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Biomedical subjects

W Norde

Publications and source records attributed to W Norde.

At least 19 recordsLinked to original sources
PubMed2001

Adsorption of IgG onto hydrophobic teflon. Differences between the F(ab) and F(c) domains.

The effect of differences in the degree of hydrophobicity of protein patches/fragments on the adsorption behaviour of the protein is investigated. The adsorption isotherm of a monoclonal mouse anti-human immunoglobulin G (isotype 2b) onto hydrophobic Teflon particles is measured using a depletion method. The adsorption-induced denaturation of the immunoglobulin as a function of the adsorbed amount is studied by differential scanning calorimetry, and the corresponding rearrangements in the secondary structure of the whole IgG molecule and its F(ab) and F(c) fragments are determined by circular dichroism spectroscopy. The effects of adsorption on the F(ab) and F(c) fragments in the intact IgG molecule occur independently. Adsorption of the whole IgG molecule leads to denaturation of the F(ab) fragments, whereas the F(c) fragment remains unperturbed; adsorption of the isolated fragments results in structural changes in both F(ab) and F(c). The surface hydrophobicity of the isolated fragments was studied by HPLC. These experiments support the hypothesis that differences in the degree of denaturation between F(ab) and F(c) are due to the higher degree of hydrophobicity of the F(ab) fragment. The adsorption-induced changes in the secondary structure are more prominent for the isolated fragments as compared to intact IgG. This is ascribed to the higher flexibility of the isolated fragment, as compared to the fragment in the whole molecule.

Adsorption↗
PubMed2001

The Adsorption-Desorption Cycle. Reversibility of the BSA-Silica System.

The reversibility of the adsorption-desorption cycle was established by comparing the thermostability (determined by differential scanning calorimetry) and secondary structure (obtained by circular dichroism spectroscopy) of BSA before adsorption, adsorbed on, and exchanged from silica particles. Circular dichroism was also measured as a function of temperature at a given wavelength. Adsorbed BSA presents a higher thermostability and a lower alpha-helix content than the native protein while it regains its conformation when released from the surface back into the solution; the homomolecular exchange is reversible.The changes in ellipticity (at a given wavelength) as a function of the temperature show that the thermal denaturation of native, adsorbed, and exchanged BSA proceeds in two steps. For the dissolved protein, the first step up to 50 degrees C involves a slight change in the structure while in the 50-90 degrees C temperature range the actual unfolding takes place. For the adsorbed BSA, the first step proceeds up to 60 degrees C and includes some intermolecular association between the adsorbed protein molecules, which may be responsible for the increased thermostability. The unfolding occurs in the 60-90 degrees C range; it is less cooperative and involves a lower enthalpy change than the native protein. Adsorbed BSA presents the same secondary structure as that observed for dissolved BSA that has passed a heating-cooling cycle. Copyright 2001 Academic Press.

Journal Article↗
PubMed2000

Adsorption of Immunoglobulin G on Core-Shell Latex Particles Precoated with Chaps.

The aim of this work is to investigate the adsorption behavior of a monoclonal antibody (immunoglobulin G, IgG) on latex particles, possessing reactive chloromethyl groups, precoated with 3-([3-cholamidopropyl]dimethylammonio-1-propanesulfonate (Chaps). The amount and reactivity of the surface chloromethyl groups were monitored by the nucleophilic attack of glycinate to the functional groups as a function of time at 22 and 36 degrees C. The extent of displacement of Chaps by IgG and the enthalpy of the process were determined under two different conditions of precoating the latex particles with Chaps, at 22 and 36 degrees C. The adsorption of IgG takes place in two steps; the first one involves physical interaction between IgG and the surface. This step is relatively fast (in the range of minutes) and independent of temperature. In the second step covalent bonding between the protein and the active surface groups occurs. This reaction is improved by raising the temperature because Chaps desorption, which exposes the reactive chloromethyl groups on the latex particles, is kinetically and thermodynamically favored at 36 degrees C and the covalent bonding of IgG is faster at 36 degrees C. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

BSA structural changes during homomolecular exchange between the adsorbed and the dissolved states.

The secondary structure and the thermostability of bovine serum albumin (BSA), before adsorption and after homomolecular displacement from silica and polystyrene particles, are studied by circular dichroism spectroscopy and differential scanning calorimetry. The structural perturbations induced by the hydrophilic silica surface are reversible, i.e. BSA completely regains the native structure and stability after being exchanged. On the other hand, the adsorption on, and subsequent desorption from, polystyrene particles causes irreversible changes in the stability and (secondary) structure of BSA. The exchanged proteins have a higher denaturation temperature and a lower enthalpy of denaturation than native BSA. The alpha-helix content is reduced while the beta-turn fraction is increased in the exchanged molecules. Both effects are more pronounced when the protein is displaced from less crowded sorbent surfaces. The irreversible surface-induced conformational change may be related to some aggregation of BSA molecules after being exposed to a hydrophobic surface.

Animals↗
PubMed2000

The unfolding/denaturation of immunogammaglobulin of isotype 2b and its F(ab) and F(c) fragments.

The unfolding and further denaturation of IgG and its F(ab) and F(c) fragments were studied both on a macroscopic and molecular level, using differential scanning calorimetry and circular dichroism spectroscopy, respectively. It was shown that the structural integrity of the F(ab) and F(c) units was retained after fragmentation of the IgG. The F(ab) fragment denatured at approximately 61 degrees C and the F(c) fragment at 71 degrees C. The structural transitions observed in the whole IgG is the sum effect of those determined for the isolated F(ab) and F(c) fragments.

Animals↗
PubMed2000

The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein.

The denaturation of immunoglobulin G was studied by different calorimetric methods and circular dichroism spectroscopy. The thermogram of the immunoglobulin showed two main transitions that are a superimposition of distinct denaturation steps. It was shown that the two transitions have different sensitivities to changes in temperature and pH. The two peaks represent the F(ab) and F(c) fragments of the IgG molecule. The F(ab) fragment is most sensitive to heat treatment, whereas the F(c) fragment is most sensitive to decreasing pH. The transitions were independent, and the unfolding was immediately followed by an irreversible aggregation step. Below the unfolding temperature, the unfolding is the rate-determining step in the overall denaturation process. At higher temperatures where a relatively high concentration of (partially) unfolded IgG molecules is present, the rate of aggregation is so fast that IgG molecules become locked in aggregates before they are completely denatured. Furthermore, the structure of the aggregates formed depends on the denaturation method. The circular dichroism spectrum of the IgG is also strongly affected by both heat treatment and low pH treatment. It was shown that a strong correlation exists between the denaturation transitions as observed by calorimetry and the changes in secondary structure derived from circular dichroism. After both heat- and low-pH-induced denaturation, a significant fraction of the secondary structure remains.

Animals↗
PubMed1999

ATR-FTIR Study of IgG Adsorbed on Different Silica Surfaces.

Thesecondary structure of adsorbed immunoglobulin G (IgG) on different silica surfaces (hydrophilic, hydrophobic, hydrophobic with preadsorbed triblock-copolymers consisting of a polypropylene oxide buoy and two polyethylene oxide chains dangling in the solution) is studied by ATR-FTIR. Some results for adsorbed bovine serum albumin (BSA) are also presented. The secondary structure of adsorbed IgG was quantified using second-derivative spectra for the input parameters of the curve-fitting analysis of the original spectra. The secondary structure of adsorbed IgG on a hydrophilic silica surface resembles that of IgG in solution (about 60% beta-sheet and almost no alpha-helix content). There is some loss in the helix content of BSA after adsorption on the hydrophilic surface, but this structural element is still the most important one in the adsorbed protein. The IR spectra of the adsorbed proteins on the hydrophobic silica surface can not be interpreted, probably because of a large contribution to the IR signal of water molecules that are exchanged against the proteins during adsorption. The presence of preadsorbed triblock-copolymers reduces the adsorbed amount and causes an effect on the adsorbed proteins similar to that exerted by ethylene glycol: a different type of beta-sheet structure in IgG and a more ordered alpha-helix structure in BSA are provoked. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

Interaction between fatty acid salts and elastin: kinetics, absorption equilibrium, and consequences for elasticity.

Elastin from bovine ligamentum nuchae is incubated in aqueous solutions of sodium salts of fatty acids (FAS). The FAS are laurate, myristate, and palmitate. Absorption of FAS in the elastin network is studied as a function of time, FAS concentration, and ionic strength. The consequences of this uptake for the elasticity of the elastin are studied by static and dynamic stress-strain measurements. Generally, distinction must be made between the initial time-dependent stage (I) and the final equilibrium stage (II). In I the initial rate of absorption follows a second-order binding mechanism, with the rate constant increasing with decreasing length of the FAS. In this regime, the elasticity modulus remains more or less unaffected. Especially in regime II the absorption of FAS is enhanced by a reduction in the cross-link density in the elastin network. This is ascribed to an osmotic pressure primarily caused by the concomitant uptake of low molecular weight ions in the elastin. The absorption equilibrium can be described by Langmuir theory. The absorption affinity increases with increasing hydrocarbon chain length of the FAS, indicating the contribution of hydrophobic interaction. Although the elasticity is not lost, the modulus is now reduced and a concomitant viscous component is developed.

Animals↗
PubMed1999

Bovine serum albumin adsorption on titania surfaces and its relation to wettability aspects.

The adsorption of bovine serum albumin (BSA) from sodium chloride solution and Hanks' balanced salt solution (HBSS) onto TiO2-silicon surfaces is studied by reflectometry in stagnation point flow. The results are compared with those obtained by dynamic contact-angle (DCA) analysis of titanium substrates. The adsorption isotherms show that the adsorbed amount of protein always is lower in HBSS, that is, in the presence of calcium and phosphate ions. This may be related to the increase in surface hydrophilicity caused by these ions, as suggested by the authors in previous works. The rate of adsorption also is lower in HBSS solutions. Comparison of the initial adsorption rates with the rate of mass transfer to the surface reveals that in both solvents only a small fraction of the protein that arrives at the surface adsorbs onto it. Electrostatic and/or conformational effects can explain the energy barrier to adsorption. The DCA analysis of high concentration (4 mg/mL) protein solutions shows a strong reduction of the contact-angle hysteresis, both in HBSS and in NaCl solutions, which confirms that the immediate adsorption of the protein to the surface forms a stable, hydrophilic film.

Adsorption↗
PubMed1999

The adsorption-induced secondary structure of beta-casein and of distinct parts of its sequence in relation to foam and emulsion properties.

Changes in the secondary structure upon adsorption of beta-casein (betaCN) and of distinct parts of its sequence were investigated by far-ultraviolet circular dichroism in order to find suggested relationships with foam and emulsion-forming and -stabilising properties of the same protein/peptides. A teflon/water interface was used as a model system for foam and emulsion interfaces. The maximum surface loads of beta-casein and its derived peptides were investigated. The main secondary structure element of all samples in solution was the unordered random coil, but upon adsorption ordered structure, especially alpha-helix, was induced. At lower pH more ordered structure was induced, just as at lower ionic strength. Apparently, both hydrophobic and hydrophilic groups influence the change of secondary structure induced at a hydrophobic interface. The results suggest that the hydrophobic C-terminal half of betaCN accounted for the high maximum surface load on teflon, while the N-terminal half of betaCN seemed to be responsible for the secondary structure induction upon adsorption. A relation between the maximum surface load and the foam-stabilising properties was found, but an influence of the secondary structure properties on the foam and emulsion-forming and -stabilising properties was not observed.

Caseins↗
PubMed1998

Structural changes of IgG induced by heat treatment and by adsorption onto a hydrophobic Teflon surface studied by circular dichroism spectroscopy.

Thermal denaturation of mouse monoclonal immunoglobulin G (isotype 1), as well as structural rearrangements resulting from adsorption on a hydrophobic Teflon surface, are studied by circular dichroism spectroscopy. Both heat-induced and adsorption-induced denaturation do not lead to complete unfolding into an extended polypeptide chain, but leave a significant part of the IgG molecule in a globular or corpuscular form. Heating dissolved IgG causes a decrease of the fractions of beta-sheet and beta-turn conformations, whereas those of random coil and, to a lesser extent, alpha-helix increase. Adsorption enhances the formation of alpha-helices and random coils, but the beta-sheet content is strongly reduced. Heating adsorbed IgG results in a gradual break-down of the alpha-helix and beta-turn contents, and a concomitant formation of beta-sheet structures. Thus, the structural changes in IgG caused by heating and by adsorption, respectively, are very different. However, after heating, the structure of adsorbed IgG approaches the structure of thermally denatured IgG in solution.

Adsorption↗
PubMed1998

Surface-induced changes in the structure and activity of enzymes physically immobilized at solid/liquid interfaces.

A proteolytic enzyme, alpha-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solutions on solid surfaces with different hydrophobicities and morphologies. With both enzymes the affinity of adsorption is larger for the more hydrophobic surface. Water-soluble, flexible oligomers grafted on the sorbent surface cause a decrease in enzyme adsorption. CD spectroscopy and differential scanning calorimetry (DSC) indicate severe structural perturbations in the enzymes resulting from adsorption. The CD spectra reflect an average of the structure of the whole protein population. The DSC data allow additional conclusions to be drawn on the heterogeneity in the conformational states of the adsorbed enzymes. The degree of structural perturbation, that is the fraction of the adsorbed molecules of which the structure is perturbed, is lower at a surface that (1) is less hydrophobic, (2) contains water-soluble flexible oligomers and (3) is more covered by the protein. The specific activities of the enzymes are decreased on adsorption, more or less following the extent of structural perturbation. Unlike in solution, in the adsorbed state the heat-induced inactivation process is not identical with the heat-induced unfolding process. Furthermore, when the enzymes are adsorbed their specific activities are much less sensitive to temperature variation.

Adsorption↗
PubMed1997

Adsorption-Induced Conformational Changes in the Serine Proteinase Savinase: A Tryptophan Fluorescence and Circular Dichroism Study.

In this paper spectroscopic data of a proteolytic enzyme adsorbed on solid-liquid interfaces are discussed. The experiments consisted of time-resolved and steady-state fluorescence of tryptophan residues and of circular dichroism (CD) which give information on the tertiary and secondary state of the protein, respectively. The spectroscopic properties are measured for the inhibited form of subtilisin 309 in situ on a hydrophilic silica surface and on a hydrophobic Teflon surface. The results are compared with those obtained for the protein in solution. In the case of fluorescence it is reasoned that the average excited-state lifetime and short internal rotation correlation times are indicative parameters for structural changes in the protein. The internal rotation is superimposed on the rotation of the adsorbed protein which is immobile on the fluorescence time scale. Fluorescence and CD both prove that the protein alters its conformation when it adsorbs at low surface coverage on hydrophobic Teflon particles. In that case the tryptophan fluorescence lifetime is shortened which is accompanied by an increase in the alpha-helix content. At monolayer coverage the protein maintains its original structure, although minor changes in fluorophore dynamics occur. On hydrophilic silica particles the results from both techniques do not point in the same direction. The fluorescence was not affected, irrespective of the surface occupation, while the CD experiments show a decrease in alpha-helix content at low surface coverage. Copyright 1997 Academic Press.

Journal Article↗
PubMed1997

Structure, Stability, and Activity of Adsorbed Enzymes

A proteolytic enzyme, alpha-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solution onto a hydrophobic Teflon surface and a hydrophilic silica surface. We investigated the influence of adsorption on the structure, the structure thermal stability and the activity of these enzymes. Probing the protein structure by circular dichroism spectroscopy indicates that Teflon promotes the formation of helical structure in alpha-chymotrypsin, but the reverse effect is found with cutinase. The perturbed protein structures on Teflon are remarkably stable, showing no heat-induced structural transitions up to 100°C, as monitored by differential scanning calorimetry. Contact with the hydrophilic silica surface leads to a loss in the helix content of both proteins. Differential scanning calorimetry points to a heterogeneous population of adsorbed protein molecules with respect to their conformational states. The fraction of the native-like conformation in the adsorbed layer increases with increasing coverage of the silica surface by the proteins. The specific enzymatic activity in the adsorbed state qualitatively correlates with the fraction of proteins in the native-like conformation.

Journal Article↗
PubMed1997

Conductivity and Dielectric Dispersion of Gram-Positive Bacterial Cells

The conductivity of bacterial cell suspensions has been studied over a wide range of ionic strengths and is interpreted in terms of their cell wall properties. The experimental data have been analyzed after improving the high kappaa double-layer theory of Fixman, by accounting for ionic mobility in the hydrodynamically stagnant layer, i.e., in the bacterial wall. Static conductivity and dielectric dispersion measurements both show that the counterions in the porous gel-like cell wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the ions in the bacterial wall are of the same order but somewhat lower than those in the bulk electrolyte solution. The occurrence of surface conductance reduces the electrophoretic mobility in electrophoresis studies. If this effect is not taken into account, the zeta-potential will be underestimated, especially at low electrolyte concentrations.

Journal Article↗
PubMed1997

X-ray photoelectron spectroscopy analysis of whole cells and isolated cell walls of gram-positive bacteria: comparison with biochemical analysis.

The surface chemical composition of whole cells and isolated cell walls of four coryneform bacteria and of a Bacillus brevis strain has been determined by X-ray photoelectron spectroscopy (XPS). The XPS data were converted into concentrations of model compounds: peptides, polysaccharides, and hydrocarbonlike compounds. The composition of the surface of B. brevis differed markedly from that of coryneforms: the peptide concentration was about twice higher in the former case, which is attributed to the presence of an S-layer at the cell surface; in contrast, the surface of coryneforms was rich in hydrocarbonlike compounds (about 40%), which was concomitant with a high water contact angle. The peptide surface concentration of the isolated cell walls of the five strains deduced from XPS data fitted well with the total peptide content determined by biochemical analysis, which supports the validity of XPS to determine the overall macromolecular composition of the bacterial cell surface. Compared to biochemical analysis of isolated cell walls, XPS analysis of whole cells provides information which concerns directly the cell surface (2- to 5-nm-thick layer) and is less subject to alteration via losses of cell wall constituents or contamination by intracellular compounds.

Actinomycetales↗
PubMed1995

Inactivation of a subtilisin in colloidal systems.

The aim of the present study is to establish the relation between the inactivation of the proteolytic enzyme Savinase and its adsorption at different types of solid-liquid interfaces. The loss of activity has been determined both in solution and in the presence of colloidal particles, which provide a surface area for adsorption of 25% of the enzyme population. Analysis of the remaining solution at different periods of incubation of the various systems shows that the intact protein is converted into autolytic degradation products at the expense of biological activity. The different particles, however, deactivate the enzymes to a different extent. Under the experimental conditions the half-life of the enzymatic activity in solution is 3.5 hours. In the presence of particles that have hydrophobic surface properties (teflon- or polystyrene latex) the half-life is reduced to 0.7 hours. On the contrary, hydrophilic silica particles stabilize the enzyme against autolysis as compared to the inactivation in solution. Polystyrene latex particles which are chemically grafted with short poly(ethylene oxide) chains ([EO]8) are, for steric reasons, also mild with respect to the reduction of enzymatic stability. It is thus concluded that the type of surface determines the mode in which the enzyme is adsorbed on a particle which, in turn, affects the autocatalytic rate.

Adsorption↗