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.

Detection and characterization by circular dichroism of a stable intermediate state formed in the thermal unfolding of papain.

The thermal unfolding of papain was studied at pH 2.6 by means of circular dichroism and difference spectroscopy. The transition curves obtained from ellipticity changes at 208 and 220 nm were biphasic, i.e., they showed two distinct successive steps, demonstrating the existence of an intermediate state of stable secondary conformation in the denaturation process. Difference-spectroscopy studies indicated that considerable exposure of aromatic side-chains is involved in both steps of the transition. Since papain has two domains in its molecular structure, our results suggest that they unfold in a successive way and rather independently. Furthermore, the structural characteristics of the intermediate state, obtained from its circular dichroism spectrum in the far-ultraviolet region, seem to point out that the second domain (residues 111-212) is the most stable part of the molecule.

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.

Circular dichroism of cysteine proteinases from papaya latex. Evidence of differences in the folding of their polypeptide chains.

Two forms of proteinase omega were isolated from a commercial preparation of chymopapain (EC 3.4.22.6) by means of cation-exchange liquid chromatography. Their circular dichroism (CD) spectra in the 182-320 nm region indicated that the two forms possess closely related structures. For comparison, we also recorded the CD spectra of chromatographically purified samples of papain (EC 3.422.2) and the most abundant form of chymopapain. According to the qualitative criteria proposed by Manavalan and Johnson (1983) Nature 305, 831-832), the spectral characteristics of papain correctly indicate that this protein belongs to the alpha + beta class. Proteinase omega is also placed in the alpha + beta category, while chymopapain seems to be an alpha/beta protein. Quantitative estimation of secondary structures yielded contents of helices and parallel beta-sheet that were higher in the case of chymopapain. Thus, the results of this work suggest that there are some differences in the folding pattern of chymopapain with respect to the other two proteinases. This proposal seems unexpected when the high amino acid sequence identity among these enzymes is considered.

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.

Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy.

The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods. The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme. From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-sheet, 15% turns and 35% of non-repetitive structures, were estimated. A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23%. The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic. Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure.

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.

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.

Isolation and characterization of a protease from the marine sponge Spheciospongia vesparia.

A protein that showed activity against proteic (casein and hide powder azure) and synthetic (BAEE and HLPA) substrates was isolated from the marine sponge Spheciospongia vesparia. The protease was purified from an aqueous extract by ammonium sulfate precipitation, gel filtration, hydrophobic and HPLC-anion exchange chromatographies. The purified protease showed a single band in SDS-PAGE minigels and had a molecular weight of 29,600, but when submitted to isoelectric focusing it showed 2 bands with isoelectric points of 4.56 and 4.43. Its catalytic action was inhibited by EDTA and 1,10-phenanthroline, so it seemed to be a metalloprotease.

Secondary structure of a crustacean neuropeptide hormone family by means of CD.

Three hormonal neuropeptides have been purified from the sinus gland of the Mexican crayfish Procambarus bouvieri by means of a single-step HPLC method: The molt-inhibiting hormone (MIH) and two isoforms of the crustacean hyperglycemic hormone (CHH-B and CHH-C). Compositional analysis and partial characterization of three neuropeptides revealed such a high degree of homology that we consider them to be members of a family. Circular dichroic spectra of the three neuropeptides showed that the secondary structure of both isoforms of the CHH are very similar, but that there are important differences in secondary structure between MIH and the CHHs, especially in helix content and in disordered regions.

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.

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.

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.

Unfolding behavior of human alpha 1-acid glycoprotein is compatible with a loosely folded region in its polypeptide chain.

The unfolding of human plasma alpha 1-acid glycoprotein (AGP) induced by heat or guanidine hydrochloride was studied under equilibrium conditions. In thermal unfolding, an intermediate state was detected by the appearance of unusual positive difference absorption bands in the 287-295-nm region, which occurred at lower temperatures than the common denaturation bands at 284 and 291 nm. The formation of this intermediate species apparently involves a local conformational change that perturbs the environment of tryptophyl residues, without affecting the secondary structure of the protein as judged from circular dichroism spectra. On the other hand, denaturation of the glycoprotein induced by guanidine hydrochloride seemed to follow a two-state model with no evidence of any intermediate species; however, the analysis of the transition curve indicated that the change in the accessibility to solvent of amino acid residues of AGP upon unfolding is significantly lower than those observed for other proteins. According to these results, it is proposed that part of the polypeptide chain in native AGP, namely, that from residue 122 to the C-terminus, may be "loosely" folded.

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.

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.

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.

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.

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.