Structural evolution of pea-derived albumins during pH and heat treatment studied with light and X-ray scattering.

Pea albumins are found in the side stream during the isolation of pea proteins. They are soluble at acidic pH and have functional properties which differ from their globulin counterparts. In this study, we have investigated the aggregation and structural changes occurring to pea albumins under different environmental conditions, using a combination of size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and small-angle X-ray scattering (SAXS). Albumins were extracted from a dry fractionated pea protein concentrate by precipitating the globulin fraction at acidic pH. The albumins were then studied at different pH (3, 4, 4.5, 7, 7.5, and 8) values. The effect of heating at 90 °C for 1, 3, and 5 min on their structural changes was investigated using SAXS. In addition, size exclusion of the albumins showed 4 distinct populations, depending on pH and heating conditions, with two large aggregates peaks (∼250 kDa): a dimer peak (∼24 kDa) containing predominantly pea albumin 2 (PA2), and a monomer peak of a molar mass of about 12 kDa (PA1). X-ray scattering intensities as a function of q were modeled as polydisperse spheres, and their aggregation was followed as a function of heating time. Albumins was most stable at pH 3, showing no aggregation during heat treatment. While albumins at pH 7.5 and 8 showed aggregation after heating, solutions at pH 4, 4.5, and 7 already contained aggregates even before heating. This work provides new knowledge on the overall structural development of albumins under different environmental conditions, improving our ability to employ these as future ingredients in foods.

Heat-induced agglomeration of water-soluble cod proteins toward gelled structures.

Cod proteins (CPs) have potential applications in designing desirable gel-based products, and this study aimed to unravel their heat-induced aggregation pattern and further probe the roles in protein gels. SDS-PAGE analysis indicated that high-precipitation-coefficient aggregates (HPCAs) of CPs aggregates were composed of considerable polymers of myosin heavy chains and actin, and their low-precipitation-coefficient aggregates (LPCAs) contained myosin light chains and tropomyosin. Studies from correlation analysis between the structure and aggregation kinetics revealed that the generation of ß-sheet and SS bonds were responsible for their spontaneous thermal aggregation induced by heating temperature and protein concentration, respectively. Additionally, as protein denaturation ratio increased, more and larger HPCAs were formed, which was evidenced driving the network formation of protein gels and resulting in higher storage modulus (G') values. These novel findings may be applicable to other animal proteins for better tailoring the manufacturing of muscle gel-based products.

Mechanisms of heat-mediated aggregation behavior of water-soluble cod protein.

Cod proteins (CPs) are considered potential functional ingredients for developing gel-based foods, but present studies on the aggregation behavior of CPs upon heating remain limited. With this respect, the heat-induced aggregation kinetics of CPs at a subunit level was investigated. Based on different centrifugal forces, CPs aggregates were divided into three fractions: large-sized, intermediary-sized, and small-sized aggregates. SDS-PAGE and diagonal SDS-PAGE indicated that myosin heavy chains exhibited a higher affinity with actin to form intermediary-sized and large-sized aggregates; tropomyosin and myosin light chains were hardly engaged in the thermal aggregation and formed small-sized aggregates. The highly-polymerized aggregates adopted considerable transitions of helix-to-sheet in protein structures, whereas the structure of small-sized aggregates featured substantial helix-coil transitions. Furthermore, molecular interactions at different heating stages were revealed. These novel insights might advance our knowledge on the heat-induced aggregation behavior of CPs and provide fundamental information for the application of CPs in gel-based foods.

Impact of the PEG length and PEGylation site on the structural, thermodynamic, thermal, and proteolytic stability of mono-PEGylated alpha-1 antitrypsin.

Conjugation to polyethylene glycol (PEG) is a widely used approach to improve the therapeutic value of proteins essentially by prolonging their body residence time. PEGylation may however induce changes in the structure and/or the stability of proteins and thus on their function(s). The effects of PEGylation on the thermodynamic stability can either be positive (stabilization), negative (destabilization), or neutral (no effect). Moreover, various factors such as the PEG length and PEGylation site can influence the consequences of PEGylation on the structure and stability of proteins. In this study, the effects of PEGylation on the structure, stability, and polymerization of alpha1-antitrypsin (AAT) were investigated, using PEGs with different lengths, different structures (linear or 2-armed) and different linking chemistries (via amine or thiol) at two distinct positions of the sequence. The results show that whatever the size, position, and structure of PEG chains, PEGylation (a) does not induce significant changes in AAT structure (either at the secondary or tertiary level); (b) does not alter the stability of the native protein upon both chemical- and heat-induced denaturation; and (c) does not prevent AAT to fully refold and recover its activity following chemical denaturation. However, the propensity of AAT to aggregate upon heat treatment was significantly decreased by PEGylation, although PEGylation did not prevent the irreversible inactivation of the enzyme. Moreover, conjugation to PEG, especially 2-armed 40 kDa PEG, greatly improved the proteolytic resistance of AAT. PEGylation of AAT could be a promising strategy to prolong its half-life after infusion in AAT-deficient patients and thereby decrease the frequency of infusions.

Tracking aggregation behaviour and gel properties induced by structural alterations in myofibrillar protein in mirror carp (Cyprinus carpio) under the synergistic effects of pH and heating.

The synergistic effect of pH and heating on the structure, aggregation behaviour and gel properties of myofibrillar protein (MP) in mirror carp (Cyprinus carpio) was evaluated. The surface hydrophobicity of the control at pH 5.0 (143.6 ± 0.3 µg) was significantly higher than that of other samples (P < 0.05). Under the same pH conditions, the decrease in total sulfhydryl content of all samples during the heating process demonstrated that covalent/non-covalent cross-linking occurred between proteins due to heat input. Moreover, the decrease in solubility and the increase in turbidity of all samples verified the fact of MP aggregation, and the changes in the elasticity index (EI) and macroscopic viscosity index (MVI) also indicated a decrease in MP fluidity upon heating treatment. Therefore, the aggregation of MP was affected by pH and heating, and the optimal three-dimensional network structure and gel properties could be formed at pH 6.0 and above 70 °C.

Ameliorative effects of L-arginine? On heat-induced phase separation of Aristichthys nobilis myosin are associated with the absence of ordered secondary structures of myosin.

This investigation aimed to study the potential mechanism of L-arginine (L-Arg) on the heat-induced phase separation phenomenon of myosin from the perspective of conformational changes of myosin. L-Arg ameliorated the phase separation of myosin after a two-step heating procedure via suppression of heat-induced aggregation of myosin. The effect of L-Arg on the heating of myosin at high temperatures (75-85 °C) was more pronounced than that in the setting stage (35-45 °C), suggesting that the ameliorative effects of L-Arg on the heat-induced phase separation of myosin are mainly attributed to the inhibition of rod-rod cross-linking between denatured myosin molecules. Additionally, L-Arg without pH modification exhibited an increased ability to suppress the gelation of myosin compared with pH modification, indicating that both pH effects and the particular structure of L-Arg play noticeable roles in the suppression of myosin gelation. Far-UV circular dichroism, intrinsic fluorescence spectroscopy and differential scanning calorimetry demonstrated that L-Arg induced the absence of ordered secondary structures of myosin molecules, especially ß-sheets, and thus generated a looser protein structure, which may represent the dominant suppression mechanisms of L-Arg on the heat-induced aggregation of myosin. This work provided support for the use of L-Arg as a food additive, and the results of this study will be attractive to the meat and beverage products.

Characterization of arginine preventive effect on heat-induced aggregation of insulin.

Aggregation of proteins can affect their efficacy, and is especially important concerning therapeutic proteins such as insulin. Use of additives such as amino acids can counteract this deleterious process. Heat-induced aggregate formation of human insulin was kinetically studied with the use of various concentrations of the protein, at different temperatures, and in the presence of EDTA by UV-visible spectrophotometry. Effect of arginine, lysine, and histidine was then tested on the process at pH 4.8 and 45 °C. Kinetic parameters of the obtained growth curves (parameters t* and t0.5 characterizing the rate of the nucleation stage and the rate of the stage of aggregate growth respectively) were computed in all these conditions, and structure of aggregates was characterized by spectrofluorimetry, and transmission electron microscopy (TEM). Presence of high concentrations of the chelator EDTA increased aggregation. Among used additives, L-arginine (50 mM) most efficiently suppresses the heat-induced amorphous aggregation of insulin, affecting parameters t0.5 and t* presumably by preserving the protein's structure, as observed by the protein intrinsic fluorescence and CD spectra, and smaller formed aggregates in TEM images and dynamic light scattering. Docking experiment and subsequent molecular dynamics simulation indicated possible sites of interaction for arginine with the B-chain of insulin.

Effects of l-arginine and l-histidine on heat-induced aggregation of fish myosin: Bighead carp (Aristichthys nobilis).

This research focused on the effects of l-arginine (l-Arg) and l-histidine (l-His) on the heat-induced aggregation of fish myosin. l-Arg/l-His increased the pH of the myosin solution from 6.82 to 8.74 and 7.24, respectively, and decreased the turbidity, aggregate size, shear modulus, and breaking force. The incorporation of l-Arg/l-His decreased the surface hydrophobicity during setting, but increased it during the two-step heating. The heat-induced aggregation of myosin was suppressed by both amino acids, with the inhibitory effect being greater for l-Arg than l-His. On one hand, the change in the pH played a critical role in suppressing the heat-induced aggregation of myosin. On the other hand, the characteristics of l-Arg/l-His themselves, such as net charges and particular R-groups, were another main contributor to aggregation suppression. Particularly, l-Arg/l-His could interact with exposed aromatic residues of myosin, and the interactions may dominate and overwhelm the burial of aromatic residues during two-step heating.

Influence of L-homoarginine as an analogue of L-arginine on the heat-induced aggregation of proteins.

The influence of l-homoarginine on the heat-induced aggregation of three model proteins, i.e. porcine, mink, and human growth hormones was investigated by circular dichroism spectroscopy. It was found that the effect of l-homoarginine as an analogue of arginine depends on the concentration of the additive as well as the protein itself. l-Homoarginine increased the onset temperature of heat-induced aggregation of both porcine and mink growth hormones. However, the formation of human growth hormone aggregates was increased at low concentrations of l-homoarginine. Only at higher concentrations of the additive was the onset temperature of human growth hormone aggregation found to increase. Additional experiments of human growth hormone melting in the presence of histidine, lysine, and sodium chloride were performed. The effect of lysine was similar as in the presence of l-homoarginine. It follows that in protein formulations low concentrations of amino acids should be used with some precaution. At low concentration of additive, depending on the charge of both protein and amino acid used, the promotion of aggregation of unfolding intermediates may occur.

ß-Lactoglobulin heat-induced aggregates as carriers of polyunsaturated fatty acids.

The aim of this work was to obtain heat-induced ß-lactoglobulin (BLG) aggregates in order to test them as carriers of a model polyunsaturated fatty acid (PUFA), linoleic acid (LA). BLG aggregates were obtained at 85 °C by varying the heating time (0-60 min) and pH of protein dispersion (6.5-7.5). Aggregates were characterised by intrinsic and extrinsic fluorescence and surface hydrophobicity (S0). Binding experiments were conducted by fluorescence spectroscopy. Results showed increased BLG aggregate S0 values which could strongly depend on the pH of aggregate formation. Aggregates obtained at pH 6.5 showed the greatest S0 values, so they could find application as LA carriers. Nevertheless, conjugation of LA to BLG aggregates showed complex behaviour depending on the aggregate producing conditions (pH, heating time and/or combination). The LA binding properties of BLG aggregates were not linked to their hydrophobic characteristics, suggesting that conjugation could require the structural preservation of the LA binding site.

Formation of heat-induced cottonseed congossypin(7S) fibrils at pH 2.0.

BACKGROUND: Heat-induced protein aggregation is important for the texture of various food products. Many types of food proteins have been found to assemble into fibrillar structures under certain conditions. We studied fibril formation of cottonseed 7S storage protein upon heating (for 0-720 min) at 90°C and pH 2.0, investigated the conversion rate, and determined the extent of thermal aggregation. RESULTS: Thioflavin-T fluorescence and Congo-red analysis indicated the formation of amyloid-like fibrils upon heating. Centrifugal filtration indicated that the conversion was very low (<10%) until congossypin concentration up to 2 mg mL(-1), and the conversion increases with increasing heating time, but levels off after longer heating times. Dynamic light scattering and atomic force microscopy showed that the extent of thermal aggregation at pH 2.0, or contour length of the worm-like and fine-stranded aggregates, progressively increased with increasing heating time. Furthermore, reducing electrophoresis analyses indicated that progressive polypeptide hydrolysis occurred upon heating. Experiments indicate that congossypin can form heat-induced amyloid-like aggregates and the conversion of congossypin monomers into fibrils increased with heating time and protein concentration. CONCLUSION: The results would be of vital importance for the utilisation of cottonseed proteins to produce thermally induced fibrillar gels with excellent properties.

Thermal denaturation and aggregation of myosin subfragment 1 isoforms with different essential light chains.

We compared thermally induced denaturation and aggregation of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different "essential" (or "alkali") light chains, A1 or A2. We applied differential scanning calorimetry (DSC) to investigate the domain structure of these two S1 isoforms. For this purpose, a special calorimetric approach was developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins. Using this approach, we revealed two calorimetric domains in the S1 molecule, the more thermostable domain denaturing in two steps. Comparing the DSC data with temperature dependences of intrinsic fluorescence parameters and S1 ATPase inactivation, we have identified these two calorimetric domains as motor domain and regulatory domain of the myosin head, the motor domain being more thermostable. Some difference between the two S1 isoforms was only revealed by DSC in thermal denaturation of the regulatory domain. We also applied dynamic light scattering (DLS) to analyze the aggregation of S1 isoforms induced by their thermal denaturation. We have found no appreciable difference between these S1 isoforms in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). Under these conditions kinetics of this process was independent of protein concentration, and the aggregation rate was limited by irreversible denaturation of the S1 motor domain.