PM-IRRAS investigation of the interaction of serum albumin and fibrinogen with a biomedical-grade stainless steel 316LVM surface

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was applied to investigate the interaction of bovine serum albumin (BSA) and fibrinogen with a biomedical-grade 316LVM stainless steel surface, in terms of the adsorption thermodynamics and adsorption-induced secondary structure changes of the proteins. Highly negative apparent Gibbs energy of adsorption values revealed a spontaneous adsorption of both proteins onto the surface, accompanied by significant changes in their secondary structure. It was determined that, at saturated surface coverages, lateral interactions between the adsorbed BSA molecules induced rather extensive secondary structure changes. Fibrinogen's two coiled coils appeared to undergo negligible secondary structure changes upon adsorption of the protein, while large structural rearrangements of the protein's globular domains occurred upon adsorption. The secondary structure of adsorbed fibrinogen was not influenced by lateral interactions between the adsorbed fibrinogen molecules. PM-IRRAS was deemed to be viable for investigating protein adsorption and for obtaining information on adsorption-induced changes in their secondary structures.     

Do protein molecules unfold in a simple shear flow?

Protein molecules typically unfold (denature) when subjected to extremes of heat, cold, pH, solvent composition, or mechanical stress. One might expect that shearing forces induced by a nonuniform fluid flow would also destabilize proteins, as when a protein solution flows rapidly through a narrow channel. However, although the protein literature contains many references to shear denaturation, we find little quantitative evidence for the phenomenon. We have investigated whether a high shear can destabilize a small globular protein to any measurable extent. We study a protein (horse cytochrome c, 104 amino acids) whose fluorescence increases sharply upon unfolding. By forcing the sample through a silica capillary (inner diameter 150-180 microm) at speeds approaching 10 m/s, we subject the protein to shear rates dv(z)/dr as large as approximately 2 x 10(5) s(-1) while illuminating it with an ultraviolet laser. We can readily detect fluorescence changes of <1%, corresponding to shifts of < approximately 0.01 kJ/mol in the stability of the folded state. We find no evidence that even our highest shear rates significantly destabilize the folded protein. A simple model suggests that extraordinary shear rates, approximately 10(7) s(-1), would be required to denature typical small, globular proteins in water.     

Circular dichroism studies on conformational changes in protein molecules upon adsorption on ultrafine polystyrene particles

The conformational changes in well-characterized model proteins [bovine ribonuclease A (RNase A), horseradish peroxidase, sperm-whole myoglobin, human hemoglobin, and bovine serum albumin (BSA)] upon adsorption on ultrafine polystyrene (PS) particles have been studied using circular dichroism (CD) spectroscopy. These proteins were chosen with special attention to molecular flexibility. The ultrafine PS particles were negatively charged and have average diameters of 20 or 30 nm. Utilization of these ultrafine PS particles makes it possible to apply the CD technique to determine the secondary structure of proteins adsorbed on the PS surface. Effects of protein properties and adsorption conditions on the extent of the changes in the secondary structure of protein molecules upon adsorption on ultrafine PS particles were studied. The CD spectrum changes upon adsorption were significant in the "soft" protein molecules (myoglobin, hemoglobin, and BSA), while they were insingnificant in the "rigid" proteins (RNase A and peroxidase). The soft proteins sustained a marked decrease in alpha-helix content upon adsorption. Moreover, the native alpha-helix content, which is given as the percentage of the alpha-helix content in the free proteins, of adsorbed BSA was found to decrease with decreasing pH and increase with increasing adsorbed amount. These observations confirm some well-known hypotheses for the confirmational chages in protein molecules upon adsorption. (c) 1992 John Wiley & Sons, Inc.     

Use of surface enhanced infrared absorption spectroscopy (SEIRA) to probe the functionality of a protein monolayer

We present a novel infrared method to investigate the functionality of a protein monolayer tethered to a metal substrate. The approach employs Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS), which renders high surface sensitivity by enhancing the signal of the adsorbed protein by up to approximately 2 orders of magnitude. We demonstrate that the electrochemically induced absorption changes of a cytochrome c monolayer can be observed with excellent signal-to-noise ratio when the protein is adhered to a modified gold surface. To probe membrane proteins, a concept is introduced for the oriented incorporation into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent is immobilized on a chemically modified gold surface via the affinity of its histidine (His)-tag to a nickel-chelating nitro-triacetic acid (NTA) surface. The protein monolayer is reconstituted into the lipid environment by detergent removal. Changing the orientation of the protein with respect to the metal surface is achieved by inserting the His-tag on either side of the membrane protein surface. Orientational control is mandatory for experiments in which electrons are injected from the electrode into the protein. The presented methodology opens new avenues to study the mechanism of the biomedically relevant class of electron and voltage-gated proteins on the atomic level.     

Substrate chemistry-dependent conformations of single laminin molecules on polymer surfaces are revealed by the phase signal of atomic force microscopy

The conformation of single laminin molecules adsorbed on synthetic substrates is directly observed making use of the phase magnitude in tapping mode atomic force microscopy (AFM). With AFM, it is not possible to differentiate the proteins on the substrate if use is made of the height signal, since the roughness of the material becomes of the same order of magnitude as the adsorbed protein, typically 10 nm height. This work shows how AFM can be exploited to reveal protein conformation on polymer materials. Different laminin morphologies are observed on a series of different copolymers based on ethyl acrylate and hydroxyethyl acrylate as a function of the surface density of -OH groups: from globular to completely extended morphologies of the protein molecules are obtained, and the onset of laminin network formation on some substrates can be clearly identified. The results stress the importance of the underlying synthetic substrate's surface chemistry for the biofunctional conformation of adsorbed proteins.     

Mobility of adsorbed proteins: a Brownian dynamics study

We simulate the adsorption of lysozyme on a solid surface, using Brownian dynamics simulations. A protein molecule is represented as a uniformly charged sphere and interacts with other molecules through screened Coulombic and double-layer forces. The simulation starts from an empty surface and attempts are made to introduce additional proteins at a fixed time interval that is inversely proportional to the bulk protein concentration. We examine the effect of ionic strength and bulk protein concentration on the adsorption kinetics over a range of surface coverages. The structure of the adsorbed layer is examined through snapshots of the configurations and quantitatively with the radial distribution function. We extract the surface diffusion coefficient from the mean square displacement. At high ionic strengths the Coulombic interaction is effectively shielded, leading to increased surface coverage. This effect is quantified with an effective particle radius. Clustering of the adsorbed molecules is promoted by high ionic strength and low bulk concentrations. We find that lateral protein mobility decreases with increasing surface coverage. The observed trends are consistent with previous theoretical and experimental studies.     

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.     

A piezoelectric quartz crystal biosensor: the use of two single cysteine mutants of the periplasmic Escherichia coli glucose/galactose receptor as target proteins for the detection of glucose

We have examined the potential utility of a glucose biosensor that employs the glucose/galactose receptor of Escherichia coli with a quartz crystal microbalance (QCM). Two different genetically engineered mutant proteins were utilized, each involving the incorporation of a single cysteine into the amino acid sequence of the protein. The proteins were immobilized on the surface of a piezoelectric crystal by a direct sulfur-gold linkage. Since the cysteines were located at different positions in the sequence, the receptors attach to the surface with different orientations. Considering only mass effects, the target sugars for this receptor are predicted to be too small to be detectable with a QCM. However, our sensors indicated measurable and reproducible frequency responses when immobilized receptor was exposed to sugar. This unexpectedly large frequency response occurs because the protein film is transformed from a viscous layer to a more rigid nondissipative film. The QCM can detect these changes because of the direct linkage of the proteins to the surface. Calculations of the frequency response expected for a viscoelastic film with different rheological characteristics support this hypothesis. This study is significant because it illustrates a widened applicability for the QCM methodology to protein systems that bind small molecules and undergo ligand-induced conformational changes.     

Hydrodynamically coupled water in surface adsorbed amino acids as a tool to study hydrated peptides

The influence of different amino acid residues on properties of a protein surface is of great interest and importance. Hydrodynamically coupled water in the amino acids has the potential to be used as a tool to study surface properties of proteins. The contribution of this coupled water fraction in design of a hydropathy scale in surface adsorbed amino acid films on solid using quartz crystal microbalance is presented in this work. This scale compares well with the hydropathy scale of Guy reported in the literature and can be correlated with the solid/liquid interfacial tension and work of adhesion of the adsorbed amino acid films. Using Graphical Representation and Analysis of Surface Properties (GRASP) the free energy of transfer from Octanol to water for the amino acids has been estimated and shows approximately an inverse relationship with the coupled water fraction. This scale has been applied in a benchmark test for a native Laminin peptide YIGSR and its mutated sequences (with mutations carried out at 'Y and 'R' positions). The experimentally measured coupled water fractions seem to compare well with that obtained from the present scale assuming the total solvent fraction to be a linear function of the amino acids in the sequence. A survey of the protein data bank showed that sets of sequences based on this scale occur in membrane insertion domain or in trans-membrane proteins suggesting that the scale is suitable to study structure-function correlation in proteins.     

Kinetics and thermodynamics of protein adsorption: a generalized molecular theoretical approach

The thermodynamics and kinetics of protein adsorption are studied using a molecular theoretical approach. The cases studied include competitive adsorption from mixtures and the effect of conformational changes upon adsorption. The kinetic theory is based on a generalized diffusion equation in which the driving force for motion is the gradient of chemical potentials of the proteins. The time-dependent chemical potentials, as well as the equilibrium behavior of the system, are obtained using a molecular mean-field theory. The theory provides, within the same theoretical formulation, the diffusion and the kinetic (activated) controlled regimes. By separation of ideal and nonideal contributions to the chemical potential, the equation of motion shows a purely diffusive part and the motion of the particles in the potential of mean force resulting from the intermolecular interactions. The theory enables the calculation of the time-dependent surface coverage of proteins, the dynamic surface tension, and the structure of the adsorbed layer in contact with the approaching proteins. For the case of competitive adsorption from a solution containing a mixture of large and small proteins, a variety of different adsorption patterns are observed depending upon the bulk composition, the strength of the interaction between the particles, and the surface and size of the proteins. It is found that the experimentally observed Vroman sequence is predicted in the case that the bulk solution is at a composition with an excess of the small protein, and that the interaction between the large protein and the surface is much larger than that of the smaller protein. The effect of surface conformational changes of the adsorbed proteins in the time-dependent adsorption is studied in detail. The theory predicts regimes of constant density and dynamic surface tension that are long lived but are only intermediates before the final approach to equilibrium. The implications of the findings to the interpretation of experimental observations is discussed.     

The density and refractive index of adsorbing protein layers

The structure of the adsorbing layers of native and denatured proteins (fibrinogen, gamma-immunoglobulin, albumin, and lysozyme) was studied on hydrophilic TiO(2) and hydrophobic Teflon-AF surfaces using the quartz crystal microbalance with dissipation and optical waveguide lightmode spectroscopy techniques. The density and the refractive index of the adsorbing protein layers could be determined from the complementary information provided by the two in situ instruments. The observed density and refractive index changes during the protein-adsorption process indicated the presence of conformational changes (e.g., partial unfolding) in general, especially upon contact with the hydrophobic surface. The structure of the formed layers was found to depend on the size of the proteins and on the experimental conditions. On the TiO(2) surface smaller proteins formed a denser layer than larger ones and the layer of unfolded proteins was less dense than that adsorbed from the native conformation. The hydrophobic surface induced denaturation and resulted in the formation of thin compact protein films of albumin and lysozyme. A linear correlation was found between the quartz crystal microbalance measured dissipation factor and the total water content of the layer, suggesting the existence of a dissipative process that is related to the solvent molecules present inside the adsorbed protein layer. Our measurements indicated that water and solvent molecules not only influence the 3D structure of proteins in solution but also play a crucial role in their adsorption onto surfaces.     

Adsorption of zein on surfaces with controlled wettability and thermal stability of adsorbed zein films

Adsorption characteristics of zein protein on hydrophobic and hydrophilic surfaces have been investigated to understand the orientation changes associated with the protein structure on a surface. The protein is adsorbed by a self-assembly procedure on a monolayer-modified gold surface. It is observed that zein shows higher affinity toward hydrophilic than hydrophobic surfaces on the basis of the initial adsorption rate followed by quartz crystal microbalance studies. Reflection absorption infrared (RAIR) spectroscopic studies reveal the orientation changes associated with the adsorbed zein films. Upon adsorption, the protein is found to be denatured and the transformation of alpha-helix to beta-sheet form is inferred. This transformation is pronounced when the protein is adsorbed on hydrophobic surfaces as compared to hydrophilic surfaces. Electrochemical techniques (cyclic voltammetry and impedance techniques) are very useful in assessing the permeability of zein film. It is observed that the zein moieties adsorbed on hydrophilic surfaces are highly impermeable in nature and act as a barrier for small molecules. The topographical features of the deposits before and after adsorption are analyzed by atomic force microscopy. The protein adsorbed on hydrophilic surface shows rod- and disclike features that are likely to be the base units for the growth of cylindrical structures of zein. The thermal stability of the adsorbed zein film has been followed by variable-temperature RAIR measurements.     

Comparison of the adsorbed conformation of barley lipid transfer protein at the decane-water and vacuum-water interface: a molecular dynamics simulation

Molecular dynamics simulation is used to model the adsorption of the barley lipid transfer protein (LTP) at the decane-water and vacuum-water interfaces. Adsorption at both surfaces is driven by displacement of water molecules from the interfacial region. LTP adsorbed at the decane surface exhibits significant changes in its tertiary structure, and penetrates a considerable distance into the decane phase. At the vacuum-water interface LTP shows small conformational changes away from its native structure and does not penetrate into the vacuum space. Modification of the conformational stability of LTP by reduction of its four disulphide bonds leads to an increase in conformational entropy of the molecules, which reduces the driving force for adsorption. Evidence for changes in the secondary structure are also observed for native LTP at the decane-water interface and reduced LTP at the vacuum-water interface. In particular, intermittent formation of short (six-residue) regions of beta-sheet is found in these two systems. Formation of interfacial beta-sheet in adsorbed proteins has been observed experimentally, notably in the globular milk protein beta-lactoglobulin and lysozyme.     

The antibody–antigen interaction at an aqueous–solid interface: A study by means of polarized infrared ATR spectroscopy

The determination of structural changes in antibodies due to their specific interaction with antigenic proteins is an important problem in understanding immunological responses. The method of polarized ATR infrared spectroscopy applied to protein films adsorbed on an appropriate solid surface can give information about the conformation of the polypeptide chains, as well as their orientation with respect to the surface. The adsorption of anti-rabbit serum albumin onto monomolecular films of rabbit serum albumin, bovine serum albumin, and ovalbumin, and of anti-ovalbumin onto films of rabbit serum albumin and ovalbumin at a Ge-aqueous interface have been studied by this technique. The intensity of the amide I absorption indicates that the strengths of binding of these three albumin proteins with anti-rabbit serum albumin is, under appropriate conditions, in the order rabbit > bovine ? ovalbumin; with anti-ovalbumin, it is ovalbumin ? rabbit. Since the frequencies of the amide I band appear near 1655 cm^?1 for all the proteins and protein complexes studied, the major contributions to their conformation comes from α-helix and random-coil structures. The average orientation of the transition moments of the amide I and A bands has been shown to be about 75° with respect to the surface normal. This indicates that the polypeptides chains are on the average approximately parallel to the surface for all the systems studied. Consequently, the effect of the specific antibody-antigen interaction on the conformation and orientation of the former seems negligible in these films.     

Characterization of medium conditioned by irradiated cells using proteome-wide, high-throughput mass spectrometry

Shedding, the release of cell surface proteins by regulated proteolysis, is a general cellular response to injury and is responsible for generating numerous bioactive molecules including growth factors and cytokines. The purpose of our work is to determine whether low doses of low-linear energy transfer (LET) radiation induce shedding of bioactive molecules. Using a mass spectrometry-based global proteomics method, we tested this hypothesis by analyzing for shed proteins in medium from irradiated human mammary epithelial cells (HMEC). Several hundred proteins were identified, including transforming growth factor beta (TGFB); however, no changes in protein abundances attributable to radiation exposure, based on immunoblotting methods, were observed. These results demonstrate that our proteomic-based approach has the sensitivity to identify the kinds of proteins believed to be released after low-dose radiation exposure but that improvements in mass spectrometry-based protein quantification will be required to detect the small changes in abundance associated with this type of insult.     

Induction of changes in the secondary structure of globular proteins by a hydrophobic surface

Circular dichroism, ellipsometry and radiolabeling techniques were employed to study the induction of changes in the secondary structure of BSA, myoglobin and cytochrome C by a hydrophobic surface. The results showed that adsorbed protein molecules lose their ordered native structure in the initial stage of adsorption and the structure appears to be a random or disordered conformation. Protein molecules adsorbed in later stages adopt a more ordered secondary structure ( helix and structure). The changes of secondary structure of globular proteins induced by a hydrophobic surface can be explained by the steric interaction between adsorbed proteins as well as by hydrophobic interactions during the adsorption process. In addition, there is obviously an intermediate stage in which the protein molecules are mainly in the structure, indicating that for certain proteins, the structure may be a more stable secondary structure than helix on the hydrophobic surface. Correspondence to: S.-F. Sui     

Protein structural perturbation and aggregation on homogeneous surfaces

We have demonstrated that globular proteins, such as hen egg lysozyme in phosphate buffered saline at room temperature, lose native structural stability and activity when adsorbed onto well-defined homogeneous solid surfaces. This structural loss is evident by alpha-helix to turns/random during the first 30 min and followed by a slow alpha-helix to beta-sheet transition. Increase in intramolecular and intermolecular beta-sheet content suggests conformational rearrangement and aggregation between different protein molecules, respectively. Amide I band attenuated total reflection/Fourier transformed infrared (ATR/FTIR) spectroscopy was used to quantify the secondary structure content of lysozyme adsorbed on six different self-assembled alkanethiol monolayer surfaces with -CH3, -OPh, -CF3, -CN, -OCH3, and -OH exposed functional end groups. Activity measurements of adsorbed lysozyme were in good agreement with the structural perturbations. Both surface chemistry (type of functional groups, wettability) and adsorbate concentration (i.e., lateral interactions) are responsible for the observed structural changes during adsorption. A kinetic model is proposed to describe secondary structural changes that occur in two dynamic phases. The results presented in this article demonstrate the utility of the ATR/FTIR spectroscopic technique for in situ characterization of protein secondary structures during adsorption on flat surfaces.     

An Auto-Catalytic Surface for Conformational Replication of Amyloid Fibrils—Genesis of an Amyloid World?

Amyloid fibrils are composed of self assembled stacked peptide or protein molecules folded and trapped in a stable cross-beta-sheet conformation. The amyloid fibrillation mechanism represents an intriguing self-catalyzed process rendering replication of a molecular conformational memory of interest for prebiotic chemistry. Herein we describe how a solid surface can be rendered auto-catalytic for fibrillation of a protein solution. We have discovered that a hydrophobic silicon or glass surface can be made to continuously fibrillate solutions of insulin monomers under stressed conditions (pH 1.6, 65°C). It was found that the surface acts as a platform for the formation of nascent seeds that induce fibril replication on and at the surface. This autocatalytic effect stems from a layer a few insulin molecules thick representing an oligomeric layer of misfolded, conformationally trapped, insulin molecules that rapidly through epitaxial growth catalyze the rate determining step (nucleation) during fibril replication. This autocatalytic layer is generated by the protein-solid surface interaction and conformational changes of the adsorbed protein during exposure at the air-water interface. The resulting autocatalytic surface thus both initiates local conformational molecular self-replication and acts as a reservoir for fibril seeds budding off into solution spreading fibril replication entities to the surrounding medium. The possibility of catalysis of the conformational replication process by minute amounts of nucleation sites located on a recruiting surface can evade the issue of dramatic concentration dependence of amyloidogenesis.     

Chemical modification probes accessibility to organic phase: proteins on surfaces are more exposed than in lyophilized powders

Chemical modification of myoglobin and cutinase suspended in n-hexane by acyl chlorides and iodine was monitored by electrospray mass spectrometry. The general rate of modification was always much faster for protein adsorbed to supports (silica or polypropylene) than for lyophilized powders. Modification rates were slower for larger acyl chlorides, particularly with lyophilized powders. About 20% of the protein molecules in lyophilized powders were modified much more quickly than the rest, a fraction consistent with those exposed on the surface of the solid. It appears that access to most of the molecules in lyophilized powders requires a very slow stage of solid-phase diffusion. This has been neglected in previous discussion of mass transfer limitation of lyophilized enzymes in organic media, and would not be revealed by the experimental evidence used to dismiss it. Studies of the effects of particle size and dilution with inactive protein are only sensitive to diffusion in liquid-filled pores, not through the solid phase. Slow solid-phase diffusion is not required for access to most support-adsorbed proteins, which is probably a major contributory factor to their enhanced catalytic efficiency in organic media. Hydration of lyophilized proteins accelerates chemical modification rates, as it does their catalytic activity. The main site of reaction of acyl chlorides in organic media is not amino groups (which are probably ion-paired), but is likely to be hydroxyl groups instead.     

Molecular mechanisms of microheterogeneity-induced defect formation in ferritin crystallization

We apply in situ atomic force microscopy to the crystallization of ferritins from solutions containing approximately 5% (w/w) of their inherent molecular dimers. Molecular resolution imaging shows that the dimers consist of two bound monomers. The constituent monomers are likely partially denatured, resulting in increased hydrophobicity of the dimer surface. Correspondingly, the dimers strongly adsorb on the crystal surface. The adsorbed dimers hinder step growth and on incorporation by the crystal initiate stacks of up to 10 triple and single vacancies in the subsequent crystal layers. The molecules around the vacancies are shifted by approximately 0.1 molecular dimensions from their crystallographic positions. The shifts strain the lattice and, as a consequence, at crystal sizes > 200 microm, the accumulated strain is resolved by a plastic deformation whereupon the crystal breaks into mosaic blocks 20-50 microm in size. The critical size for the onset of mosaicity is similar for ferritin and apoferritin and close to the value for a third protein, lysozyme; it also agrees with theoretical predictions. Trapped microcrystals in ferritin and apoferritin induce strain with a characteristic length scale equal to that of a single point defect, and, as a consequence, trapping does not contribute to the mosaicity. The sequence of undesired phenomena that include heterogeneity generation, adsorption, incorporation, and the resulting lattice strain and mosaicity in this and other proteins systems, could be avoided by improved methods to separate similar proteins species (microheterogeneity) or by increasing the biochemical stability of the macromolecules against oligomerization.