Effect of different treatments on the ability of α-lactalbumin to form nanoparticles.

Nanoparticles of bovine α-lactalbumin (α-LA) prepared by desolvation and glutaraldehyde crosslinking are promising carriers for bioactive compounds in foods. The objective of this work was to study the effect of changes in hydrophobic interactions by using different desolvating agents (acetone, ethanol, or isopropanol) and the use of a heat or high-pressure treatment step before the desolvation process on the size, structure, and properties of α-LA nanoparticles. In all cases, a high average particle yield of 99.63% was obtained. Smaller sizes (152.3 nm) can be obtained with the use of acetone as the desolvating agent and without any pretreatment. This is the first time that α-LA nanoparticles in the size range of 100 to 200 nm have been obtained. These nanoparticles, with an isoelectric point of 3.61, are very stable at pH values >4.8, based on their ζ-potential, although their antioxidant activity is weak. The use of the desolvating agent with the smallest polarity index (isopropanol) produced the largest particles (293.4 to 324.9 nm) in all cases. These results support the idea that controlling hydrophobic interactions is a means to control the size of α-LA nanoparticles. No effect of pretreatment on nanoparticle size could be detected. All types of nanoparticles were easily degraded by the proteolytic enzymes assayed.

Effects of high hydrostatic pressure on the structure of bovine alpha-lactalbumin.

The effects of high hydrostatic pressure (HHP) processing (at 200 to 600 MPa, 25 to 55 degrees C, and from 5 to 15 min) on some structural properties of alpha-lactalbumin was studied in a pH range of 3.0 to 9.0. The range of HHP processes produced a variety of molten globules with differences in their surface hydrophobicity and secondary and tertiary structures. At pH values of 3 and 5, there was a decrease in the alpha-helix content concomitant with an increase in beta-strand content as the pressure increased. No changes in molecular size due to HHP-induced aggregation were detected by sodium dodecyl sulfate-PAGE. All samples showed higher thermostability as the severity of the treatment increased, indicating the formation of a less labile structure related to the HHP treatment.

Thermostability of native and pegylated Myceliophthora thermophila laccase in aqueous and mixed solvents.

A commercial preparation of laccase (EC 1.10.3.2), cloned from Myceliophthora thermophila and expressed in Aspergillus oryzae (MtL), was purified and modified by conjugation with poly(ethylene glycol) (M(r) = 5000) and is labeled PEG-MtL. Native enzyme was found to have a molecular mass of 80 kDa, as determined by gel filtration, and 110 kDa, by SDS-PAGE. The oxidative dimerization of 2,6-dimethoxyphenol (DMP) to produce the corresponding dibenzoquinone was catalyzed by MtL in a manner comparable to that for a diffusion-controlled reaction (k(cat)/K(M) approximately = 10(8) M(-)(1) s(-)(1) and E(a) approximately = 18 kJ M(-)(1)). PEG-MtL was found, by TNBS titration, to have blocked 54% of lysine groups; its hydrodynamic and charge properties were different from those of MtL. Catalytic efficiency (k(cat)/K(M)) of PEG-MtL was similar to that of MtL with DMP as substrate; however, k(cat)/K(M) was 2-fold reduced for the reaction in which 2',2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is oxidized to form a radical cation. E(a) values were similar in both enzyme preparations when assayed in buffered solutions. Far-UV CD spectra were similar for MtL and PEG-MtL and consistent with a protein rich in beta-sheet structure with negligible content of alpha-helices. A blue shift of near-UV CD spectrum for PEG-MtL as compared to MtL was consistent with the decreased polarity of the tyrosyl side chains upon PEG conjugation. Also the blue band of the copper active site was shifted from lambda approximately 610 nm (MtL) to lambda approximately 575 nm (PEG-MtL). Scanning microcalorimetry showed small denaturation enthalpies (6.3 and 7.5 J g(-)(1) for MtL and PEG-MtL, respectively), indicating the high stability of the beta-sheet folding pattern of laccases. However, PEG-MtL proved to be more stable, its half-denaturation temperature being 2 degrees C higher than that of MtL. In 30% alcohol, pegylated laccase showed slower enzyme-activity decay rates than the unmodified enzyme; this behavior was caused by a decrease in the activation entropy of the denaturation reaction. Results can be explained by entropic stabilization by PEG conjugation because of the restricted motion of some surface amino acid side chains, which results in a more stable active site.

Temperature-induced denaturation and renaturation of triosephosphate isomerase from Saccharomyces cerevisiae: evidence of dimerization coupled to refolding of the thermally unfolded protein.

The thermal denaturation of the dimeric enzyme triosephosphate isomerase (TIM) from Saccharomyces cerevisiae was studied by spectroscopic and calorimetric methods. At low protein concentration the structural transition proved to be reversible in thermal scannings conducted at a rate greater than 1.0 degrees C min(-1). Under these conditions, however, the denaturation-renaturation cycle exhibited marked hysteresis. The use of lower scanning rates lead to pronounced irreversibility. Kinetic studies indicated that denaturation of the enzyme likely consists of an initial first-order reaction that forms thermally unfolded (U) TIM, followed by irreversibility-inducing reactions which are probably linked to aggregation of the unfolded protein. As judged from CD measurements, U possesses residual secondary structure but lacks most of the tertiary interactions present in native TIM. Furthermore, the large increment in heat capacity upon denaturation suggests that extensive exposure of surface area occurs when U is formed. Above 63 degrees C, reactions leading to irreversibility were much slower than the unfolding process; as a result, U was sufficiently long-lived as to allow an investigation of its refolding kinetics. We found that U transforms into nativelike TIM through a second-order reaction in which association is coupled to the regain of secondary structure. The rate constants for unfolding and refolding of TIM displayed temperature dependences resembling those reported for monomeric proteins but with considerably larger activation enthalpies. Such large temperature dependences seem to be determinant for the occurrence of kinetically controlled transitions and thus constitute a simple explanation for the hysteresis observed in thermal scannings.

Stereochemical metrics of lectin-carbohydrate interactions: comparison with protein-protein interfaces.

A global census of stereochemical metrics including interface size, hydropathy, amino acid propensities, packing and hydrogen bonding was carried out on 32 x-ray-elucidated structures of lectin-carbohydrate complexes covering eight different lectin families. It is shown that the interactions at primary binding subsites are more efficient than at other subsites. Another salient behavior found for primary subsites was a marked negative correlation between the interface size and the polar surface content. It is noteworthy that this demographic rule is delineated by lectins with unrelated phylogenetic origin, indicating that independent interface architectures have evolved through common optimization paths. The structural properties of lectin-carbohydrate interfaces were compared with those characterizing a set of 32 protein homodimers. Overall, the analysis shows that the stereochemical bases of lectin-carbohydrate and protein-protein interfaces differ drastically from each other. In comparison with protein-protein complexes, lectin-carbohydrate interfaces have superior packing efficiency, better hydrogen bonding stereochemistry, and higher interaction cooperativity. A similar conclusion holds in the comparison with protein-protein heterocomplexes. We propose that the energetic consequence of this better interaction geometry is a larger decrease in free energy per unit of area buried, feature that enables lectins and carbohydrates to form stable complexes with relatively small interface areas. These observations lend support to the emerging notion that systems differing from each other in their stereochemical metrics may rely on different energetic bases.

Structural bases of lectin-carbohydrate affinities: comparison with protein-folding energetics.

We have made a comparative structure based analysis of the thermodynamics of lectin-carbohydrate (L-C) binding and protein folding. Examination of the total change in accessible surface area in those processes revealed a much larger decrease in free energy per unit of area buried in the case of L-C associations. According to our analysis, this larger stabilization of L-C interactions arises from a more favorable enthalpy of burying a unit of polar surface area, and from higher proportions of polar areas. Hydrogen bonds present at 14 L-C interfaces were identified, and their overall characteristics were compared to those reported before for hydrogen bonds in protein structures. Three major factors might explain why polar-polar interactions are stronger in L-C binding than in protein folding: (1) higher surface density of hydrogen bonds; (2) better hydrogen-bonding geometry; (3) larger proportion of hydrogen bonds involving charged groups. Theoretically, the binding entropy can be partitioned into three main contributions: entropy changes due to surface desolvation, entropy losses arising from freezing rotatable bonds, and entropic effects that result from restricting translation and overall rotation motions. These contributions were estimated from structural information and added up to give calculated binding entropies. Good correlation between experimental and calculated values was observed when solvation effects were treated according to a parametrization developed by other authors from protein folding studies. Finally, our structural parametrization gave calculated free energies that deviate from experimental values by 1.1 kcal/mol on the average; this amounts to an uncertainty of one order of magnitude in the binding constant.

Estimating the degree of expansion in the transition state for protein unfolding: analysis of the pH dependence of the rate constant for caricain denaturation.

The thermal denaturation of caricain (the most alkaline of papain-related proteinases) was studied in acid media. Under all conditions tested, caricain denatured irreversibly following a single first-order reaction that involves simultaneous loss of secondary and tertiary structures. Besides, variation of the rate constant with temperature gave linear Eyring's plots. Thus, despite its irreversibility, this process resembles the kinetics of reversible protein unfolding. Due to the basicity of caricain, all of the carboxylates in the native protein interact with nearby positively charged groups. Then, it may be thought that pK values of titratable sites are mainly influenced by interactions of this type. Accordingly, we set up a simple electrostatic perturbation model, based on charge-charge interactions at distances not larger than 10 A, which reproduces reasonably well the titration curve of native caricain. Because the pH dependence of the activation free energy for unfolding (DeltaG()) can be related to differences in the protonation behavior of the native (N) state with respect to the transition (TS) state, the model was further used to analyze the experimental DeltaG() vs pH curve. Results from this analysis suggest that there is an increase of about 3 A in the average ion-pair distance when N globally expands to form TS. Alternatively, if the expansion were restricted to only one molecular domain, the structure of this domain in TS would be highly disordered. In either case, it is probable that the solvent-accessible area augments significantly during the expansion.

pH dependence of the activation parameters for chymopapain unfolding: influence of ion pairs on the kinetic stability of proteins.

We studied the irreversible thermal denaturation of chymopapain, a papain-related cysteine proteinase. It was found that this process follows simple first-order kinetics under all conditions tested. Rate constants determined by monitoring ellipticity changes at 220 or 279 nm are essentially identical, indicating that denaturation involves global unfolding of the protein. Enthalpies (DeltaH(double dagger)) and entropies (DeltaS(double dagger)) of activation for unfolding were determined at various pH values from the temperature dependence of the rate constant. In the pH range 1.1-3.0, a large variation of both DeltaH(double dagger) and DeltaS(double dagger) was observed. For the few proteins studied so far (lysozyme, trypsin, barnase) it is known that activation parameters for unfolding vary little with pH. It is proposed that this contrasting behavior of chymopapain originates from the numerous ion pairs - especially those with low solvent accessibilities - present in its molecular structure. In contrast, fewer, more exposed ion pairs are present in the other proteins mentioned above. Our results were analyzed in terms of differences in the protonation behavior of carboxylic groups between the transition (TS) and native (N) states of the protein. For this purpose, a model of independently titrating sites was assumed, which explained reasonably well the pH dependence of activation parameters, as well as the protonation properties of native chymopapain. According to these calculations, pK values of carboxyls in TS are shifted 0.6-0.9 units upwards with respect to those in N. In addition, some groups in TS appear to be protonated with unusually large enthalpy changes.

Spectroscopic and thermodynamic evidence for a complex denaturation mechanism of bovine beta-lactoglobulin A.

We present a spectroscopic and calorimetric study on the thermal denaturation of bovine beta-lactoglobulin (beta-lg) variant A. Spectroscopic data allowed detection of a stable intermediate emerging from structural modifications restricted to local regions of the native molecule. It is suggested that this kind of intermediate could be a common property of lipocalins. Using the same set of parameters that has successfully related thermodynamics and structural properties of other proteins, it is shown that the thermally denatured state of beta-lg retains a significant amount of buried hydrophobic surface area. Thus, despite being a small protein composed of a single structural domain, beta-lg exhibits a complex unfolding mechanism, comprising at least two other species different from the native and completely unfolded states.

New insights into the molecular basis of lectin-carbohydrate interactions: a calorimetric and structural study of the association of hevein to oligomers of N-acetylglucosamine.

Isothermal titration calorimetry was used to characterize thermodynamically the association of hevein, a lectin from the rubber tree latex, with the dimer and trimer of N-acetylglucosamine (GlcNAc). Considering the changes in polar and apolar accessible surface areas due to complex formation, we found that the experimental binding heat capacities can be reproduced adequately by means of parameters used in protein-unfolding studies. The same conclusion applies to the association of the lectin concanavalin A with methyl-alpha-mannopyranoside. When reduced by the polar area change, binding enthalpy values show a minimal dispersion around 100 degrees C. These findings resemble the convergence observed in protein-folding events; however, the average of reduced enthalpies for lectin-carbohydrate associations is largely higher than that for the folding of proteins. Analysis of hydrogen bonds present at lectin-carbohydrate interfaces revealed geometries closer to ideal values than those observed in protein structures. Thus, the formation of more energetic hydrogen bonds might well explain the high association enthalpies of lectin-carbohydrate systems. We also have calculated the energy associated with the desolvation of the contact zones in the binding molecules and from it the binding enthalpy in vacuum. This latter resulted 20% larger than the interaction energy derived from the use of potential energy functions.

Thermal denaturation of glutathione reductase from cyanobacterium Spirulina maxima.

The thermal unfolding of glutathione reductase (NAD[P]H:GSSG oxidoreductase EC 1.6.4.2.) from cyanobacterium Spirulina maxima was monitored by differential scanning calorimetry and circular dichroism at neutral pH. Covalent cross-linking of enzyme at different temperatures revealed dimer as the species undergoing the thermal transition. A single endotherm was observed, but its thermodynamic parameters showed dependence on the scan rate. In the transition zone, aggregation of the dimeric species was observed. Analysis of the enzyme heated at 80 degrees C revealed that the resultant species retained a high content of secondary structure. The addition of low concentrations of guanidinium hydrochloride resulted in a full cooperative thermal transition. A model in which the dimeric protein undergoes a partial unfolding in a kinetically controlled fashion is proposed, such that the experimental value of delta H(cal) results from the simultaneous occurrence of endothermic and exothermic events.

Effect of irreversibility on the thermodynamic characterization of the thermal denaturation of Aspergillus saitoi acid proteinase.

The thermal denaturation of the acid proteinase from Aspergillus saitoi was studied by CD and differential scanning calorimetry (DSC). This process seemed to be completely irreversible, as protein samples that were heated to temperatures at which the transition had been completed and then cooled at 25 degrees C did not show any reversal of the change in the CD signal. Similar results were obtained with DSC. Nevertheless, we were able to detect the presence of reversibly unfolded species in experiments in which the enzyme solution was heated to a temperature within the transition region, followed by rapid cooling at 25 degrees C. Accordingly, the denaturation of behaviour of the acid proteinase seems to be consistent with the existence of one (or more) reversible unfolding transition followed by an irreversible step. The van't Hoff enthalpy, delta HvH, which corresponds to the reversible transition was calculated from extrapolation to infinite heating rate as 310 kJ.mol-1. This parameter was also determined from direct estimation of the equilibrium constant at several temperatures (delta HvH = 176 kJ.mol-1). Comparison of the average delta HvH with the calorimetric enthalpy (delta Hcal. = 770 kJ.mol-1) gave a value of 3.2 for the delta Hcal./delta HvH ratio, indicating that the molecular structure of the enzyme is probably formed by three or four cooperative regions, a number similar to that of the acid proteinase, pepsin. It should be noted that a completely different conclusion would be obtained from a straightforward analysis of the calorimetric curves, disregarding the effect of irreversibility on the denaturation process.

Metal content and conformation of the metalloprotease from the marine sponge Spheciospongia vesparia.

We have recently purified a protease from the marine sponge Spheciospongia vesparia. It consists of a single nonglycosylated polypeptide chain with a molecular weight of 29 600 and has one free thiol group. Metal analysis revealed the presence of zinc at 2.02 +/- 0.05 g-atoms per mole of protein, as measured by atomic absorption spectroscopy. The circular dichroism spectrum in the far UV region (183-259 nm) indicates that the sponge protease contains appreciable amounts of beta sheet. This enzyme resembles very much an aminopeptidase from Aeromonas proteolytica concerning activity and some physiochemical characteristics.

The thermal denaturation of stem bromelain is consistent with an irreversible two-state model.

The thermal denaturation of bromelain, a cysteine proteinase from the papain family, was studied by means of circular dichroism (CD) and differential scanning calorimetry (DSC). It was found that this process is completely irreversible and apparently follows a simple two-state mechanism of the type N-->D. The activation energy, E, that characterizes this reaction was calculated by the use of different approaches: (i) the effect of heating rate on the temperature at which the transition is half completed; (ii) analysis of individual transition curves; (iii) kinetic studies at fixed temperatures; and (iv) single DSC tracings. The obtained values for E were rather similar to one another, varying from 164 to 226 kJ/mol. In comparison, the total calorimetric enthalpy change was 334 kJ/mol. When a more complex mechanism is considered (N<-->U-->D), which takes into account the presence of a reversibly unfolded state (U), our results suggest that the rate-limiting step is precisely the formation of U. Calculation of the corresponding activation enthalpy and entropy also seems to support this proposal.

Denaturing behavior of glutathione reductase from cyanobacterium Spirulina maxima in guanidine hydrochloride.

The influence of guanidine hydrochloride (Gdn-HCl) on glutathione reductase from Spirulina maxima has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, thiol groups accessibility, and gel filtration chromatography. It was found that the denaturation process involves several intermediate states. At low, Gdn-HCl concentrations (Cm = 0.4 M), reductase activity was fully lost. However, below 3 M Gdn-HCl, this inhibition was freely reversible upon removal of the denaturing agent. Gel filtration experiments revealed that this reversible inhibition was not due to dissociation of the tetrameric enzyme. Structural studies strongly suggest that the conformation of this intermediate state is similar to that of native enzyme. A model in which a local region of the polypeptide chain assumes an extended conformation (D. T. Haynie, and E. Freire, Proteins 16,115-140) is proposed for the reversibly inactivated enzyme. Between 3 and 4 M Gdn-HCl (Cm = 3.5), the enzyme activity was irreversibly lost, this inhibition being concomitant with the loss of ellipticity, changes in both wavelength and intensity at the maximum of fluorescence emission, and dissociation of the enzyme into unfolded monomers; these results reveal that gross changes in the protein conformation occur under these conditions. At 4 M Gdn-HCl an equilibrium exists between the denatured forms of dimer and monomer, which is completely shifted toward the unfolded monomers at 5 M Gdn-HCl. Irreversibility in the Gdn-HCl-induced denaturation of S. maxima glutathione reductase was not due to aggregation of the unfolded enzyme.

The thermal unfolding of hevein, a small disulfide-rich protein.

Differential scanning calorimetry was used to study the thermal unfolding of hevein, a 43-residue disulfide-rich protein whose three-dimensional structure has been determined by X-ray diffraction. In the range pH 2.0-3.7 this process was approximately 75% reversible as judged by repeated scans on the same sample. The ratios of van'tr Hoff to calorimetric enthalpies were considerably larger than one, suggesting that intermolecular cooperation is involved in the unfolding of this protein. Alternatively, it is possible that the partial irreversibility of this process may cause distortions of the endotherm that affect the calculation of the van't Hoff enthalpy. Experimental changes in heat capacity and enthalpy were compared with those calculated from polar and nonpolar surface areas buried in the native state. It was found that when the unfolded state is represented as an extended chain without disulfide cross-links, experimental and calculated parameters agree well. However, if the unfolded protein is modeled with the presence of disulfide bridges, the agreement between the two sets of parameters is lost. The entropy change/residue at 112 degrees C is considerably smaller than the average value for globular proteins, thus suggesting that, as expected, disulfide bonds strongly influence the entropy of the unfolded state of this protein.

Circular dichroism studies of acid proteinases from Aspergillus niger and Aspergillus awamori.

Acid proteinases produced by strains of Aspergillus niger and Aspergillus awamori were isolated by means of ethanol precipitation, gel filtration and anion-exchange high resolution chromatography. In each case, the purified proteinase showed a single band in polyacrylamide gel electrophoresis. Their molecular weights were almost identical (approx. 45,000). However, the proteinase from Aspergillus awamori contained 16% of neutral hexoses while the other enzyme (Aspergillus niger) showed negligible amounts of these carbohydrates. Both enzymes displayed circular dichroism spectra that share a number of features with that of penicillopepsin. This suggests that proteinases from Aspergilli possess the structural folding pattern typical of aspartic proteinases. Proteolytic-activity pH optima were different, thus distinguishing one enzyme from another. This variation seems to be related to the particular resistance of the proteinases to acid denaturation, as indicated by changes in their circular dichroism spectra when the pH is decreased.

Circular dichroism of stem bromelain: a third spectral class within the family of cysteine proteinases.

Two forms of stem bromelain (EC 3.4.22.4) were isolated from commercial, crude and chromatographically purified preparations of the enzyme by means of gel-filtration and cation-exchange liquid chromatography. These forms possess nearly identical secondary and tertiary structures, as judged from their circular dichroism (c.d.) spectra. The spectral characteristics of stem bromelain suggest that this enzyme belongs to the alpha + beta protein class, as other cysteine proteinases do. In agreement with these results, quantitative estimation of secondary structures yielded amounts similar to those for papain and proteinase omega. However, the bromelain c.d. curve is clearly distinguishable from those reported for papain and proteinase omega, on one hand, and that of chymopapain, on the other. Thus, it is apparent that there are at least three types of c.d. spectra associated with the family of cysteine proteinases.

Spectrochemical evidence for the presence of a tyrosine residue in the allosteric site of glucosamine-6-phosphate deaminase from Escherichia coli.

The interaction of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli with its allosteric activator, N-acetyl-D-glucosamine 6-phosphate, was studied by different spectrophotometric methods. Analysis of the circular-dichroism differential spectra produced by the binding of the allosteric activator or the competitive inhibitor 2-amino-2-deoxy-D-glucitol 6-phosphate (a homotropic ligand displacing the allosteric equilibrium to the R conformer), strongly suggests the presence of tyrosine residues at or near the allosteric site, although a conformational effect cannot be ruled out. The involvement of a single tyrosine residue in the N-acetyl-D-glucosamine-6-phosphate binding site of glucosamine-6-phosphate deaminase was supported by spectrophotometric pH titrations performed in the presence or absence of the homotropic and heterotropic ligand. In these experiments, a single titrated tyrosine residue is completely protected by saturation with the allosteric activator; this group is considerably acidic (pK 8.75). The analysis of the amino acid sequence of the deaminase using a set of indices for the prediction of surface accessibility of amino acid residues, suggests that the involved residue may be Tyr121 or Tyr254.

Cooperativity in the unfolding transitions of cysteine proteinases. Calorimetric study of the heat denaturation of chymopapain and papain.

Differential scanning calorimetry (DSC) was employed to study the thermal unfolding of chymopapain (EC 3.4.22.6) and papain (EC 3.4.22.2), two highly homologous cysteine proteinases from Carica papaya. Under all pH conditions used, both enzymes showed irreversible thermal denaturation. However, results from experiments performed at two different scanning rates suggest that interpretation of data in terms of equilibrium thermodynamics is not unreasonable. For papain, the ratio of calorimetric (delta Hcal) to van't Hoff (delta HvH) enthalpies approximated to 2.0. This value indicates that papain domains unfold almost independently, as it has been reported previously. In contrast, chymopapain displayed a more cooperative behavior with a delta Hcal to delta HvH ratio of 1.3-1.4. DSC curves were analyzed in terms of a mechanism that includes domain-domain interactions. The results showed a negligible interdomain free energy in the case of papain, but a significant value of approx. 1.0 kcal/mol (1 cal = 4.184 J) for chymopapain. These two proteins also differed in the unfolding heat-capacity change, delta Cp, which suggests that their native structures bury different amounts of nonpolar surface area.