Concentrated Protein Solutions Research Articles

Protein-Protein Interactions in Crowded Cellular Environments

Explicit solvent fully atomistic molecular dynamics simulations of chymotrypsin 2 in concentrated protein solutions are described to better understand the role of protein-protein interactions in crowded cellular environments. The effect of protein crowding on protein stability and self-diffusion is discussed and compared with experimental data. An energetic analysis is presented to quantitatively analyze the components of the free energy of crowding, i.e. the transfer free energy between dilute solvent and crowded environments.

Time dependence of lysozyme adsorption on end-grafted polymer layers of variable grafting density and length.

A combined experimental and theoretical approach establishes the long-lived nature of protein adsorption on surfaces coated with chemically grafted macromolecules. Specifically, we monitor the time dependence of adsorption of lysozyme on surfaces comprising polymer assemblies made of poly(2-hydroxyethyl methacrylate) brushes grafted onto flat silica surfaces such that they produce patterns featuring orthogonal and gradual variation of the chain length (N) and grafting density (σ). We show that in the kinetically controlled regime, the amount of adsorbed protein scales universally with the product σN, while at equilibrium the amount of adsorbed protein is governed solely by σ. Surprisingly, for moderate concentrations of protein in solution, adsorption takes more than 72 h to reach an equilibrium, or steady state. Our experimental findings are corroborated with predictions using molecular theory that provides further insight into the protein adsorption phenomenon. The theory predicts that the universal behavior observed experimentally should be applicable to polymers in poor and theta solvents and to a limited extent also to good solvent conditions. Our combined experimental and theoretical findings reveal that protein adsorption is a long-lived phenomenon, much longer than generally assumed. Our studies confirm the previously predicted important differences in behavior for the kinetic versus thermodynamic control of protein adsorption.

Protein adsorption in three dimensions

Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and initially adsorbed protein. Interphase protein concentration CI increases as VI decreases, resulting in slow reduction in interfacial energetics. Steady state is governed by a net partition coefficient P=(CI/CB). In the process of occupying space within the interphase, adsorbing protein molecules must displace an equivalent volume of interphase water. Interphase water is itself associated with surface-bound water through a network of transient hydrogen bonds. Displacement of interphase water thus requires an amount of energy that depends on the adsorbent surface chemistry/energy. This “adsorption–dehydration” step is the significant free energy cost of adsorption that controls the maximum amount of protein that can be adsorbed at steady state to a unit adsorbent surface area (the adsorbent capacity). As adsorbent hydrophilicity increases, adsorbent capacity monotonically decreases because the energetic cost of surface dehydration increases, ultimately leading to no protein adsorption near an adsorbent water wettability (surface energy) characterized by a water contact angle θ→65°. Consequently, protein does not adsorb (accumulate at interphase concentrations greater than bulk solution) to more hydrophilic adsorbents exhibiting θ<65°. For adsorbents bearing strong Lewis acid/base chemistry such as ion-exchange resins, protein/surface interactions can be highly favorable, causing protein to adsorb in multilayers in a relatively thick interphase. A straightforward, three-component free energy relationship captures salient features of protein adsorption to all surfaces predicting that the overall free energy of protein adsorption ΔGadso is a relatively small multiple of thermal energy for any surface chemistry (except perhaps for bioengineered surfaces bearing specific ligands for adsorbing protein) because a surface chemistry that interacts chemically with proteins must also interact with water through hydrogen bonding. In this way, water moderates protein adsorption to any surface by competing with adsorbing protein molecules. This Leading Opinion ends by proposing several changes to the protein-adsorption paradigm that might advance answers to the three core questions that frame the “protein-adsorption problem” that is so fundamental to biomaterials surface science.

Influence of β-lactoglobulin and β-casein on Listeria innocua inactivation by pulsed light

The effect of β-lactoglobulin and β-casein on the pulsed light (PL) inactivation of Listeria innocua was evaluated. For low protein concentrations (β-lactoglobulin and β-casein up to 10 mg/mL), the lowest fluences applied (0.2 J/cm(2)) induced more than 7 Log reductions in cell counts. However, higher fluences were needed to induce similar reduction in L. innocua counts when this bacterium was suspended in solutions of higher protein concentration. The fluence required to induce similar microbial inactivation was lower in β-casein than in β-lactoglobulin solutions. For all protein solutions, the inactivation curves followed first order kinetics. The specific inactivation rate for L. innocua inactivation in protein solutions depended on the quantity of light transmitted in the range 230-290 nm by protein solutions. This work shows that PL technology could be used for the decontamination of high protein content solutions like whey or even higher protein concentration solutions.

Exploring the Piezophilic Behavior of Natural Cosolvent Mixtures

Proteins are only marginally stable and are hence very sensitive to environmental conditions, such as high and low temperatures or high hydrostatic pressures. In nature, living organisms are able to compensate for extreme environmental conditions and hence rescue proteins from denaturation by using osmolytes. Organic osmolytes are accumulated under anhydrobiotic, thermal, and pressure stresses. Among those osmolytes are amino acids, polyols and sugars (e.g., glycerol and trehalose), methylamines such as trimethylamine-Noxide (TMAO), and urea. TMAO has been found to enhance protein folding and ligand binding most efficiently. On the other hand, urea, a highly concentrated waste product in mammalian kidneys, is a perturbant. It is also a major organic osmolyte in marine elasmobranch fishes. Interestingly, TMAO has been found to counteract perturbations imposed by urea and hydrostatic pressure in deep-sea animals, most effectively at a 2:1 urea:TMAO ratio. In the deep sea, hydrostatic pressures up to the 1 kbar (100 MPa) range prevail, and living organisms have to cope with such extreme environmental conditions. High hydrostatic pressure generally destabilizes the protein structure, inhibits polymerization of proteins and ligand binding. Interestingly, TMAO has been shown to largely offset these pressure effects. In fact, it was found that the amount of TMAO in the cells of a series of marine organisms increases linearly with the depth of the ocean. For that reason, TMAO is thought to serve as pressure counteractant. The term “piezolyte” has been coined for such kind of cosolute. About the underlying mechanism of stabilization by TMAO at ambient pressure conditions several experimental and theoretical (molecular dynamics simulations) articles have been published in recent years. TMAO is largely excluded from the protein surface and enhances the water structure causing greater organization through more and stronger hydrogen bonding among water molecules. However, the mechanism of this “chemical chaperon” at high hydrostatic pressure (HHP) conditions is still unclear. To yield a deeper understanding of this phenomenon, we determined the intermolecular interaction of dense protein solutions in the absence and presence of cosolvent mixtures of TMAO and urea also under HHP conditions. Small-angle Xray scattering (SAXS) experiments on dense lysozyme solutions have been carried out in the pressure range from 1 bar up to 4 kbar. The SAXS technique accurately monitors structural alterations of the protein solution and yields quantitative information on the state-dependent protein– protein interaction potential. As lysozyme is a highly stable protein, pressure-induced effects will only be attributed to changes in the protein–protein interaction of the native protein and how this is influenced by osmolytes. No pressureinduced unfolding of the protein occurs in the pressure range covered. Complementary thermodynamic data, that is, the temperature of unfolding and the volume change upon unfolding of the protein, were obtained by differential scanning (DSC) and pressure perturbation calorimetry (PPC), respectively. To verify that the protein is folded at all solution conditions studied, SAXS measurements on diluted lysozyme solutions (cP= 10 mgmL ) were carried out in the whole pressure range covered. For diluted protein solutions, the scattering intensity I(q) is proportional to the form factor P(q) (q= (4p/l)sin(V/2) is the wave vector transfer, l the wavelength of the X-rays, and V the scattering angle), which depends on the structure and size of the protein. For dilute lysozyme solutions, the radius of gyration of the particle, Rg, could be determined. We found a constant Rg value of (15.1 0.4) up to 4 kbar, indicating the absence of unfolding even at the highest pressure applied. In the case of concentrated protein solutions, the interaction between the particles gives rise to an additional scattering contribution. This SAXS signal can be described as the product of the form factor and an effective structure factor, which is related to the intermolecular structure factor S(q). To relate the structure factor to the protein–protein interaction potential, statistical mechanical model approaches have to be employed. Here, the mean-spherical approximation (MSA) in combination with the DLVO (Derjaguin– Landau–Verwey–Overbeek) potential V(r) has been used. The pair potential V(r) is given as the sum of a hard sphere potential VHS(r), a repulsive screened Coulomb-like potential VSC(r) and an attractive Yukawian potential VY(r), which is frequently used to describe protein–protein interactions (for details, see the Supporting Information). [*] Y. Zhai, Prof. Dr. R. Winter Fakult t Chemie, TU Dortmund Physikalische Chemie—Biophysikalische Chemie Otto-Hahn Str. 6, 44227 Dortmund (Germany) E-mail: roland.winter@tu-dortmund.de

Protein assembly at the air-water interface studied by fluorescence microscopy.

Protein assembly at the air-water interface (AWI) occurs naturally in many biological processes and provides a method for creating biomaterials. However, the factors that control protein self-assembly at the AWI and the dynamic processes that occur during adsorption are still underexplored. Using fluorescence microscopy, we investigated assembly at the AWI of a model protein, human serum albumin minimally labeled with Texas Red fluorophore. Static and dynamic information was obtained under low subphase concentrations. By varying the solution protein concentration, ionic strength, and redox state, we changed the microstructure of protein assembly at the AWI accordingly. The addition of pluronic surfactant caused phase segregation to occur at the AWI, with fluid surfactant domains and more rigid protein domains revealed by fluorescence recovery after photobleaching experiments. Protein domains were observed to coalesce during this competitive adsorption process.

Label-acquired magnetorotation as a signal transduction method for protein detection: aptamer-based detection of thrombin.

This paper presents a new signal transduction method, called label-acquired magnetorotation (LAM), for the measurement of the concentration of proteins in solution. We demonstrate the use of LAM to detect the protein thrombin using aptamers, with a limit of detection of 300 pM. LAM is modeled after a sandwich assay, with a 10 μm nonmagnetic "mother" sphere as the capture component and with 1 μm magnetic "daughter" beads as the labels. The protein-mediated attachment of daughter beads to the mother sphere forms a rotating sandwich complex. In a rotating magnetic field, the rotational frequency of a sandwich complex scales with the number of attached magnetic beads, which scales with the concentration of the protein present in solution. This paper represents the first instance of the detection of a protein using LAM.

Characterization of N-acetyltryptophan degradation products in concentrated human serum albumin solutions and development of an automated high performance liquid chromatography–mass spectrometry method for their quantitation

N-acetyltryptophan (NAT) has long been used as a stabilizer in some protein solutions, such as human serum albumin, to prevent oxidative protein degradation. However, the fate of NAT has not been discussed in literature. Two NAT degradation products have been observed in concentrated albumin solutions (20% and 25%) and identified as 1-acetyl-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid and 1-acetyl-3a,8a-dihydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid. To monitor the levels of these two previously unidentified NAT degradation products in concentrated albumin solutions, a fully automated method, incorporating online size exclusion chromatography (SEC) trapping and reversed-phase high performance liquid chromatography–mass spectrometry (HPLC–MS) with multiple reaction monitoring (MRM) analysis, has been developed and validated for their quantitative analysis. The method does not require an internal standard. The only sample manipulation is to obtain an albumin concentration of 4% in all standards and test HPLC samples. A limit of quantitation (LOQ) as low as 20 ng/mL has been achieved for both compounds. This method can readily be adopted for the quantitative determination of other small molecules in concentrated protein solutions.

Equilibrium clusters in concentrated lysozyme protein solutions

We have studied the structure of salt-free lysozyme at 293K and pH 7.8 using molecular simulations and experimental SAXS effective potentials between proteins at three volume fractions, ϕ=0.012, 0.033, and 0.12. We found that the structure of lysozyme near physiological conditions strongly depends on the volume fraction of proteins. The studied lysozyme solutions are dominated by monomers only for ϕ⩽0.012; for the strong dilution 70% of proteins are in a form of monomers. For ϕ=0.033 only 20% of proteins do not belong to a cluster. The clusters are mainly elongated. For ϕ=0.12 almost no individual particles exits, and branched, irregular clusters of large extent appear. Our simulation study provides new insight into the formation of equilibrium clusters in charged protein solutions near physiological conditions.

Structural Basis of Rnd1 Binding to Plexin Rho GTPase Binding Domains (RBDs)

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.

A comparison between semi-theoretical and empirical modeling of cross-flow microfiltration using ANN

The applicability of semi-empirical and artificial neural network (ANN) modeling techniques for predicting the characteristics of a microfiltration system was assessed. Flux decline under various operating parameters in cross-flow microfiltration of BSA (bovine serum albumin) was measured. Two hydrophobic membranes were used: PES (polyethersulfone) and MCE (mixed cellulose ester) with average pore diameters of 0.22 μm and 0.45 μm, respectively. The experiments were carried out to investigate the effect of protein solution concentration and pH, trans-membrane pressure (TMP), cross-flow velocity (CFV), and membrane pore size on the trend of flux decline and membrane rejection at constant trans-membrane pressure and ambient temperature. Subsequently, the experimental flux data were modeled using both classical pore blocking and feed forward ANN models. Semi-empirical models based on classic mechanisms of fouling have been proposed. It was shown that these mechanisms could predict the microfiltration flux for a specified period of processing time; while through appropriate selection of ANN parameters such as the network structure and training algorithm, the ANN-based models are competent in modeling membrane filtration systems for all operating conditions and the entire filtration time with desired accuracy.

Protein adsorption and transport in cation exchangers with a rigid backbone matrix with and without polymeric surface extenders

We compare the properties and protein adsorption characteristics of two polymeric cation exchangers: UNOsphere S, which has an open macroporous architecture, and Nuvia S, which is based on a very similar backbone matrix but contains sulfonated polymeric surface extenders. A monoclonal IgG and lysozyme were used as model adsorbates. The characteristic pore sizes, determined by inverse size exclusion chromatography, were about 140 nm for UNOsphere S, and only about 10 nm for Nuvia S, indicating that the polymeric extenders occupy a substantial portion of the base matrix pores. Greater exclusion limits were found for Nuvia S in 1 M NaCl and for a similar matrix containing uncharged surface extenders, suggesting that the polymeric extenders collapse partially at high ionic strength or when they are uncharged. Large equilibrium binding capacities were obtained for Nuvia S, approaching 320 ± 10 mg/mL of particle volume for both proteins in comparison with the UNOsphere S values of 170 ± 10 and 120 ± 10 mg/mL for lysozyme and IgG, respectively. Much higher adsorption rates were also found for Nuvia S, and the rate was nearly independent of protein concentration in solution. Confocal laser scanning microscopy showed very sharp intraparticle protein concentration profiles for UNOsphere S, consistent with a pore diffusion mechanism but diffuse concentration profiles for Nuvia S, consistent with a solid diffusion mechanism. The improved capacity and transport afforded by the polymeric extenders provide substantial potential benefits for bioprocess applications without sacrificing the desirable flow properties of the backbone matrix.

The Potential of Different Saline Solution on the Extraction of the Moringa oleifera Seed's Active Component for Water Treatment

The main objective of the present work is to study the efficiency in terms of removing color and turbidity of raw water in order to obtain drinking water. For this purpose, different coagulant solutions were obtained utilizing different salts, NaCl, KCl and MgCl2, in concentration 1M, distilled water combined with the Moringa oleifera Lam seed. Each coagulant solution obtained was studied with concentrations ranging from 25 to 300 mg/L of Moringa in salt solution. The tests were performed in the “Jar Test,” and the efficiency of the process was assessed in terms of color and turbidity. The results show no difference in the coagulation for extracts using the salts KCl, NaCl and MgCl2 1 Molar. The best results were found employing the coagulant solutions extracted with salt, in various concentration ranges (125 to 300 mg/L); as the concentration of protein in solution becomes higher, the greater is its power as a coagulant. The lowest content of protein was found in the solution extracted with water, which consequently had the lowest values of color and turbidity removal. Finally, the results obtained by the present work show that the seed of Moringa oleifera Lam is a great alternative for use as a coagulant in drinking water treatment systems.

A model for fouling of plate-and-frame heat exchangers in food industry

Fouling of plate-and-frame heat exchangers is considered a major problem in food industries. The presence of microorganisms creates the problem. They grow inside the microchannels of heat exchanger's plates. This leads to a rise in the pressure drop across the exchanger and consequently costs billions of dollars every year due to cleaning operations and even sometimes need replacement of exchanger. This article demonstrates the effects of major factors in fouling formation including fluid velocity, temperature and particulate concentration in plate-and-frame heat exchangers used in milk industry. The variation of the overall heat transfer coefficient is determined after fouling formation. Then, to demonstrate the heat and hydraulic effects in a plate heat exchanger, the concept of ‘fouling threshold model’ which was developed for quantifying and mitigating fouling in crude oil processing is extended to model the fouling of 1% Whey protein concentration (WPC) solution. Using this model, curves for fouling threshold of milk are determined and therefore the sensitive exchangers prone to fouling can be identified easily for plant engineers to cleaning program. The predicted values given by the proposed model are compared with experimental results with quite good agreement. In addition, the effects of fouling are added to the model through the overall heat transfer coefficient correlation. © 2011 Curtin University of Technology and John Wiley & Sons, Ltd.

Structure-Function Analysis of Grass Clip Serine Protease Involved in Drosophila Toll Pathway Activation

Grass is a clip domain serine protease (SP) involved in a proteolytic cascade triggering the Toll pathway activation of Drosophila during an immune response. Epistasic studies position it downstream of the apical protease ModSP and upstream of the terminal protease Spaetzle-processing enzyme. Here, we report the crystal structure of Grass zymogen. We found that Grass displays a rather deep active site cleft comparable with that of proteases of coagulation and complement cascades. A key distinctive feature is the presence of an additional loop (75-loop) in the proximity of the activation site localized on a protruding loop. All biochemical attempts to hydrolyze the activation site of Grass failed, strongly suggesting restricted access to this region. The 75-loop is thus proposed to constitute an original mechanism to prevent spontaneous activation. A comparison of Grass with clip serine proteases of known function involved in analogous proteolytic cascades allowed us to define two groups, according to the presence of the 75-loop and the conformation of the clip domain. One group (devoid of the 75-loop) contains penultimate proteases whereas the other contains terminal proteases. Using this classification, Grass appears to be a terminal protease. This result is evaluated according to the genetic data documenting Grass function.

Integrated forward osmosis–membrane distillation (FO–MD) hybrid system for the concentration of protein solutions

For the first time, an integrated forward osmosis–membrane distillation (FO–MD) hybrid system has been demonstrated for the concentration of protein solutions, specifically a bovine serum albumin (BSA) solution. A hydrophilic polybenzimidazole (PBI) nanofiltration hollow fiber membrane and a hydrophobic polyvinylidene fluoride-polytetrafluoroethylene (PVDF-PTFE) hollow fiber membrane were fabricated and employed in the FO and MD processes, respectively. A concentrated NaCl solution was employed as the draw solute to dehydrate proteins in FO, while distillate water is a by-product during the re-concentration of diluted NaCl draw solution in MD. To determine suitable operating conditions for the hybrid system, independent characterizations were carried out for both FO and MD processes using different NaCl concentrations as draw solutes in FO and different feed temperatures in MD. It was found that the integrated system is stable in continuous operation when the dehydration rate across the FO membrane is the same as the water vapor rate across the MD membrane. Simple mathematical models consistent with the experimental results were also developed for the FO and the FO–MD hybrid systems. The newly developed FO–MD hybrid system is promising for the concentration of pharmaceuticals/protein solutions in the foreseeable future.

Effects of arginine on heat‐induced aggregation of concentrated protein solutions

Arginine is one of the commonly used additives to enhance refolding yield of proteins, to suppress aggregation of proteins, and to increase solubility of proteins, and yet the molecular interactions that contribute to the role of arginine are unclear. Here, we present experiments, using bovine serum albumin (BSA), lysozyme (LYZ), and β-lactoglobulin (BLG) as model proteins, to show that arginine can enhance heat-induced aggregation of concentrated protein solutions, contrary to the conventional belief that arginine is a universal suppressor of aggregation. Results show that the enhancement in aggregation is caused only for BSA and BLG, but not for LYZ, indicating that arginine's preferential interactions with certain residues over others could determine the effect of the additive on aggregation. We use this previously unrecognized behavior of arginine, in combination with density functional theory calculations, to identify the molecular-level interactions of arginine with various residues that determine arginine's role as an enhancer or suppressor of aggregation of proteins. The experimental and computational results suggest that the guanidinium group of arginine promotes aggregation through the hydrogen-bond-based bridging interactions with the acidic residues of a protein, whereas the binding of the guanidinium group to aromatic residues (aggregation-prone) contributes to the stability and solubilization of the proteins. The approach, we describe here, can be used to select suitable additives to stabilize a protein solution at high concentrations based on an analysis of the amino acid content of the protein.

Mobility in Amorphous Biomaterials: Microstructure, Temperature, and Interaction Among Components

Mobility in amorphous solid sugars and proteins influences the rates of chemical reactions and physical processes; we have studied the relation between mobility, structure, and rate in amorphous sugar and protein films. Phosphorescence emission and intensity decay from erythrosin B were used to monitor the temperature dependence of mobility in glucose/glycerol films. Glycerol plasticized at both low (mole ratio 1/10 glycerol/glucose) and high (mole ratio 1/2) glycerol content. The IR hydrogen bond bandwidth increased with increasing glycerol, suggesting that the strength of the bond became more widely distributed with added plasticizer. An increase with temperature in the hydrogen bond peak frequency indicated a decrease in the number of hydrogen bonds at higher temperatures. AFM images reveal no evidence for phase separation in the microstructure of films. We have also prepared thin films from zein dissolved in 70% ethanol/water by spreading and drying concentrated protein solutions on quartz slides; films contained 25 % (w/w) glycerol and different contents of the antioxidants propyl gallate and octyl gallate. The oxygen diffusion and mobility of these films were measured using phosphorescence from dispersed erythrosin B. Although both antioxidants increased the mobility of the zein matrix to about the same extent, octyl gallate, but not propyl gallate, dramatically increased the permeability of oxygen. AFM imaging indicated that propyl gallate induced aggregation of zein complexes, which could lead to more condensed film. We used a fluorescein model for photobleaching to test the performance of these antioxidants in the zein films. In this model fluorescein was used to generate singlet oxygen, which then attacks the probe to destroy fluorescence, while antioxidant clears the singlet oxygen to protect fluorescein from being destructed. Results indicate that propyl gallate provided better protection against photobleaching than octyl gallate at low concentrations.

Decolorization kinetics of sludge protein solution by 60Co γ-ray irradiation/H2O2 oxidation

In order to carry out decolorization, sludge protein solution, a dark brown close to black solution from activated sludge, was subjected to 60Co γ-ray irradiation in the presence of hydrogen peroxide. UV/Vis spectrophotometric method was used to investigate the effect of H2O2 on the coloration apparent kinetics and rate constants of sludge protein solution under γ-ray irradiation. In addition, the effects of irradiation dose, initial sludge protein solution concentration, and pH value on the decolorization efficiency of sludge protein solution were studied. Results showed that the decolorization apparent kinetics of sludge protein solution was a first-order reaction. The solution decolorization percentage increased with the increase of irradiation dose or the decrease of initial sludge protein solution concentration. The examination results of pH value showed that the sludge protein solution could be more efficiently decolorized in alkaline media than in acid media. Moreover, sensory evaluation and foamability analysis indicated that irradiated samples under H2O2 oxidation showed better sensory score and foamability.

Hollow capsules formed by the self-assembly of caseins on sessile air bubbles

This paper introduces an easy method to produce size-controllable hollow capsules by incubating air bubbles in high concentrated protein solutions. We use the Axis-symmetric drop shape analysis (ADSA) system to keep the bubble size constant as well as to set the initial sizes of objective air bubbles. Dynamic surface tension results indicate that protein molecules start to assemble on those air bubbles surfaces as soon as the air bubbles have been injected into the protein solutions; while “empty bag” like structures come forth when air bubbles begin to shrink after long time incubation. Upon abruptly volume vibration, aged air bubbles exhibit the propensity of resisting the forced dilatation/shrinkage while the fresh air bubbles in the same solution tend to collapse immediately. Parallel ellipsometry experiments confirm that protein molecules are able to form multilayer films at the planer air/water interfaces after such a long time adsorption. Brewster Angel Microscopy results reveal that the aged interfacial protein films are much more rigid than the fresh films, indicating that protein molecules may slowly form cross-link structures over time. We believe that protein molecules assemble a well-organized multilayer shell on air bubble surface after long time incubation; such kind of shells yield the mechanic stability of the air bubbles and inhibit the material exchange across the interfaces of the air bubbles. We hope this report can be a positive inspiration to excite the potential for future development in drug encapsulation techniques.