Thermal inactivation, denaturation and aggregation processes of papain-like proteases.

The investigation into the behavior of ficin, bromelain,  papain under thermal conditions holds both theoretical and practical significance. The production processes of medicines and cosmetics often involve exposure to high temperatures, particularly during the final product sterilization phase. Hence, it's crucial to identify the "critical" temperatures for each component within the mixture for effective technological regulation. In light of this, the objective of this study was to examine the thermal inactivation, aggregation, and denaturation processes of three papain-like proteases: ficin, bromelain, papain. To achieve this goal, the following experiments were conducted: (1) determination of the quantity of inactivated proteases using enzyme kinetics with BAPNA as a substrate; (2) differential scanning calorimetry (DSC); (3) assessment of protein aggregation using dynamic light scattering (DLS) and spectrophotometric analysis at 280 nm. Our findings suggest that the inactivation of ficin and papain exhibits single decay step which characterized by a rapid decline, then preservation of the same residual activity by enzyme stabilization. Only bromelain shows two steps with different kinetics. The molecular sizes of the active and inactive forms are similar across ficin, bromelain, and papain. Furthermore, the denaturation of these forms occurs at approximately the same rate and is accompanied by protein aggregation.

The Low-Waste Grafting Copolymerization Modification of Chitosan Is a Promising Approach to Obtaining Materials for Food Applications.

Chitosan takes second place of the most abundant polysaccharides naturally produced by living organisms. Due to its abundance and unique properties, such as its polycationic nature, ability to form strong elastic porous films, and antibacterial potential, it is widely used in the food industry and biomedicine. However, its low solubility in both water and organic solvents makes its application difficult. We have developed an environmentally friendly method for producing water-soluble graft copolymers of chitosan and poly (N-vinylpyrrolidone) with high grafting efficiency and a low yield of by-products. By using AFM, SEM, TGA, DSC, and XRD, it has been demonstrated that the products obtained have changed properties compared to the initial chitosan. They possess a smoother surface and lower thermal stability but are sufficient for practical use. The resulting copolymers have a higher viscosity than the original chitosan, making them a promising thickener and stabilizer for food gels. Moreover, the copolymers exhibit an antibacterial effect, suggesting their potential use as a component in smart food packaging.

Underused Marine Resources: Sudden Properties of Cod Skin Gelatin Gel.

The main object of this work was to characterize the structure and properties of laboratory-made fish gelatin from cod skin in comparison with known commercial gelatins of fish and mammalian origin. This is one way we can contribute to the World Food Program and characterize foodstuff resources from alternative natural sources. Our research was based on the combination of an expanded set of complementary physical-chemical methods to study the similarities and distinctions of hydrogels from traditional and novel gelatin sources from underused marine resources. In this work, we have compared the morphology, supramolecular structure and colloid properties of two commercial (mammalian and fish) gelatins with gelatin we extracted from cold-water cod skin in laboratory conditions. The obtained results are novel, showing that our laboratory-produced fish gelatin is much closer to the mammalian one in terms of such parameters as thermal stability and strength of structural network under temperature alterations. Especially interesting are our experimental observations comparing both fish gelatins: it was shown that the laboratory-extracted cod gelatin is essentially more thermally stable compared to its commercial analogue, being even closer in its rheological properties to the mammalian one.

Protein translational diffusion as a way to detect intermolecular interactions.

In this work, we analyze the information on the protein intermolecular interactions obtained from macromolecular diffusion. We have shown that the most hopeful results are given by our approach based on analysis of protein translational self-diffusion and collective diffusion obtained by dynamic light scattering and pulsed-field gradient NMR (PFG NMR) spectroscopy with the help of Vink's approach to analyze diffusion motion of particles by frictional formalism of non-equilibrium thermodynamics and the usage of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloid particles interactions in electrolyte solutions. Early we have shown that integration of Vink's theory with DLVO provides a reliable basis for uniform interpreting of PFG NMR and DLS experiments on concentration dependence of diffusion coefficients. Basic details of theoretical and mathematical procedures and a broad analysis of experimental attestation of proposed conception on proteins of various structural form, size, and shape are presented. In the present review, the main capabilities of our approach obtain the details of intermolecular interactions of proteins with different shapes, internal structures, and mass. The universality of Vink's approach is experimentally shown, which gives the appropriate description of experimental results for proteins of complicated structure and shape.

Ions-Induced Alginate Gelation According to Elemental Analysis and a Combinatorial Approach.

This study considers the potential of elemental analysis of polysaccharide ionotropic gels in elucidating the junction zones for different divalent cations. The developed algorithm ensures the correct separation of contributions from physically adsorbed and structure-forming ionic compounds, with the obtained results scaled to alginate C12 block. Possible versions of chain association into dimers and their subsequent integration into flat junction zones were analyzed within the framework of the "egg-box" model. The application of combinatorial analysis made it possible to derive theoretical relations to find the probability of various types of egg-box cell occurrences for alginate chains with arbitrary monomeric units ratio µ = M/G, which makes it possible to compare experimental data for alginates of different origins. Based on literature data and obtained chemical formulas, the possible correspondence of concrete biopolymer cells to those most preferable for filling by alkaline earth cations was established. The identified features of elemental composition suggest the formation of composite hydrated complexes with the participation of transition metal cations. The possibility of quantitatively assessing ordered secondary structures formed due to the physical sorption of ions and molecules from environment, correlating with the sorption capabilities of Me2+ alginate, was established.

Sulfated Polysaccharides as a Fighter with Protein Non-Physiological Aggregation: The Role of Polysaccharide Flexibility and Charge Density.

Proteins can lose native functionality due to non-physiological aggregation. In this work, we have shown the power of sulfated polysaccharides as a natural assistant to restore damaged protein structures. Protein aggregates enriched by cross-ß structures are a characteristic of amyloid fibrils related to different health disorders. Our recent studies demonstrated that model fibrils of hen egg white lysozyme (HEWL) can be disaggregated and renatured by some negatively charged polysaccharides. In the current work, using the same model protein system and FTIR spectroscopy, we studied the role of conformation and charge distribution along the polysaccharide chain in the protein secondary structure conversion. The effects of three carrageenans (κ, ι, and λ) possessing from one to three sulfate groups per disaccharide unit were shown to be different. κ-Carrageenan was able to fully eliminate cross-ß structures and complete the renaturation process. ι-Carrageenan only initiated the formation of native-like ß-structures in HEWL, retaining most of the cross-ß structures. In contrast, λ-carrageenan even increased the content of amyloid cross-ß structures. Furthermore, κ-carrageenan in rigid helical conformation loses its capability to restore protein native structures, largely increasing the amount of amyloid cross-ß structures. Our findings create a platform for the design of novel natural chaperons to counteract protein unfolding.

Complexation of Bromelain, Ficin, and Papain with the Graft Copolymer of Carboxymethyl Cellulose Sodium Salt and N-Vinylimidazole Enhances Enzyme Proteolytic Activity.

This study investigates the features of interactions between cysteine proteases (bromelain, ficin, and papain) and a graft copolymer of carboxymethyl cellulose sodium salt with N-vinylimidazole. The objective is to understand the influence of this interactions on the proteolytic activity and stability of the enzymes. The enzymes were immobilized through complexation with the carrier. The interaction mechanism was examined using Fourier-transform infrared spectroscopy and flexible molecular docking simulations. The findings reveal that the enzymes interact with the functional groups of the carrier via amino acid residues, resulting in the formation of secondary structure elements and enzyme's active sites. These interactions induce modulation of active site of the enzymes, leading to an enhancement in their proteolytic activity. Furthermore, the immobilized enzymes demonstrate superior stability compared to their native counterparts. Notably, during a 21-day incubation period, no protein release from the conjugates was observed. These results suggest that the complexation of the enzymes with the graft copolymer has the potential to improve their performance as biocatalysts, with applications in various fields such as biomedicine, pharmaceutics, and biotechnology.

Effects of Homogeneous and Heterogeneous Crowding on Translational Diffusion of Rigid Bovine Serum Albumin and Disordered Alfa-Casein.

Intracellular environment includes proteins, sugars, and nucleic acids interacting in restricted media. In the cytoplasm, the excluded volume effect takes up to 40% of the volume available for occupation by macromolecules. In this work, we tested several approaches modeling crowded solutions for protein diffusion. We experimentally showed how the protein diffusion deviates from conventional Brownian motion in artificial conditions modeling the alteration of medium viscosity and rigid spatial obstacles. The studied tracer proteins were globular bovine serum albumin and intrinsically disordered α-casein. Using the pulsed field gradient NMR, we investigated the translational diffusion of protein probes of different structures in homogeneous (glycerol) and heterogeneous (PEG 300/PEG 6000/PEG 40,000) solutions as a function of crowder concentration. Our results showed fundamentally different effects of homogeneous and heterogeneous crowded environments on protein self-diffusion. In addition, the applied "tracer on lattice" model showed that smaller crowding obstacles (PEG 300 and PEG 6000) create a dense net of restrictions noticeably hindering diffusing protein probes, whereas the large-sized PEG 40,000 creates a "less restricted" environment for the diffusive motion of protein molecules.

Phase Behavior of Aqueous Mixtures of Sodium Alginate with Fish Gelatin: Effects of pH and Ionic Strength.

The phase behavior of aqueous mixtures of fish gelatin (FG) and sodium alginate (SA) and complex coacervation phenomena depending on pH, ionic strength, and cation type (Na+, Ca2+) were studied by turbidimetric acid titration, UV spectrophotometry, dynamic light scattering, transmission electron microscopy and scanning electron microscopy for different mass ratios of sodium alginate and gelatin (Z = 0.01-1.00). The boundary pH values determining the formation and dissociation of SA-FG complexes were measured, and we found that the formation of soluble SA-FG complexes occurs in the transition from neutral (pHc) to acidic (pHφ1) conditions. Insoluble complexes formed below pHφ1 separate into distinct phases, and the phenomenon of complex coacervation is thus observed. Formation of the highest number of insoluble SA-FG complexes, based on the value of the absorption maximum, is observed at рHopt and results from strong electrostatic interactions. Then, visible aggregation occurs, and dissociation of the complexes is observed when the next boundary, pHφ2, is reached. As Z increases in the range of SA-FG mass ratios from 0.01 to 1.00, the boundary values of рНc, рHφ1, рHopt, and рHφ2 become more acidic, shifting from 7.0 to 4.6, from 6.8 to 4.3, from 6.6 to 2.8, and from 6.0 to 2.7, respectively. An increase in ionic strength leads to suppression of the electrostatic interaction between the FG and SA molecules, and no complex coacervation is observed at NaCl and CaCl2 concentrations of 50 to 200 mM.

Aggregation of Amyloidogenic Peptide Uperin-Molecular Dynamics Simulations.

Uperin 3.5 is a remarkable natural peptide obtained from the skin of toadlets comprised of 17 amino acids which exhibits both antimicrobial and amyloidogenic properties. Molecular dynamics simulations were performed to study the ß-aggregation process of uperin 3.5 as well as two of its mutants, in which the positively charged residues Arg7 and Lys8 have been replaced by alanine. All three peptides rapidly underwent spontaneous aggregation and conformational transition from random coils to beta-rich structures. The simulations reveal that the initial and essential step of the aggregation process involves peptide dimerization and the formation of small beta-sheets. A decrease in positive charge and an increase in the number of hydrophobic residues in the mutant peptides lead to an increase in the rate of their aggregation.

Ion-Induced Polysaccharide Gelation: Peculiarities of Alginate Egg-Box Association with Different Divalent Cations.

Structural aspects of polysaccharide hydrogels based on sodium alginate and divalent cations Ba2+, Ca2+, Sr2+, Cu2+, Zn2+, Ni2+ and Mn2+ was studied using data on hydrogel elemental composition and combinatorial analysis of the primary structure of alginate chains. It was shown that the elemental composition of hydrogels in the form of freezing dried microspheres gives information on the structure of junction zones in the polysaccharide hydrogel network, the degree of filling of egg-box cells by cations, the type and magnitude of the interaction of cations with alginate chains, the most preferred types of alginate egg-box cells for cation binding and the nature of alginate dimers binding in junction zones. It was ascertained that metal-alginate complexes have more complicated organization than was previously desired. It was revealed that in metal-alginate hydrogels, the number of cations of various metals per C12 block may be less than the limiting theoretical value equal to 1 for completely filled cells. In the case of alkaline earth metals and zinc, this number is equal to 0.3 for calcium, 0.6 for barium and zinc and 0.65-0.7 for strontium. We have determined that in the presence of transition metals copper, nickel and manganese, a structure similar to an egg-box is formed with completely filled cells. It was determined that in nickel-alginate and copper-alginate microspheres, the cross-linking of alginate chains and formation of ordered egg-box structures with completely filled cells are carried out by hydrated metal complexes with complicated composition. It was found that an additional characteristic of complex formation with manganese cations is the partial destruction of alginate chains. It has been established that the existence of unequal binding sites of metal ions with alginate chains can lead to the appearance of ordered secondary structures due to the physical sorption of metal ions and their compounds from the environment. It was shown that hydrogels based on calcium alginate are most promising for absorbent engineering in environmental and other modern technologies.

The Ability of Some Polysaccharides to Disaggregate Lysozyme Amyloid Fibrils and Renature the Protein.

The deposition of proteins in the form of insoluble amyloid fibril aggregates is linked to a range of diseases. The supramolecular architecture of such deposits is governed by the propagation of ß-strands in the direction of protofilament growth. In the present study, we analyze the structural changes of hen egg-white lysozyme fibrils upon their interactions with a range of polysaccharides, using AFM and FTIR spectroscopy. Linear anionic polysaccharides, such as κ-carrageenan and sodium alginate, are shown to be capable to disaggregate protofilaments with eventual protein renaturation. The results help to understand the mechanism of amyloid disaggregation and create a platform for both the development of new therapeutic agents for amyloidose treatment, and the design of novel functional protein-polysaccharide complex-based nanomaterials.

Carboxymethyl Cellulose-Based Polymers as Promising Matrices for Ficin Immobilization.

The present work is devoted to research on the interaction between carboxymethyl cellulose sodium salt and its derivatives (graft copolymer of carboxymethyl cellulose sodium salt and N,N-dimethyl aminoethyl methacrylate) with cysteine protease (ficin). The interaction was studied by FTIR and by flexible molecular docking, which have shown the conjugates' formation with both matrices. The proteolytic activity assay performed with azocasein demonstrated that the specific activities of all immobilized ficin samples are higher in comparison with those of the native enzyme. This is due to the modulation of the conformation of ficin globule and of the enzyme active site by weak physical interactions involving catalytically valuable amino acids. The results obtained can extend the practical use of ficin in biomedicine and biotechnology.

Regulation of Intersubunit Interactions in Homotetramer of Glyceraldehyde-3-Phosphate Dehydrogenases upon Its Immobilization in Protein-Kappa-Carrageenan Gels.

Polysaccharides, being biocompatible and biodegradable polymers, are highly attractive as materials for protein delivery systems. However, protein-polysaccharide interactions may lead to protein structural transformation. In the current study, we analyze the structural adjustment of a homotetrameric protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), upon its interactions with both flexible coil chain and the rigid helix of κ-carrageenan. FTIR spectroscopy was used to probe the secondary structures of both protein and polysaccharide. Electrostatically driven protein-polysaccharide interactions in dilute solutions resulted in an insoluble complex formation with a constant κ-carrageenan/GAPDH ratio of 0.2, which amounts to 75 disaccharide units per mole of protein tetramer. Upon interactions with both coiled and helical polysaccharides, a weakening of the intersubunit interactions was revealed and attributed to a partial GAPDH tetramer dissociation. In turn, protein distorted the helical conformation of κ-carrageenan when co-gelled. Molecular modeling showed the energy favorable interactions between κ-carrageenan and GAPDH at different levels of oligomerization. κ-Carrageenan binds in the region of the NAD-binding groove and the S-loop in OR contact, which may stabilize the OP dimers. The obtained results highlight the mutual conformational adjustment of oligomeric GAPDH and κ-carrageenan upon interaction and the stabilization of GAPDH's dissociated forms upon immobilization in polysaccharide gels.

Recent Advances in Protein-Protein Interactions.

Protein-protein interactions (PPIs) lead to formation of complexes and aggregates between a pair or multiple protein molecules [...].

Fibrin-Rhamnogalacturonan I Composite Gel for Therapeutic Enzyme Delivery to Intestinal Tumors.

Therapy of colorectal cancer with protein drugs, including targeted therapy using monoclonal antibodies, requires the preservation of the drug's structure and activity in the gastrointestinal tract or bloodstream. Here, we confirmed experimentally the fundamental possibility of creating composite protein-polysaccharide hydrogels based on non-degrading rhamnogalacturonan I (RG) and fibrin as a delivery vehicle for antitumor RNase binase. The method is based on enzymatic polymerization of fibrin in the presence of RG with the inclusion of liposomes, containing an encapsulated enzyme drug, into the gel network. The proposed method for fabricating a gel matrix does not require the use of cytotoxic chemical cross-linking agents and divalent cations, and contains completely biocompatible and biodegradable components. The process proceeds under physiological conditions, excluding the effect of high temperatures, organic solvents and ultrasound on protein components. Immobilization of therapeutic enzyme binase in the carrier matrix by encapsulating it in liposomes made from uncharged lipid made it possible to achieve its prolonged release with preservation of activity for a long time. The release time of binase from the composite carrier can be regulated by variation of the fibrin and RG concentration.

Novel Biocatalysts Based on Bromelain Immobilized on Functionalized Chitosans and Research on Their Structural Features.

Enzyme immobilization on various carriers represents an effective approach to improve their stability, reusability, and even change their catalytic properties. Here, we show the mechanism of interaction of cysteine protease bromelain with the water-soluble derivatives of chitosan-carboxymethylchitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan, chitosan sulfate, and chitosan acetate-during immobilization and characterize the structural features and catalytic properties of obtained complexes. Chitosan sulfate and carboxymethylchitosan form the highest number of hydrogen bonds with bromelain in comparison with chitosan acetate and N-(2-hydroxypropyl)-3-trimethylammonium chitosan, leading to a higher yield of protein immobilization on chitosan sulfate and carboxymethylchitosan (up to 58 and 65%, respectively). In addition, all derivatives of chitosan studied in this work form hydrogen bonds with His158 located in the active site of bromelain (except N-(2-hydroxypropyl)-3-trimethylammonium chitosan), apparently explaining a significant decrease in the activity of biocatalysts. The N-(2-hydroxypropyl)-3-trimethylammonium chitosan displays only physical interactions with His158, thus possibly modulating the structure of the bromelain active site and leading to the hyperactivation of the enzyme, up to 208% of the total activity and 158% of the specific activity. The FTIR analysis revealed that interaction between N-(2-hydroxypropyl)-3-trimethylammonium chitosan and bromelain did not significantly change the enzyme structure. Perhaps this is due to the slowing down of aggregation and the autolysis processes during the complex formation of bromelain with a carrier, with a minimal modification of enzyme structure and its active site orientation.

κ-Carrageenan Hydrogel as a Matrix for Therapeutic Enzyme Immobilization.

During the last few decades, polysaccharide hydrogels attract more and more attention as therapeutic protein delivery systems due to their biocompatibility and the simplicity of the biodegradation of natural polymers. The protein retention by and release from the polysaccharide gel network is regulated by geometry and physical interactions of protein with the matrix. In the present work, we studied the molecular details of interactions between κ-carrageenan and three lipases, namely the lipases from Candida rugosa, Mucor javanicus, and Rhizomucor miehei-which differ in their size and net charge-upon protein immobilization in microparticles of polysaccharide gel. The kinetics of protein release revealed the different capability of κ-carrageenan to retain lipases, which are generally negatively charged; that was shown to be in line with the energy of interactions between polysaccharides and positively charged epitopes on the protein surface. These data create a platform for the novel design of nanocarriers for biomedical probes of enzymatic origin.

Supramolecular Structure and Mechanical Performance of κ-Carrageenan-Gelatin Gel.

In this work, by means of complex physicochemical methods the structural features of a composite κ-carrageenan-gelatin system were studied in comparison with initial protein gel. The correlation between the morphology of hydrogels and their mechanical properties was demonstrated through the example of changes in their rheological characteristics. The experiments carried out with PXRD, SAXS, AFM and rheology approaches gave new information on the structure and mechanical performance of κ-carrageenan-gelatin hydrogel. The combination of PXRD, SAXS and AFM results showed that the morphological structures of individual components were not observed in the composite protein-polysaccharide hydrogels. The results of the mechanical testing of initial gelatin and engineered κ-carrageenan-gelatin gel showed the substantially denser parking of polymer chains in the composite system due to a significant increase in intermolecular protein-polysaccharide contacts. Close results were indirectly followed from the SAXS estimations-the driving force for the formation of the common supramolecular structural arrangement of proteins and polysaccharides was the increase in the density of network of macromolecular chains entanglements; therefore, an increase in the energy costs was necessary to change the conformational rearrangements of the studied system. This increase in the macromolecular arrangement led to the growth of the supramolecular associate size and the growth of interchain physical bonds. This led to an increase in the composite gel plasticity, whereas the enlargement of scattering particles made the novel gel system not only more rigid, but also more fragile.

The dielectric response of hydrated water as a structural signature of nanoconfined lichen melanins.

Lichens are unique symbiotic organisms from a mutually beneficial alliance of fungi and algae/cyanobacteria that successfully survive extreme temperatures and drought conditions. Most probably such extraordinary vitality of lichens is underlain by melanins, one of the main structural and chemical lichen components, and their mutual relationship with residual water. In this paper, we propose mechanisms, which allow lichens to store up the extra water in their structure. Melanins that are constituents of the cortical lichen layer and presumably contribute to unique water-lichen interactions are chosen for physical experiments in a wide temperature domain. Two melanin pigments extracted from different lichens are studied here - eumelanin from Lobaria pulmonaria and allomelanin from Cetraria islandica. To investigate the inner melanin structure and water-melanin interactions, FTIR and BDS techniques are applied. The BDS technique was used in a wide temperature region of 123-293 K for melanins with various hydration levels. The relaxation processes related to the confinement of supercooled water - in melanins are observed and discussed in details. At medium and high hydration levels, the relaxation process in two melanins of different chemical compositions and supramolecular structures exhibits a well-known crossover that was already observed in many types of confinements. The analysis of FTIR and BDS results helps to clarify the lichen-water interaction processes.