Band 221, Heft 1

The adsorption of flexible collagen I triple helices with lengths of 8 nm in aqueous solution on a partially hydroxylated rutile layer (pH = 7.4) is modelled by classical molecular dynamics simulation with trajectories of up to 5 ns lengths. The carboxyl groups of glutamic and aspartic acids form hydrogen bonds with surface protons, which result in stable contact points at 300 K, if the bond length is smaller than 2 Å. Lysine side chains bind to surface hydroxyl groups. In spite of rotational motion around the C–N figure axis and opening and closing of hydrogen bonds, the amino groups are immobilized at the surface at N–O distances below 4 Å. In all runs the proteins have few contact points to the surface even though it is perfectly planar in our simulation. We suggest that the experimentally observed increase of adsorption energy and area per molecule over time may be associated to an increase of the number of contact points, which induces spreading of the adsorbate.

The structural properties of the salivary proteins, acidic proline rich PRP-1 and statherin, adsorbed onto negatively charged surfaces have been studied by Monte Carlo simulations and ellipsometry. It is shown that both proteins adsorb to negatively charged surfaces, although their net charges are negative. Experimentally, an initial fast mass-controlled film build-up was detected for both proteins, and plateaus were reached within 10 min. The isotherm shape and the adsorbed amounts were similar for PRP-1 to hydrophilic and hydrophobic surfaces, while statherin adsorbs to a greater extent to the hydrophobic surface. These results could be explained from the simulation results by considering the proteins as diblock polyampholytes. It has also been shown that the adsorption of PRP-1 to a negatively charged surface may be purely electrostatically driven, while pure electrostatic interaction is not sufficient to drive adsorption of statherin, i.e. , an extra short-ranged attractive interaction is necessary to account for the experimental observations.

In various applications it is desired that biological cells or protein molecules are immobilized at surfaces. Examples are enzymes or cells in bioreactors and biosensors, immuno-proteins in solid-state diagnostics and proteinaceous farmacons in drug delivery systems. In order to retain biological activity, the structural integrity of the immobilized bio-compounds should be preserved. In other cases immobilization of cells and proteins should be avoided. Adsorption of proteins from biofluids is considered to be the first event in the biofouling process. Subsequently, bacterial and/or other biological cells ( e.g. , blood platelets, erythrocytes) deposit on the adsorbed protein layer and a biofilm is formed. This causes great problems in areas as diverse as biomedicine, food processing and the marine environment. A generic approach to influence the magnitude of the interaction between a particle ( e.g. , a cell or a globular protein molecule) and a sorbent material is to manipulate both the long- and short-range interaction forces by grafting soluble polymers or oligomers onto the sorbent surface. Application of oligomers of ethylene oxide (EO) prevents the particles from making intimate contact with the surface. Thus, adsorbed enzymes may retain their native structure and, hence, their enzymatic activity. Another interesting example is the steering effect of pre-adsorbed polymers of EO (PEO) on the orientation of subsequently depositing anisotropic particles. For instance, IgG molecules may be forced in the right orientation and conformation in the interstitial spaces between the PEO chains, therewith doubling the specific antigen binding capacity. By far the greatest part of recent research on modifying surfaces by grafting soluble polymers (usually PEO) aims at the prevention of protein adsorption and/or adhesion of biological cells. Suppression of particle deposition depends primarily on two characteristics of the polymer layer: (a) the grafting density, and (b) the extension of the polymer layer into the solution. The efficacy of grafted PEO layers to reduce protein adsorption and microbial adhesion is illustrated for blood plasma proteins, saliva proteins and a number of bacterial and yeast cells.

The acquired pellicle is the thin salivary film that covers all oral surfaces, formed by selective adsorption of primarily salivary proteins. Several cationic proteins, such as lactoferrin, lactoperoxidase, lysozyme and histatin 5 have been identified in the pellicle. This study focused on the adsorption of lactoperoxidase and histatin 5, to investigate their possible importance in the initial salivary film formation. The adsorption was investigated by means of in situ ellipsometry, to both pure (hydrophilic) and methylated (hydrophobized) silica substrates, at concentrations relevant in saliva. The adsorption was investigated in terms of surface area per molecule and influence of surface wettability. Mass transport controlled adsorption in relation to the initial adsorption from human whole saliva and glandular saliva was also investigated. Results showed that lactoperoxidase adsorbed in larger amounts on pure silica compared to methylated surfaces. Histatin 5 adsorbed to the same extent on the two types of surfaces, but to a lesser extent compared to lactoperoxidase. The mass transport calculated adsorption rates of lactoperoxidase and histatin 5 showed that histatin 5 might potentially have a significant role in the initial adsorption from saliva, whereas lactoperoxidase may also adsorb but is not a dominating component.

Controlling protein adsorption to surfaces is an important issue in biotechnology, biomedicine and basic sciences. Proteins readily adsorb non-specifically to a variety of surfaces in a process known as biofouling, which is a major problem in the biomedical, environmental and industrial fields that can be solved by pre-treating the surfaces with adsorption-resistant materials. Additionally, surfaces can be structured and functionalized so as to immobilize proteins in specific patterns, orientations and/or densities, which is important in such areas as biosensors, biocatalysis, biotechnology, protein microarrays and single molecule biophysics. Here we review the various surface modifications and bioconjugation techniques that have been used in these areas. We also introduce novel fluorescence microscopy techniques, which have been employed to analyze the resistance of the surfaces to non-specific adsorption with detection limits down to the single molecule. A huge advantageous property of fluorescence microscopy is its sensitivity to the structure of individual, immobilized proteins by using Förster resonance energy transfer (FRET). Thus one can examine if the surface coating maintains the folded, functional form of specifically immobilized proteins. We then describe several important advances in studying slow timescale biophysical processes such as protein folding of single, specifically immobilized proteins. Future directions into which this field is expected to progress are also discussed.

The folding of amyloid β (1-40) peptide into β-sheet containing fibrils, which are the main components of deposits and plaques in some neurodegenerative diseases, is thought to play a causative role in Alzheimer's disease. The peptide is amphiphilic and therefore surface active. Interactions with surfaces can play an important role in the secondary structure changes. Langmuir monolayers of zwitterionic (DMPC, DPPC, DMPE, DPPE) as well ionic (DMPG, DPPG, DPTAP, DSTAP) phospholipids have been used to study the influence of the peptide on the lipid packing and, vice versa , the influence of phospholipid monolayers on the peptide secondary structure by infrared reflection absorption spectroscopy and grazing incidence X-ray diffraction. The peptide adsorbs at the air/water (buffer) interface and penetrates into uncompressed phospholipid monolayers. After a special pre-treatment, the peptide forms predominantly a random-coil secondary structure in solution but adopts a β-sheet conformation, which is almost parallel oriented to the surface in the adsorbed state. The peptide does not influence the condensed phospholipid monolayer structure. In contrast to Aβ insertion into zwitterionic or anionic phospholipid monolayers, a non β-sheet structure was detected during the first stage of Aβ insertion into positively charged monolayers. At high lateral pressure, the peptide is squeezed out of the monolayer. It desorbs completely from zwitterionic monolayers and charged monolayers on buffer, but remains adsorbed in β-sheet conformation at charged monolayers on water. In contrast, Aβ remains in a fluid (disordered) and charged monolayer on buffer even above 30 mN m -1 . This observation shows that a combination of hydrophobic and electrostatic interactions is necessary to keep the peptide in the adsorbed state at high pressure on buffer.

We discuss the interaction of proteins dissolved in aqueous solution with spherical polyelectrolyte brushes (SPB). The SPB consist of a solid core particle of colloidal dimensions ( ca. 100 nm in diameter) onto which long polyelectrolyte chains have been grafted. Immersed in aqueous solution of proteins these SPB will take up high amounts of protein if the ionic strength is low. At high ionic strength, however, virtually no protein will enter into the brush layer attached to the surface of the core particles. We show that bovine serum albumin (BSA) bound at low ionic strength will gradually be released upon raising the salt concentration in the solution in a well-controlled manner: For each raise of the ionic strength in solution there is a well-defined amount of protein that is released. We show that BSA adsorbed to a conventional carboxylated latex will not be released if treated in the same manner. All findings, namely the uptake of protein as well as the controlled release can be explained by the “counterion release force”: Patches of positive charge on the surface of the proteins which are immersed in the brush layer become multivalent counterions of the polyelectrolyte chains thus releasing a concomitant number of counter- and coions. Release of counterions as induced by the adsorption of proteins is hence the main driving force for the polyelectrolyte-mediated protein adsorption (PMPA).

In-situ ATR FTIR spectroscopy was used to study protein adsorption at polyelectrolyte multilayers (PEM). PEM were fabricated by consecutively adsorbing poly(ethyleneimine) (PEI) and poly(acrylic acid) (PAC) analogously to the technique introduced by Decher at silicon internal reflection elements in an in-situ ATR cell. The thickness of PEM of PEI/PAC could be varied by pH value, concentration and number of adsorption steps. At this PEM system adsorption of human serum albumin was studied under electrostatically attractive conditions in dependence of the thickness. Evidence was found, that HSA was predominantly bound in the uncompensated outermost layer region rather than in the bulk phase of the PEM.

Our approach was to encapsulate the model protein ribonuclease A (RNase A) in mesoporous silica MCM-48 (Mobil Composition of Matter No. 48) with glasslike wall structure and well-defined pores to create a confined hydrophilic microenvironment. The differential scanning calorimetric (DSC) method was employed to evaluate the stability of the confined protein as a function of pH and at various co-solvent conditions. Due to a significant stabilization effect, the immobilized protein exhibits a large shift (about 28 ºC at pH 5.5) in the unfolding temperature in confined space of about 25 Å. The stabilization against temperature-induced unfolding is further enhanced with the protein immobilized at higher pH, and the maximum loading of protein has been observed near its isoelectric point. It was also found that the addition of kosmotropic (glycerol) and chaotropic (urea) co-solvents has different impacts on protein immobilization and unfolding scenario.

The adsorption of water in the mesoporous silica material with cylindrical pores of uniform diameter, Controlled Pore Glass 10-75 (CPG), was studied by 1 H-MAS solid state NMR spectroscopy. From the NMR spectra it is evident that inside the mesopores of the silica different water environments exist, which are characterized by their individual chemical shift. All observed hydrogen atoms are either surface –SiOH groups or hydrogen bonded water molecules. It is found that there exist some stronger bound water molecules on the surface which are not removable even by heating at a vacuum pump. As a tentative assignment these water molecules are attributed to surface defects or inaccessible cavities in the CPG 10-75. At intermediate water filling levels, the principal signal is a single NMR line with continuously varying chemical shift. This finding is interpreted as the result of a radial water filling mechanism. That is, the filling of the pore grows from the pore surface towards the pore axis. Finally it is shown that water is a sensor for surface and structural inhomogeneity and that a coexistence of inner pore and outer bulk water exists in the system.