Participation of cysteine 30 residue in the folding process of ovalbumin evaluated in a refolding experiment using cysteine mutants.

To understand the role of cysteine (SH) residues in the folding of hen ovalbumin (OVA), SH-mutated OVAs, in which each SH residue was replaced by alanine (C11A, C30A, C367A, and C382A), were prepared. SDS-PAGE analysis under non-reducing conditions showed that the C11A and C30A mutants produced a disulfide (SS) isomer in addition to a protein with a native SS bond (Cys73-Cys120). The susceptibility to elastase digestion suggested that the Cys73 residue in the SS isomer participates as a counterpart of the SS bond. Upon refolding of the SH-mutated OVAs under the denatured and SS-reduced states, only C30A failed to refold into an intact form. This indicated that the Cys30 residue plays an important role in correct refolding. To confirm this, each of the four SH-mutated OVAs, in which the original SS-forming sites (C11/73/120A, C30/73/120A, C73/120/367A, and C73/120/382A) were deleted, was constructed and expressed. The C11/73/120A and C30/73/120A mutants formed no SS form, in contrast to C73/120A as a control. Thus, we concluded that Cys30 participates in the correct folding of OVA, and that its SS bond (Cys11-Cys30) is transiently generated during the early folding stage to avoid misfolding, and then the native SS form of OVA is regenerated through SH-SS exchanges.

Interfacial and enhanced emulsifying behavior of phosphorylated ovalbumin.

Improvement of emulsifying properties by phosphorylation could have a wide potential application in food industry. In this study, ovalbumin (OVA) was phosphorylated under wet-heating in the presence of sodium tripolyphosphate. Phosphorylated OVA (P-OVA) with low, middle, high phosphorus content were obtained with reaction time increased. Their enhanced emulsification capacity and the mechanism were investigated. Compared with native OVA (N-OVA), the emulsifying activity and stability of P-OVA were increased by 26% and 109% (P < 0.05), respectively. The structure studies (Fourier transform infrared spectroscopy, circular dichroism spectra, scanning electron microscope) demonstrated that introduction of phosphate groups to OVA increased the molecular flexibility of P-OVA. The absolute values of surface zeta-potential and surface hydrophobicity were increased as the phosphorus content increased, which indicated that the phosphate group inhibited protein aggregation. And it caused a large amount absorption of protein on the surface of the oil droplets, which ultimately improved the emulsion stability. Both particle size and microscopic results of emulsion showed that the particle size of OVA reduced as the degree of phosphorylation increased, which improved the emulsifying ability.

Effective hard particle model for the osmotic pressure of highly concentrated binary protein solutions.

The experimentally measured concentration dependence of the osmotic pressure of an equimolar mixture of hen egg ovalbumin and bovine serum albumin at pH 7.0 and 25 degrees C in the presence of 0.15 M NaCl is shown to be quantitatively accounted for by a model in which each protein species is represented by an effective hard sphere. The size of this sphere is determined by analysis of the concentration dependence of the osmotic pressure of the isolated protein.

Interaction and incorporation of ovalbumin with stearic acid monolayer: Langmuir-Blodgett film formation and deposition.

In this communication, the surface activity of the ovalbumin (OVA) at the air/water interface was studied to establish the nature of the interaction with the stearic acid (SA) monolayer, based on Langmuir-Blodgett (LB) technique. The interaction was monitored by studying the time (t) variation of surface pressure (pi) at constant area (A). The growth of pi with time indicates a positive association between the SA and the OVA molecules. The surface compressibility analysis has been performed to specify the phase transition of OVA-SA mixed monolayer. Incorporation/association of OVA within the SA monolayer led to noteworthy changes in surface compressibility and was surface pressure as well as protein concentration dependent. Both the hydrophobic and the Vander wall type interactions are found to be responsible for the association. The quenching of tyrosine band in tryptophan excitation spectrum is observed in steady-state fluorescence spectroscopy. This suggests that the tyrosine is the probable binding site with SA. Due to incorporation of SA, the energy transfer from tyrosine to tryptophan is hindered. At higher pressure, OVA tend to squeeze out from the SA monolayer. The high-resolution field emission scanning electron microscope (FE-SEM) image confirms this observation. Aggregated protein structure observed at high pressure indicates unfolding of protein.

Using TEM to couple transient protein distribution and release for PLGA microparticles for potential use as vaccine delivery vehicles.

In the development of tunable PLGA microparticles as vaccine delivery vehicles, it is important to understand the drug distribution within the microparticle over time as well as the long-term release of the drug during polymer degradation. This study addresses the transient 3-D drug distribution in PLGA microparticles during in vitro degradation. Specifically, poly (lactide-co-glycolide) (PLGA 75:25) microparticles containing ovalbumin (OVA) as a model protein were fabricated by double-emulsion (w/o/w) method. The microparticles were incubated at 37 degrees C and 250 rpm in PBS buffer (pH 7.4) over a 100-day period. The in vitro polymer erosion, transient protein distribution profiles and protein release behaviors were investigated. Protein release profiles were determined via spectrophotometry using a BCA assay for the solution. Transmission electron microscopy (TEM) images were obtained for the OVA-loaded microparticles before and during degradation (0 day, 30 days and 60 days), and the corresponding 3-D constructions were developed. From the 3-D constructions, the overall protein distribution of the entire microparticle was vividly reflected. Pixel number analysis of the TEM images was used to quantify transient protein distribution. The transient protein release obtained from the TEM analysis was in good agreement with the BCA analysis. This technique provides an additional tool in helping develop polymer matrices for tunable delivery vehicles in vaccination and other drug delivery scenarios.

The effect of gamma-irradiation on PLGA/PEG microspheres containing ovalbumin.

Poly(ethylene glycol) (PEG) and sodium chloride (NaCl) are excipients used in PLGA microsphere preparation to stabilize proteins and reduce their burst release. No information is till now available in the literature on the effect due to the use of such excipients on the biopharmaceutical performance of gamma-irradiated microparticulate systems. On this purpose, different batches of microspheres containing ovalbumin (OVA) were prepared by using a PLGA 50:50 (average Mr: 13000), different amounts of PEG (Mr: 400 or 4000) and/or sodium chloride. The non-irradiated and irradiated microspheres were characterized in terms of morphology (SEM, particle size distribution), OVA and PEG content and in vitro OVA release. Radiolysis mechanisms of OVA and OVA loaded microspheres were investigated by EPR analysis. Gamma irradiation affects either microsphere morphology or the release of OVA as a function of the amount of PEG, and the use of NaCl. Irradiation significantly reduces release rate of protein from the microspheres containing 15% and 30% of PEG and from controls (microspheres without additives), while no significative effect on protein release rate is highlighted on microspheres containing lower amounts of PEG. EPR investigation shows that increasing amounts of PEG up to 30% have a perturbation effect on OVA radiolysis path.

Irreversible self-assembly of ovalbumin into fibrils and the resulting network rheology.

The self-assembly of ovalbumin into fibrils and resulting network properties were investigated at pH 2, as a function of ionic strength. Using transmission electron microscopy (TEM), the effect of ovalbumin concentration on the contour length was determined. The contour length was increasing with increasing ovalbumin concentration. TEM micrographs were made to investigate the effect of ionic strength on the contour length. In the measured ionic strength regime (0.01-0.035 M) fibrils of approximately equal length (+/-200 nm) were observed. TEM micrographs showed that the contour length of the fibrils, versus time after dilution, remained constant, which indicates that the self-assembly of ovalbumin is irreversible. Using the results of rheological measurements, we observed a decreasing critical percolation concentration with increasing ionic strength. We explain this result in terms of an adjusted random contact model for charged semiflexible fibrils. Hereby, this model has now been proven to be valid for fibril networks of beta-lg, BSA and, currently, for ovalbumin.

Refolding of urea denatured ovalbumin with three phase partitioning generates many conformational variants.

Three phase partitioning is a process in which mixing t-butanol with ammonium sulphate with a protein solution leads to the formation of three phases. Generally, the interfacial protein precipitate (formed between upper t-butanol rich and lower aqueous phase) can be easily dissolved back in aqueous buffers. In case of ovalbumin, this led to a precipitate which was insoluble in aqueous buffers. This precipitate when solubilized with 8 M urea and subjected to three phase partitioning under various conditions led to many refolded soluble conformational variants of ovalbumin. One of these showed trypsin inhibitory activity, had marginally higher ß-sheet content and had higher surface hydrophobicity (both with respect to native ovalbumin). Scanning electron microscopy and Atomic force microscopy of this preparation showed a thread like structure characteristic of amyloid fibrils. The behaviour of ovalbumin during three phase partitioning makes it a valuable system for gaining further understanding of protein aggregation.

Role of the N-terminal amphiphilic region of ovalbumin during heat-induced aggregation and gelation.

Ovalbumin (OVA), a major globular protein in egg white, forms semiflexible fibrillar aggregates during heat-induced gelation. The N-terminal amphiphilic region (pN1-22) of OVA is removed after treatment with pepsin at pH 4 to leave a large OVA fragment (pOVA). The conformation and thermal stability of pOVA and OVA are similar, but the rheological strength of the heat-induced gel of pOVA is much lower compared to that of OVA. The aggregation rate of pOVA, which forms spherical aggregates, was lower than that of OVA. These results suggest that the N-terminal amphiphilic region of OVA facilitates the α-to-ß conformational transition, which triggers OVA fibril formation. Heat treatment of OVA in the presence of pN1-22 consistently resulted in the formation of straight fibrils. The strength of OVA and collagen gels was increased when prepared in the presence of pN1-22, suggesting that pN1-22 may be used to control the properties of protein gels.

Evaluation of the influence of albumin on the mineralization of a glass by Atomic Force Microscopy.

Bioactive glasses have been used as a graft material that can stimulate the formation of a new bone. In vitro tests usually give sensible indications about the potential bioactivity of these glasses. In the present work the influence of egg albumin on the formation of a Ca-P precipitate on a glass of the system SiO(2)-CaO-MgO-P(2)O(5) was evaluated. The samples were immersed in simulated body fluid (SBF) that simulates the composition of human plasma, with and without albumin. After immersion in this solution for 7 and 14 days, the glass was characterized by X-Ray Diffraction (XRD) and Atomic Force Microscopy (AFM). AFM results of the samples after immersion in SBF with albumin show the development of a precipiate formed from the solution/substrate reaction. Glasses immersed in albumin-free SBF exhibit the formation of a thin layer easily detached from the substrate. XRD results indicate that the precipitate is essentially amorphous, evolving to octacalcium phosphate. As the formation of an adherent precipitate on the glass samples only occurred when the substrate was immersed in SBF with albumin, it is suggested that albumin improves the mineralization on the glasses.

Surface characterization by atomic force microscopy of sterilized PLGA microspheres.

Atomic force microscopy (AFM) is recognized a suitable and powerful technique for surface and morphological analysis. Even if until now this technique has not been frequently used in the pharmaceutical field, it can contribute to an accurate morphologic characterization of microspheres and nanospheres. In this work, atomic force microscopy has been used to perform the surface characterization of sterilized microspheres. The aim is to investigate the morphologic modifications induced by gamma irradiation on poly(lactide-co-glycolide) microspheres loaded with ovalbumin and to compare the results obtained by AFM to those obtained by scanning electron microscopy (SEM). The results obtained show that, with respect to SEM, AFM can give some additional information regarding the modifications induced by gamma-irradiation on microspheres surface morphology. The significant changes in surface roughness after irradiation are indicative of damage due to gamma-irradiation. The unchanged surface roughness values calculated for microspheres containing PEG in their matrix, suggest that this polymer exerts a protective effect towards gamma-irradiation.

Effect of lysine modification on the secondary structure of ovalbumin.

In order to determine the effect of chemical modification of the epsilon-amino groups on the secondary structure of ovalbumin, we prepared six acetylated (17, 36, 54, 70, 82, and 98%) and four succinylated derivatives (25, 50, 72, and 97%) of the protein. Native ovalbumin and the acylated derivatives were homogeneous as revealed by the electrophoretic pattern. The UV-absorption and fluorescence spectra changed progressively with the extent of modification. However, circular dichroic (CD) studies indicated that acylation of 15 of the 20 lysine residues had little effect on the secondary structure of ovalbumin. Acylation of the remaining five lysine residues resulted in a fairly severe change in the secondary structure. The alpha-helical content decreased from about 31% in the native state to 16.5% in the 97% succinylated ovalbumin and to 21.5% in the 98% acetylated derivative. A comparison of these data with the spectral and hydrodynamic data of Qasim and Salahuddin (1978) suggested that the secondary structure of ovalbumin is more resistant to acylation than is the tertiary structure and, thus, the tertiary and the secondary structures are, to some extent, mutually independent. Raising the pH to 11.2 did not alter the secondary structure of ovalbumin and increasing the ionic strength by more than 20-fold did not reverse the loss of helical structure in 97% succinylated protein. These two observations suggest that the change in secondary structure upon maximal acylation may not only involve electrostatic effects, but also certain other factors, such as steric hindrance due to the entering bulky groups.

Mobile reactive centre of serpins and the control of thrombosis.

Two protease inhibitors in human plasma play a key part in the control of thrombosis: antithrombin inhibits coagulation and the plasminogen activator inhibitor PAI-1 inhibits fibrinolysis, the dissolving of clots. Both inhibitors are members of the serpin family and both exist in the plasma in latent or inactive forms. We show here that the reactive centre of the serpins can adopt varying conformations and that mobility of the reactive centre is necessary for the function of antithrombin and its binding and activation by heparin; the identification of a new locked conformation explains the latent inactive state of PAI-1. This ability to vary conformation not only allows the modulation of inhibitory activity but also protects the circulating inhibitor against proteolytic attack. Together these findings explain the retention by the serpins of a large and unconstrained reactive centre as compared to the small fixed peptide loop of other families of serine protease inhibitors.

Natural and artificial carbohydrate-glued protein aggregates.

Carbohydrate gluing (which may have a carbohydrate-lectin binding mechanism) was first recognized as a major contributor in the supramolecular assembly of annelid giant Hb from the marine-worm P. aibuhitensis. Although this assembly obviously also relies on protein-protein interactions, the authors tested the application of carbohydrate gluing in the assembly of a protein aggregate using a lectin and a carbohydrate-containing protein. The resultant aggregate was a mixture of the protein aggregate and the ingredient proteins. The significance of the method is that the assembly of the aggregate can be controlled by using a hapten sugar. This controllability, in conjunction with newly developing glyco-technology, has great potential for the construction of arbitrary protein molecules into a regular protein aggregate, thereby providing sophisticated functions.

Absence of large-scale conformational change upon limited proteolysis of ovalbumin, the prototypic serpin.

1H and 31P NMR spectroscopies have been used to examine the effects of limited proteolysis with subtilisin Carlsberg on the global conformation of ovalbumin and on the local environment of phosphoserine 344, a residue two positions removed from the site of proteolysis. Such limited proteolysis has been shown to result in excision of a hexapeptide from the region of the protein that, in other serine protease inhibitors (serpins), contains the reactive center. Based on the structure of the related serpin alpha 1-antitrypsin, it has been predicted that phosphoserine 344 should undergo a large change in environment upon proteolysis of ovalbumin (Löbermann, H., Tokuoka, R., Deisenhofer, J., and Huber, R. (1984) J. Mol. Biol. 177, 531-550). Proteolysis of ovalbumin produces a small upfield shift (0.15 ppm) of the 31P resonance of phosphoserine 344. In addition, the pKa of phosphoserine 344 is raised by 0.1 pH unit. At pH 8.5, phosphoserine 344 in cleaved ovalbumin (plakalbumin) is as accessible to hydrolysis by Escherichia coli alkaline phosphatase as it is in native ovalbumin. 1H NMR shows that dephosphorylation of serine 344 has an imperceptible effect on the protein's conformation. Similarly, little effect on conformation is seen by 1H NMR upon proteolysis of ovalbumin. These findings suggest that ovalbumin does not undergo a marked conformational change analogous to that inferred for the related members of the serpin superfamily, alpha 1-antitrypsin and antithrombin III, nor do the residues close to the site of proteolysis appear to change environment from that of an exposed loop to a buried strand of beta-sheet. These findings are not consistent with the hypothesis of Carrell and Owen ((1985) Nature 317, 730-732) for the role of the exposed loop in serpins of directly facilitating conformational change upon cleavage of the loop. Instead, it is proposed that cleavage of the exposed loop alters the solvent accessibility of residues formerly covered by the loop and that this provides the thermodynamic impetus for conformational change, perhaps by disruption of a salt bridge crucial to the integrity of the native structure.

Circular dichroic study of conformational changes in ovalbumin induced by modification of sulfhydryl groups and disulfide reduction.

Sulfhydryl groups of ovalbumin were chemically modified under denaturing conditions in the absence and presence of dithiothreitol, and effects on the secondary structure of the protein were investigated by circular dichroic (CD) measurements. The contents of alpha-helix, beta-structure, and "random coil" (unordered, nonrepetitive structure) were estimated by simulation of the CD spectra and using the parameters established by Chen et al. The principal findings were these: (1) Modification of the four free sulfhydryl groups [with 5,5'-dithiobis(2-nitrobenzoic acid), iodoacetate, or iodoacetamide] caused ovalbumin molecule to unfold partially and to undergo primarily helix-to-beta structure transition. (2) Cleavage of the disulfide bond did not lead to a further conformational change in the sulfhydryl-modified ovalbumin. (3) The remaining helical structure existed in a destabilized state with increased chain flexibility, as the modified protein was very susceptible to denaturation by guanidine and urea. (4) Further evidence for increased chain flexibility was provided by the finding that sodium dodecyl sulfate (SDS) induced helix formation in the sulfhydryl-modified, but not native, ovalbumin. And (5), since both nonreduced and reduced proteins, with their sulfhydryl groups blocked, displayed similar transitions in solutions of guanidine, urea, and SDS suggested that the single disulfide bond did not physically constrain the ovalbumin molecule.

Structure and dynamics of egg white ovalbumin adsorbed at the air/water interface.

The molecular properties of egg white ovalbumin adsorbed at the air/water interface were studied using infrared reflection absorption spectroscopy (IRRAS) and time-resolved fluorescence anisotropy (TRFA) techniques. Ovalbumin adsorbed at the air/water interface adopts a characteristic partially unfolded conformation in which the content of the beta-sheet is 10% lower compared to that of the protein in bulk solution. Adsorption to the interface leads to considerable changes in the rotational dynamics of ovalbumin. The results indicate that the end-over-end mobility of the ellipsoidal protein becomes substantially restricted. This is likely to reflect a preferential orientation of the protein at the interface. Continuous compression of surface layers of ovalbumin causes local aggregation of the protein, resulting in protein-network formation at the interface. The altered protein-protein interactions contribute to the strong increase in surface pressure observed.