Plant proteins and their colloidal state

Plant proteins are characterized by a complex colloidal state in their physiological environment. The main reasons are related to the multiple functions of plant proteins as well as the different architectures encountered in the plant cells from various sources. During extraction process to produce ingredients, plant proteins reorganize in several native or denatured colloidal states depending on the energy and the physico–chemical changes applied to the system. In most cases, an equilibrium between the native (soluble monomers or oligomers) and denatured (mostly insoluble) oligomeric/aggregated states is reached. Further, processing of the plant protein ingredients during food production, introducing new hydrophobic phases (e.g., gas, oil), energy (pressure, temperature, shear), and physico–chemical conditions (pH, ionic salts) will lead to a final colloidal state, specific to the structural features of the considered final food product.     

Determination of pore/protein size via electrophoresis and slit sieve model

We used electrophoresis for three purposes: (i) estimation of the mean pore size of polyacrylamide gels via measuring electrophoretic mobility of globular proteins of known sizes in combination with simple sieve (cylindrical and slit) models; (ii) determination of the average size of protein molecules (native or denatured) by the use of the same models; (iii) monitoring the changes in molecular dimensions of proteins in the course of their denaturation. Both models yield results that are in good agreement with those found via the more elaborate techniques (considering the principal differences involved). The approach provides a direct and convenient way of monitoring the variations in protein sizes during the course of their denaturation in gels having a gradient of denaturants, and possibly the number of conformational states involved in the process, a facet that is quite unique and useful. The simpler slit model seems to yield better results in the latter case and is moreover supported by the recently reported data on electrophoresis of DNA molecules through the 1 microm slits of a microbrush matrix made of micropillars arranged in a hexagonal lattice.     

OBSERVATION OF DNA PARTIAL DENATURATION BY ATOMIC FORCE MICROSCOPY

Atomic force microscopy was used to investigate the DNA-cetyltrimethylammonium bromide (CTAB) complexes adsorbed on highly ordered pyrolytic graphite (HOPG). These complexes, at low concentrations, can automatically spread out on the surface of HOPG. The DNA-CTAB complexes display a typically extended structure rather than a globular structure. Partially denaturated DNA produced by binding CTAB to DNA is directly observed by AFM with high resolution. The three-dimensional resolution of partially denaturated DNA obtained by AFM is not available by any other technique at present.     

Dynamic High Pressure Micro-fluidization Effects on Structure and Physico-chemical Properties of Egg White Protein

The authors dealt with the effects of dynamic high pressure micro-fluidization(untreated and 20―160 MPa treated) on the structure and physico-chemical properties of egg white solutions[6%(mass fraction) or 61.2 mg protein/mL]. Micro-fluidization treatment resulted in a decrease in mean particle size,and little change in solubility. It was found that the residual denaturation enthalpy was decreased with the increase of pressure from 20 to 100 MPa and from 120 to 160 MPa,reaching into the maximum at 160 MPa. ...     

Denaturation of Adenosine Deaminase with Urea and Guanidine Hydrochloride

The denaturation effect of urea and guanidine hydrochloride on (lie adenosine deaminase has been investigated spectrophotometrically at the two temperatures of 27 °C and 37 °C at pH = 7.50, phosphate buffer (55 mM). A simple, reversible two stale transition, N ?? D, was used to analyze the denaturation process from which conformational stability was estimated using three different methods, namely, the linear extrapolation method (LEM), Tanford's model (TM), and the denaturant binding method (DBM). A good agreement was observed among these methods. The results from free energy of denaturation at zero concentration of denaturant, ΔG°_H2O, show the fragile conformation for adenosine deaminase molecule.     

Study of cellulase enzymes self-assembly in aqueous-acetonitrile solvent: Viscosity measurements

The present study extends the viscosity measurements performed by Ghaouar et al. [Physica B, submitted for publication.] to study the conformational change of the cellulase enzymes in aqueous-acetonitrile mixture. We aim to investigate: (i) the denaturation process by measuring the specific viscosity for temperatures varying between 25 and 65 °C and acetonitrile concentrations between 0% and 50%, (ii) the enzyme–enzyme interaction by calculating the Huggins coefficient and (iii) the enzyme sizes by following the hydrodynamic radius for various temperatures. The precipitation of cellulases versus acetonitrile concentration is also considered. We show from all physical quantities measured in this work that the precipitation and the denaturation processes of cellulase enzymes exist together.     

Pressure stability of insulin: A fourier transform infrared spectroscopy study

High hydrostatic pressure can induce partially unfolded protein conformations that are highly aggregation prone under conditions that normally favour the native state. We investigated the pressure stability of insulin at pH 2 with Fourier transform infrared spectroscopy. We do not observe any cooperative change up to 13 kbar. All spectral changes in the amide I area are fully reversible and are assumed to arise from the elastic effect of pressure, which causes no conformational changes. Moreover, insulin has no higher temperature-induced aggregation tendency when pressure pre-treated.     

Dominant structural factors for complexation and denaturation of proteins using carboxylic acid receptors

Complexation accompanied by denaturation of protein with synthetic carboxylic acid receptors was investigated, to evaluate the key factors for recognition of proteins. The synthetic receptors used were tetraphenylporphyrin (TPP) derivatives and receptors bearing multiple (2–8) carboxylic acid groups. The complexation behavior was quantified from the absorption in the far UV CD spectrum attributed to the secondary structure of the protein. TPP derivatives bearing multiple carboxylic acid groups in the side chains exhibited higher affinity than other receptors that were smaller and had fewer carboxylic acid groups. As the degree of complexation was influenced by the pH and ionic strength in aqueous solution, electrostatic interaction was one of the most important factors for the recognition of proteins. Complexation was also estimated by observation of fluorescence quenching of the TPP derivatives. The stoichiometry of the complexes between lysozyme and the porphyrins was investigated by quantitative analysis of the denaturation using CD spectra. From the results of Job plots and slope analysis for the amount of denatured protein, formation of 1:1 complexes was confirmed. The equilibrium association constants (K_ass) for lysozyme and the TPP receptors ranged from 0.6 × 10⁶ to 1.1 × 10⁶ M⁻¹. The lytic activity of lysozyme was partially lost in the presence of anionic TPP derivatives, due to complexation and denaturation.     

Structural analysis and binding domain of albumin complexes with natural dietary supplement humic acid

Humic acid, a natural ionic molecule, is rapidly being recognized as one of the crucial elements in our modern diets of the new century. A biophysical protocol utilizing circular dichroism (CD), steady state and time-resolved fluorescence for the investigation of the complexation of the humic acid to the staple in vivo transporter, human serum albumin (HSA), as a model for protein-humic substances, is proclaimed. The alterations of CD and three-dimensional fluorescence suggest that the polypeptide chain of HSA partially folded after complexation with humic acid. The data of fluorescence emission displayed that the binding of humic acid to HSA is the formation of HSA-humic acid complex with an association constant of 10⁴ M⁻¹; this corroborates the fluorescence lifetime measurements that the static mechanism was operated. The precise binding domain of humic acid in HSA has been verified from the denaturation of albumin, hydrophobic ANS displacement, and site-specific ligands; subdomain IIA (Sudlow's site I) was earmarked to possess high-affinity for humic acid. The observations are relevant for other albumin-humic substance systems when the ligands have analogous configuration with humic acid.     

Characterization of BSA unfolding and aggregation using a single-capillary viscometer and dynamic surface tension detector

A dynamic surface tension detector (DSTD) has been equipped with an additional pressure sensor for simultaneous viscosity measurements, as a detector for flow injection analysis. The viscosity measurement is based on a single capillary viscometer (SCV) placed in parallel configuration with the DSTD. The viscometer in the optimized conditions consists of a PEEK capillary (i.d. = 0.25 mm, L = 75 cm) kept at constant temperature using a thermostatic bath, which leads on the two sides to the two arms of a differential piezoelectric pressure transducer with a range of 0-35 psi. The DSTD, described previously, measures the changing pressure across the liquid/air interface of 2 μL drops repeatedly forming at the end of a capillary.SCV performance has been evaluated by measuring dynamic viscosity of water/glycerol mixtures analysed in flow injection and comparing the results with the values reported in the literature. The detection limits of SCV and DSTD, calculated as 3σ of the blank, were 0.012 cP and 0.6 dyn cm⁻¹, respectively.The FI-SCV-DSTD system has been applied to the study of temperature-induced denaturation/aggregation process in bovine serum albumin (BSA).The results have been supported and discussed with respect to BSA conformational analysis performed using Fourier Transform infrared spectroscopy.