Andrés Hernández-Arana

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.

Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins

In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins.

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‐α‐mannopyranoside. When reduced by the polar area change, binding enthalpy values show a minimal dispersion around 100°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. Proteins 29:467–477, 1997.

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.

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.

Purification and characterization of several digestive proteases from the blue abalone, Haliotis fulgens

Abstract Several proteases were found in an aqueous extract from the hepatopancreas of blue abalone. Four, called PH1, PH2, PH3 and PH4, were purified by ammonium sulfate precipitation, low pressure DEAE-Sepharose chromatography and anion exchange HPLC. All these proteases were active on protein substrates (casein and hide powder azure); they showed different activities with synthetic substrates and were inhibited by different compounds. PH1, PH2 and PH3 seemed to be serine-like proteases (PH1, chymotrypsin-like; PH2 and PH3, trypsin-like) while protease PH4 had carboxypeptidase behavior. The purified enzymes showed single bands in SDS-PAGE and IEF minigels showing relative molecular masses of 28,100, 29,500, 32,000 and 30,000, and isoelectric points of 6.3, 3.6, 4.0 and 3.8, respectively. PH1, PH2 and PH3 have common features in their amino acid contents. These enzymes possess different secondary structures as judged from their circular dichroism spectra.

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.

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.

PURIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS OF ASCLEPAIN G FROM ASCLEPIAS GLAUCESCENS H.B.K.

Abstract Ten asclepain forms were found to be present in the latex of milkweed (Asclepias glaucescens H.B.K.) plants. Four of them were purified by high performance liquid chromatography on a cation exchange column, and characterized. Asclepains Ag3, Ag6, Ag7 and Ag8 were isolated as homogeneous proteins of very similar molecular weights (approx. 23 000) with isoelectric points greater than 9.0. These forms possess nearly identical secondary structures, as judged from their circular dichroism spectra. Interestingly, one of these forms (Ag3) markedly differed from the other three forms with respect to its specificity toward ester and amide substrates.

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.