess the binding characteristics of potential cellulose binding domains for the construction of a synthetic cellulase over a wide range of temperatures (20 degrees to 120 degrees C). Aromatic, planar chemical structures appear to be key indicators of cellulose adsorption. Indole, the side-chain of the amino acid tryptophan, has been shown to reversibly adsorb to cellulose at temperatures between 30 degrees and 120 degrees C. Trypan blue, a polyaromatic, planar molecule, was shown to be irreversibly adsorbed to cotton cellulose at temperatures of <120 degrees C on the time scale of the experiments. These results confirm the importance of hydrophobic cellulose and the cellulose-binding component of cellulolytic enzymes and cellulolytic enzyme mimetics.