Morphology and properties of soy protein isolate thermoplastics reinforced with chitin whiskers

Environmentally friendly thermoplastic nanocomposites were successfully developed using a colloidal suspension of chitin whiskers as a filler to reinforce soy protein isolate (SPI) plastics. The chitin whiskers, having lengths of 500 +/- 50 nm and diameters of 50 +/- 10 nm on average, were prepared from commercial chitin by acid hydrolysis. The dependence of morphology and properties on the chitin whiskers content in the range from 0 to 30 wt % for the glycerol plasticized SPI nanocomposites was investigated by dynamic mechanical thermal analysis, scanning electron microscopy, swelling experiment, and tensile testing. The results indicate that the strong interactions between fillers and between the filler and SPI matrix play an important role in reinforcing the composites without interfering with their biodegradability. The SPI/chitin whisker nanocomposites at 43% relative humidity increased in both tensile strength and Young's modulus from 3.3 MPa for the SPI sheet to 8.4 MPa and from 26 MPa for the SPI sheet to 158 MPa, respectively. Further, incorporating chitin whisker into the SPI matrix leads to an improvement in water resistance for the SPI based nanocomposites.     

Morphology and properties of soy protein and polylactide blends

Blends of soy protein (SP) and a semicrystalline polylactide (PLA) were prepared using a twin-screw extruder. The melt rheology, phase morphology, mechanical properties, water resistance, and thermal and dynamic mechanical properties were investigated on specimens prepared by injection molding of these blends. The melt flowability of soy-based plastics was improved through blending with PLA. Scanning electron microscopy revealed that a co-continuous phase structure existed in the blends with soy protein concentrate (SPC) to PLA ratios ranging from 30:70 to 70:30. SPC/PLA blends showed fine co-continuous phase structures, while soy protein isolate (SPI)/PLA blends presented severe phase coarsening. At the same SP to PLA ratios, SPC/PLA blends demonstrated a higher tensile strength than SPI/PLA blends. The water absorption of soy plastics was greatly reduced by blending with PLA. The compatibility was improved by adding 1-5 phr poly(2-ethyl-2-oxazoline) (PEOX) in the blends, and the resulting blends showed an obvious increase in tensile strength and a reduction in water absorption for SPI/PLA blends. The compatibility between SP and PLA was evaluated by mechanical testing, dynamic mechanical analysis (DMA), water absorption, and scanning electron microscopy (SEM) experiments. Differential scanning calorimetry (DSC) revealed that PLA in the blends was mostly amorphous in the injection molded articles, and SP accelerated the cold crystallization and could increase the final crystallinity of PLA in the blends.     

Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles

Coulombic interactions between charges on the surface of proteins contribute to stability. It is difficult, however, to estimate their importance by protein engineering methods because mutation of one residue in an ion pair alters the energetics of many interactions in addition to the coulombic energy between the two components. We have estimated the interaction energy between two charged residues, Asp-12 and Arg-16, in an alpha-helix on the surface of a barnase mutant by invoking a double-mutant cycle involving wild-type enzyme (Asp-12, Thr-16), the single mutants Thr----Arg-16 and Asp----Ala-12, and the double mutant Asp----Ala-12, Thr----Arg-16. The changes in free energy of unfolding of the single mutants are not additive because of the coulombic interaction energy. Additivity is restored at high concentrations of salt that shield electrostatic interactions. The geometry of the ion pair in the mutant was assumed to be the same as that in the highly homologous ribonuclease from Bacillus intermedius, binase, which has Asp-12 and Arg-16 in the native enzyme. The ion pair does not form a hydrogen-bonded salt bridge, but the charges are separated by 5-6 A. The mutant barnase containing the ion pair Asp-12/Arg-16 is more stable than wild type by 0.5 kcal/mol, but only a part of the increased stability is attributable to the electrostatic interaction. We present a formal analysis of how double-mutant cycles can be used to measure the energetics of pairwise interactions.     

Electrostatic stabilization in four-helix bundle proteins.

Charge substitutions generated by site-directed mutagenesis at the termini of adjacent anti-parallel alpha-helices in a four-helix bundle protein were used to determine a precise value for the contribution of indirect charge-charge interactions to overall protein stability, and to simulate the electrostatic effects of alpha-helix macrodipoles. Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti-parallel helices in the four-helix bundle cytochrome b562 from Escherichia coli. Previously, theoretical calculations of helix macrodipole interactions using modeled four-helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol. Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges. At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti-parallel helix pair. Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude. The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect. Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti-parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.     

Protein stabilization by salt bridges: concepts, experimental approaches and clarification of some misunderstandings

Salt bridges in proteins are bonds between oppositely charged residues that are sufficiently close to each other to experience electrostatic attraction. They contribute to protein structure and to the specificity of interaction of proteins with other biomolecules, but in doing so they need not necessarily increase a protein's free energy of unfolding. The net electrostatic free energy of a salt bridge can be partitioned into three components: charge-charge interactions, interactions of charges with permanent dipoles, and desolvation of charges. Energetically favorable Coulombic charge-charge interaction is opposed by often unfavorable desolvation of interacting charges. As a consequence, salt bridges may destabilize the structure of the folded protein. There are two ways to estimate the free energy contribution of salt bridges by experiment: the pK(a) approach and the mutation approach. In the pK(a) approach, the contribution of charges to the free energy of unfolding of a protein is obtained from the change of pK(a) of ionizable groups caused by altered electrostatic interactions upon folding of the protein. The pK(a) approach provides the relative free energy gained or lost when ionizable groups are being charged. In the mutation approach, the coupling free energy between interacting charges is obtained from a double mutant cycle. The coupling free energy is an indirect and approximate measure of the free energy of charge-charge interaction. Neither the pK(a) approach nor the mutation approach can provide the net free energy of a salt bridge. Currently, this is obtained only by computational methods which, however, are often prone to large uncertainties due to simplifying assumptions and insufficient structural information on which calculations are based. This state of affairs makes the precise thermodynamic quantification of salt bridge energies very difficult. This review is focused on concepts and on the assessment of experimental methods and does not cover the vast literature.     

Structure and properties of soy protein/poly(butylene succinate) blends with improved compatibility

A novel environmentally friendly thermoplastic soy protein/polyester blend was successfully prepared by blending soy protein isolate (SPI) with poly(butylene succinate) (PBS). To improve the compatibility between SPI and PBS, the polyester was pretreated by introducing different amounts of urethane and isocyanate groups before blending. The blends containing pretreated PBS showed much finer phase structures because of good dispersion of polyester in protein. Consequently, the tensile strength and modulus of blends increased obviously. A lower glass transition temperature of protein in the blends than that of the pure SPI, which was caused by the improvement of the compatibility between two phases, was observed by dynamic mechanical analyzer (DMA). The hydrophobicity, water resistance, and moisture absorption at different humidities of the blends were modified significantly due to the incorporation of PBS.     

Chitosan-soyprotein interaction as determined by thermal unfolding experiments

Chitosan interaction with soybean beta-conglycinin beta(3) was investigated by thermal unfolding experiments using CD spectroscopy. The negative ellipticity of the protein was enhanced with rising solution temperature. The transition temperature of thermal unfolding of the protein (T(m)) was 63.4 degrees C at pH 3.0 (0.15 M KCl). When chitosan was added to the protein solution, the T(m) value was elevated by 7.7 degrees C, whereas the T(m) elevation upon addition of chitosan hexamer (GlcN)(6) was 2.2 degrees C. These carbohydrates appear to interact with the protein stabilizing the protein structure, and the interaction ability could be evaluated from the T(m) elevation. Similar experiments were conducted at various pHs from 2.0 to 3.5, and the T(m) elevation was found to be enhanced in the higher pH region. We conclude that chitosan interacts with beta-conglycinin through electrostatic interactions between the positive charges of the chitosan polysaccharide and the negative charges of the protein surface.     

Affinity-repulsion chromatography. Principle and application to lectins

The interactions of proteins with their immobilized ligands in an electrically charged microenvironment were studied. The binding of lectins to erythrocytes and to affinity matrices was used as a model system. Lectins bind and agglutinate erythrocytes in the presence of at least 10 mM NaCl or 1 mM CaCl2, but not in deionized water. The salt dependence of the agglutination process is due to the ability of salts to provide counterions neutralizing the forces of repulsion between the electrostatic charges of similar sign present on the erythrocyte cell surface and on the lectins. The same salt dependence is observed for the binding of lectins to affinity matrices. These observations are the basis of a protein separation process coined affinity-repulsion chromatography in which the electrostatic charges present, or purposely introduced, on affinity matrices are exploited and allow the elution, by electrostatic repulsion, of proteins carrying electrostatic charges of the same sign as that of the matrix. In this process, proteins are loaded on the affinity matrix in a salt solution and eluted with deionized water. Affinity-repulsion chromatography has been successfully applied here to the isolation of several lectins. Its physicochemical basis, merits, and potential applications are discussed.     

Electrostatic potentials and electrostatic interaction energies of rat cytochrome b5 and a simulated anion-exchange adsorbent surface.

Electrostatic potentials were determined for the soluble tryptic core of rat cytochrome b5 (using a structure derived from homology modeling) and a simulated anion-exchange surface through application of the linearized finite-difference Poisson-Boltzmann equation with the simulation code UHBD. Objectives of this work included determination of the contributions of the various charged groups on the protein surface to electrostatic interactions with a simulated anion-exchange surface as a function of orientation, separation distance, and ionic strength, as well as examining the potential existence of a preferred contact orientation. Electrostatic interaction free energies for the complex of the model protein and the simulated surface were computed using the electrostatics section of UHBD employing a 110(3) grid. An initial coarse grid spacing of 2.0 A was required to obtain correct boundary conditions. The boundary conditions of the coarse grid were used in subsequent focusing steps until the electrostatic interaction free energies were relatively independent of grid spacing (at approximately 0.5 A). Explicit error analyses were performed to determine the effects of grid spacing and other model assumptions on the electrostatic interaction free energies. The computational results reveal the presence of a preferred interaction orientation; the interaction energy between these two entities, of opposite net charge, is repulsive over a range of orientations. The electrostatic interaction free energies appear to be the summation of multiple fractional interactions between the protein and the anion-exchange surface. The simulation results are compared with those of ion-exchange adsorption experiments with site-directed mutants of the recombinant protein. Comparisons of the results from the computational and experimental studies should lead to a better understanding of electrostatic interactions of proteins and charged surfaces.     

Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents.

The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.     

Kinetics of reduction of high redox potential ferredoxins by the semiquinones of Clostridium pasteurianum flavodoxin and exogenous flavin mononucleotide. Electrostatic and redox potential effects

We have measured the ionic strength dependence of the rate constants for the electron-transfer reactions of flavin mononucleotide (FMN) and flavodoxin semiquinones with 10 high redox potential ferredoxins (HiPIP's). The rate constants were extrapolated to infinite ionic strength by using a theoretical model of electrostatic interactions developed in our laboratory. In all cases, the sign of the electrostatic interaction was the same as the protein net charge, but the magnitudes were much smaller. The results are consistent with a model in which the electrical charges are approximately uniformly distributed over the HiPIP surface and in which there are both short- and long-range electrostatic interactions. An electrostatic field calculation for Chromatium vinosum HiPIP is consistent with this. The presumed site of electron transfer includes that region of the protein surface to which the iron-sulfur cluster is nearest and appears to be relatively hydrophobic. The principal short-range electrostatic interaction would involve the negative charge on the iron-sulfur cluster. For some net negatively charged proteins, this effect is magnified, and for net positively charged HiPIP's, it is counterbalanced. The rate constants extrapolated to infinite ionic strength can be correlated with redox potential differences between the reactants, as has previously been shown for cytochrome-flavin semiquinone reactions. Both electrostatic and redox potential effects are magnified for the flavodoxin semiquinone as compared to the FMN semiquinone-HiPIP reactions. This was also observed previously for the flavin semiquinone-cytochrome reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

About factors providing the fast protein-protein recognition in processes of complex formation

A package of programs for the examination of areas of subunit contacts (interface) in protein-protein (PP) complexes has been created and used for a detailed study of amino acid (AA) composition and interface structure in a large number of PP complexes from Brookhaven database (PBD). It appeared that in about 75% of the complexes, the AA composition of the subunit surface is not important. This suggests that, along with the surface AA composition, interactions between AA from the inner parts of protein globules may play a significant role in PP recognition. Such interactions between relatively distant AA residues can only be of electrostatic nature and contribute to the total electric field of the protein molecule. The configuration of the electric field itself appears to determine the PP recognition. The total electric field created by protein molecules can be calculated as a result of superimposition of the fields created by the protein multipole (i.e. by the totality of partial electric charges assigned to each atom of the molecule). We performed preliminary calculations for the distant electrostatic interaction of ribonuclease subunits in a vacuum. The results reveal that the effect of the electric fields of the protein multipole is strong enough to orient protein molecules prior to their Brown collision.     

Protein stability and electrostatic interactions between solvent exposed charged side chains

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.     

Electrostatic contributions to the kinetics and thermodynamics of protein assembly

The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D(9k) reconstitution was measured in the presence of Ca(2+) using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K(A) = 1.3 x 10(10) M(-1); K(D) = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.     

The Formation and Disaggregation of Soy Protein Isolate Fibril: Effects of pH

To identify the effects of charged states on the formation and disaggregation of soy protein isolate (SPI) fibril, we studied the thermal aggregation behaviors of the constituent peptides of SPI fibril (CPSF) at various pH values (2–10) and investigated the structural changes of SPI fibril with increasing pH (2–11). Results showed that CPSF would assemble into diverse shapes at different pH values, among which the aggregates contained multiple β-sheet structures at pH less than 6, but these β-sheets were stacked to form fibrils only at pH 2. The damages from the increased pH to SPI fibril structure could be roughly divided into two stages, as follows: when pH was less than or equal to 6, the morphology of fibrils changed markedly due to electrostatic neutralization; at pH larger than 6, the fibrils suffered great losses in β-sheet, causing its structure to disintegrate rapidly. This study could provide theoretical reference to improve the pH stability of SPI fibril from the aspects of preparation and structural protection of the fibril.

     

Brownian dynamics simulation of the lateral distribution of charged membrane components

Brownian dynamics simulations were performed to study the contribution of electric interactions between charged membrane components to their lateral distribution in a two-dimensional viscous liquid (bilayer lipid membrane). The electrostatic interaction potential was derived from an analytical solution of the linearized Poisson-Boltzmann equation for point charges in an electrolyte solution — membrane — electrolyte solution system. Equilibrium as well as dynamic quantities were investigated. The lateral organization of membrane particles, modelled by mobile cylinders in a homogeneous membrane separating two electrolyte solutions was described by spatial distribution functions, diffusion coefficients and cluster statistics. Disorder, local order and crystal-like arrangements were observed as a function of the particle charge, the closest possible distances between the charges and the particle density. The simulations revealed that the system is very sensitive to the position of the charges with respect to the electrolyte solution — membrane interface. Electrostatic interactions of charges placed directly on the membrane surface were almost negligible, whereas deeper charges demonstrated pronounced interaction. Biologically relevant parameters corresponded at most to local and transient ordering. It was found that lateral electric forces can give rise to a preferred formation of clusters with an even number of constituents provided that the closest possible charge-charge distances are small. It is concluded that lateral electrostatic interactions can account for local particle aggregations, but their impact on the global arrangement and movement of membrane components is limited. Correspondence to: D. Walther     

Interactions between adsorbed hydrogenated soy phosphatidylcholine (HSPC) vesicles at physiologically high pressures and salt concentrations

Using a surface force balance, we measured normal and shear interactions as a function of surface separation between layers of hydrogenated soy phosphatidylcholine (HSPC) small unilamellar vesicles (SUVs) adsorbed from dispersion at physiologically high salt concentrations (0.15 M NaNO₃). Cryo-scanning electron microscopy shows that each surface is coated by a close-packed HSPC-SUV layer with an overlayer of liposomes on top. A clear attractive interaction between the liposome layers is seen upon approach and separation, followed by a steric repulsion upon further compression. The shear forces reveal low friction coefficients (μ = 0.008–0.0006) up to contact pressures of at least 6 MPa, comparable to those observed in the major joints. The spread in μ-values may be qualitatively accounted for by different local liposome structure at different contact points, suggesting that the intrinsic friction of the HSPC-SUV layers at this salt concentration is closer to the lower limit (μ = ∼0.0006). This low friction is attributed to the hydration lubrication mechanism arising from rubbing of the hydrated phosphocholine-headgroup layers exposed at the outer surface of each liposome, and provides support for the conjecture that phospholipids may play a significant role in biological lubrication.     

Electrostatic Interactions in Aminoglycoside-RNA Complexes

Electrostatic interactions often play key roles in the recognition of small molecules by nucleic acids. An example is aminoglycoside antibiotics, which by binding to ribosomal RNA (rRNA) affect bacterial protein synthesis. These antibiotics remain one of the few valid treatments against hospital-acquired infections by Gram-negative bacteria. It is necessary to understand the amplitude of electrostatic interactions between aminoglycosides and their rRNA targets to introduce aminoglycoside modifications that would enhance their binding or to design new scaffolds. Here, we calculated the electrostatic energy of interactions and its per-ring contributions between aminoglycosides and their primary rRNA binding site. We applied either the methodology based on the exact potential multipole moment (EPMM) or classical molecular mechanics force field single-point partial charges with Coulomb formula. For EPMM, we first reconstructed the aspherical electron density of 12 aminoglycoside-RNA complexes from the atomic parameters deposited in the University at Buffalo Databank. The University at Buffalo Databank concept assumes transferability of electron density between atoms in chemically equivalent vicinities and allows reconstruction of the electron densities from experimental structural data. From the electron density, we then calculated the electrostatic energy of interaction using EPMM. Finally, we compared the two approaches. The calculated electrostatic interaction energies between various aminoglycosides and their binding sites correlate with experimentally obtained binding free energies. Based on the calculated energetic contributions of water molecules mediating the interactions between the antibiotic and rRNA, we suggest possible modifications that could enhance aminoglycoside binding affinity.     

Soy Protein Microparticles for Enhanced Oral Ibuprofen Delivery: Preparation,Characterization, and <Emphasis Type="Italic">In Vitro</Emphasis> Release Evaluation

The objective of this work was to evaluate soy protein isolate (SPI) and acylated soy protein (SPA) as spray-drying encapsulation carriers for oral pharmaceutical applications. SPI acylation was performed by the Schotten–Baumann reaction. SPA, with an acylation rate of 41%, displayed a decrease in solubility in acidic conditions, whereas its solubility was unaffected by basic conditions. The drug encapsulation capacities of both SPI and SPA were tested with ibuprofen (IBU) as a model poorly soluble drug. IBU-SPI and IBU-SPA particles were obtained by spray-drying under eco-friendly conditions. Yields of 70 to 87% and microencapsulation efficiencies exceeding 80% were attained for an IBU content of 20 to 40% w/w, confirming the excellent microencapsulation properties of SPI and the suitability of the chemical modification. The in vitro release kinetics of IBU were studied in simulated gastrointestinal conditions (pH 1.2 and pH 6.8, 37°C). pH-sensitive release patterns were observed, with an optimized low rate of release in simulated gastric fluid for SPA formulations, and a rapid and complete release in simulated intestinal fluid for both formulations, due to the optimal pattern of pH-dependent solubility for SPA and the molecular dispersion of IBU in soy protein. These results demonstrate that SPI and SPA are relevant for the development of pH-sensitive drug delivery systems for the oral route.     

Spatial optimization of electrostatic interactions between the ionized groups in globular proteins

A model approach is suggested to estimate the degree of spatial optimization of the electrostatic interactions in protein molecules. The method is tested on a set of 44 globular proteins, representative of the available crystallographic data. The theoretical model is based on macroscopic computation of the contribution of charge–charge interactions to the electrostatic term of the free energy for the native proteins and for a big number of virtual structures with randomly distributed on protein surface charge consetellations (generated by a Monte-Carlo technique). The statistical probability of occurrence of random structures with electrostatic energies lower than the energy of the native protein is suggested as a criterion for spatial optimization of the electrostatic interactions. The results support the hypothesis that the folding process optimizes the stabilizing effect of electrostatic interactions, but to very different degree for different proteins. A parallel analysis of ion pairs shows that the optimization of the electrostatic term in globular proteins has increasingly gone in the direction of rejecting the repulsive short contacts between charges of equal sign than of creating of more salt bridges (in comparison with the statistically expected number of shortrange ion pairs in the simulated random structures). It is observed that the decrease in the spatial optimization of the electrostatic interactions is usually compensated for by an appearance of disulfide bridges in the covalent structure of the examined proteins. © 1994 Wiley-Liss, Inc.