Bio-based hydrogels prepared by cross-linking of microbial poly(gamma-glutamic acid) with various saccharides

Novel bio-based hydrogels were prepared by cross-linking of microbial poly(gamma-glutamic acid) (PGA) with saccharides such as glucose, maltotriose, and cyclodextrin (CD) in the presence of water-soluble carbodiimide in dimethyl sulfoxide (DMSO) by one-pot synthesis at 25 degrees C for 24 h. The degradation of the gels in alkaline solution (pH 9) at 37 degrees C was also investigated. The PGA gels cross-linked with various neutral saccharides were obtained in relatively high recovery yields by use of a base like 4,4-(dimethylamino)pyridine. The PGA gel cross-linked by glucose showed the highest water absorption of 3000 g/g. The PGA gels cross-linked by CDs showed higher water absorption than those cross-linked by the corresponding linear saccharides. It was revealed that the water absorption of the PGA gel was affected by the cross-linker content and also the structure of cross-linkers as they had an effect on the cross-linking density of the PGA gel. The PGA gels were hydrolyzed under alkaline condition (pH 9) at 37 degrees C. The degradation rate was higher when the cross-linker content of the gel was lower.     

Barley pathogenesis-related proteins with fungal cell wall lytic activity inhibit the growth of yeasts.

Proteins from intercellular fluid extracts of chemically stressed barley (Hordeum vulgare L.) leaves were separated by native polyacrylamide gel electrophoresis at alkaline or acid pH. Polyacrylamide gels contained Saccharomyces cerevisiae (bakers' yeast) or Schizosaccharomyces pombe (fission yeast) crude cell walls for assaying yeast wall lysis. In parallel, gels were overlaid with a suspension of yeasts for assaying growth inhibition by pathogenesis-related proteins. The same assays were also performed with proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. In alkaline native polyacrylamide gels, only one band corresponding to yeast cell wall lytic activity was found to be inhibitory to bakers' yeast growth, whereas in acidic native polyacrylamide gels one band inhibited the growth of both yeasts. Under denaturing nonreducing conditions, one band of 19 kD inhibited the growth of both fungi. The 19-kD band corresponded to a basic protein after two-dimensional gel analysis. The 19-kD protein with yeast cell wall lytic activity and inhibitory to both yeasts was found to be different from previously reported barley chitosanases that were lytic to fungal spores. It could be different from other previously reported lytic antifungal activities related to pathogenesis-related proteins.     

In-gel precipitation of enzymatically released phosphate

The phosphate precipitation reaction using ammonium molybdate and triethylamine under low pH has been applied to gel-based assays for detecting phosphate-releasing enzymes. The sensitivity of the assay is 10 pmol Pi/mm2 of 1.5-mm-thick gel. The assay is applicable to enzymes with a wide range of optimal pH, from acid (pH 4.5) to alkaline phosphatase (pH 9.7), and to enzymes that use acid-labile substrates such as apyrase and glutamine synthetase. Using a negative staining approach, maltose phosphorylase, a phosphate-consuming enzyme, can also be detected. The assay was used to detect glutamine synthetase isoforms, separated by nondenaturing polyacrylamide gel electrophoresis from crude maize extracts. For downstream applications such as staining gels for proteins, the gels with precipitate should be incubated in 10 mM dithiothreitol or beta-mercaptoethanol until the precipitate is dissolved and then thoroughly washed in water. In comparison to calcium phosphate precipitation or the phosphomolybdate-malachite green method, this method is more sensitive. It is a very simple, rapid, versatile, reproducible, and inexpensive method that could be a useful tool in enzymological studies.     

Gelation Phenomena Induced by Alkali-alcohol Treatment of 7S and 11S Components in Soybean Globulins

Transparent gels containing about 2% protein were obtained by mixing alkaline dope solution of 7S or 11S soybean proteins with alcohol. The 7S component showed the ability to form a stronger gel than the 11S. This phenomenon depended on pH and alcohol concentration. In 66 % ethanol, the viscosity of the 7S and 11S reached maxima at pH 11.4 and 11.2, respectively. Above these pH levels where further unfolding and dissociation into subunits of the protein molecules occur, the viscosity decreased rather. The effectiveness of alcohol to increase viscosity increased in the order; n-butanol < tert-butanol < n-propanol < iso-propanol < ethanol < methanol. Alcohols having minor hydrophobicity were more effective for increasing viscosity, but ethylene glycol was ineffective. The addition of NaCl or 2-mercaptoethanol to ethanol-mixed alkaline dope solutions resulted in the remarkable increment of the viscosity, especially for the 7S.     

Visualization of acid phosphatase activity on nitrocellulose filters following electroblotting of polyacrylamide gels

A method for visualizing acid phosphatase isoenzymes by activity staining on nitrocellulose filters after electroblotting of proteins fractionated on nondenaturing polyacrylamide gels is described. Reproducible results were obtained when 25 mM Tris-192 mM glycine was used as the transfer buffer instead of 0.7% acetic acid, 50 mM sodium acetate, pH 4, or 0.14 M acetic acid--0.35 M beta-alanine, pH 4.3. Dot-blot analysis of banana fruit extracts on nitrocellulose filters revealed that a minimum of 5 x 10(-3) units (nmol p-nitrophenyl phosphate hydrolyzed g-1.h-1) of acid phosphatase activity can be detected. This method can be suitable for screening a large number of biological samples for monitoring acid phosphatase activity.     

Sodium dodecyl sulfate-gel electrophoresis: staining of polypeptides using heavy metal salts

Water-soluble salts of several heavy metals were examined for their ability to stain polypeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Brief gel exposure (5 min or less) to cobaltous acetate or chlorides of copper, nickel, and zinc produced negatively stained protein patterns that were qualitatively indistinguishable from those of parallel gels stained with Coomassie blue R-250. Protein patterns could be visualized less than 1 min after treatment of gels with zinc chloride; the threshold of detection was estimated at about 10-12 ng protein on standard-size slab gels. Test samples including human erythrocyte membranes, sialoglycoprotein (glycophorin) extracts, and commercial molecular weight protein standards were used to establish the scope of these stains. Protein patterns visualized by the heavy metal salts were compared and contrasted with profiles seen with three widely used silver stains. Proteins from gels treated with copper or zinc chloride could be easily recovered by simple diffusion; this makes feasible both analytical and preparative electrophoretic applications of the staining procedure. A mechanism is proposed to explain the observed protein staining by heavy metal salts.     

Isolation of quercetin's salts and studies of their physicochemical properties and antioxidant relationships

Hydroxyflavones in alkaline solutions show high free radical scavenging activities. Quercetin, one of these hydroxyflavones may be submitted to chemical reactions yielding a mixture of mono-, di- and tri-sodium salts. These salts were recovered after solubilization and stepwise precipitation in methylalcohol/ethylacetate solvents. The different salts were analyzed using sodium emission spectrophotometry and nuclear magnetic resonance to determine the number of acid hydrogens at pH10 and the position of these acid hydrogens. Our study demonstrates that among the three salts of quercetin, the di-sodium compound is endowed with the more efficient scavenging properties in a phosphate buffer at physiological pH7.4. Physicochemical parameters and free hydroxyl radical scavenging activity relationships were also determined, allowing to explain the mechanisms whereby hydroxyl groups exert their radical scavenging activities.     

Fibrillar beta-lactoglobulin gels: Part 3. Dynamic mechanical characterization of solvent-induced systems

Oscillatory shear rheometry has been used to study the gelation of beta-lactoglobulin at ambient in 50% v/v trifluoroethanol (TFE)/pH 7 aqueous buffer and in 50% v/v ethanol (EtOH)/water at pH 2. In contrast to what was found on heating aqueous solutions at pH 2 (Part 2 of this series), a more expected "chemical gelation"-like profile was found with modulus components G' and G' ' crossing over as the gels formed and then with G' ' passing through a maximum. In addition, for the EtOH system, there was a significant modulus increase at long time, suggestive of a more complex two-step aggregation scheme. Modulus-concentration relationships were obtained for both systems by extrapolating cure data to infinite time. For the TFE gels, this data was accurately described by classical branching theory, although it could also be approximated by a constant power--law relationship. Only the latter described the modulus--concentration data for the gels in ethanol, but there were problems here of greater frequency dependence of the modulus values and much less certain extrapolation. Gel times for the TFE systems showed higher power laws in the concentration than could be explained by the branching theory in its simplest form being similar, in this respect, to the heat-set systems at pH 2. Such power laws were harder to establish for the EtOH gels as for these there was evidence of gel time divergence close to a critical concentration. Reduced G'/G'inf versus t/tgel data were difficult to interpret for the gels in ethanol, but for the TFE system they were consistent with previous results for the heat-set gels and approximated master curve superposition. The frequency and temperature dependences of the final gel moduli were also studied. In general, the networks induced by alcohols appeared more flexible than those obtained by heating.     

Effects of Anions on the Thiol-dependent Gelation of Conalbumin

The thiol-dependent gelation of conalbumin was highly dependent on both pH and the buffers used. The hardness of the gels was the maximum at pH 8.0 ~8.8. At pH 8.0, it was several time greater in sodium phosphate buffer than in Tris-HCl buffer. Comparison of the effects of inorganic and organic salts revealed that the difference in gel hardness was caused by the difference in anion species. The hardness of the thiol-dependent gel formed under different anion conditions was inversely correlated with the rate of gel formation. Scanning electron microscopic study showed that the gel formed in sodium phosphate appeared as a sponge-like, uniform network structure, while that in Tris-HCl was coarse.     

Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients

A reproducible high-resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two-dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra-zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra-zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2-8.2 range is now comparable to the widely used acidic ultra-zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra-zoom gels, the depth of analysis of two-dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.     

Identification of human red cell glutamate-pyruvate transaminase (GPT) phenotypes by isoelectric focusing.

Isoenzymes of human red cell glutamate-pyruvate transaminase (GPT) were resolved by isoelectric focusing (IEF) of hemolysates in polyacrylamide gels at pH 5.0-7.0. The bands of enzyme activity required both alpha-ketoglutarate and L-alanine in the staining mixture for visualization, indicating that the bands were not lactate dehydrogenase or glutamate dehydrogenase. Phenotyping of 41 individuals by IEF, including types GPT 1, 2A, 1-2A, 1-2B, and 2A-2B, agreed with the typing results obtained by electrophoresis in starch gels and in polyacrylamide gels at acid and alkaline pH. Analysis of one kindred demonstrated autosomal codominant transmission of the rare GPT*2B gene through 3 generations. IEF facilitates phenotyping by permitting identification of the GPT types on a single gel with a considerable reduction in time and cost. Although no new variants were found in this investigation, IEF may be more powerful for the recognition of presently undetected variants of GPT.     

Biochemical isolation and physiological identification of the egg- laying hormone in Aplysia californica

It has been determined that the bag cells of Aplysia californica produce two polypeptide species that comigrate on electrophoretic gels containing sodium dodecyl sulfate. By this separation procedure both species can be assigned a molecular weight of approximately 6,000. One of these molecules has an Rf of 0.65 on alkaline discontinuous electrophoresis gels, an isoelectric point at pH 4.8, a gel filtration molecular weight of approximately 12,000, and has no known biological function. The other does not enter alkaline disk gels, has an isoelectric point at approximately pH 9.3, shows a gel filtration molecular weight consistent with that determined by SDS gel electrophoresis, and is the egg-laying hormone.     

Effect of physicochemical parameters on the formation of chitosan-based gels

The possibility for forming physical gels based on Pchelozan (bee chitosan with a molecular weight of 230 kDa and an acetylation degree of 26-65%) has been demonstrated. Conditions for obtaining the gels (1% solution in 1% glycolic acid, 25 degrees C, pH 5.5-7.5) were selected. The effects of (1) the concentration of the original solution, (2) the degree of acetylation of Pchelozan, and (3) the value of pH on the process of gel formation, were studied. The gels obtained may be classified with reversible toxotropic systems. These gels are stable for a long time when stored within a temperature range of 18-55 degrees C. The gel with a degree of acetylation of 34% was characterized by irreversible syneresis.     

Preparation of immobilized enzymes by N-vinylpyrrolidone and the general properties of the glucoamylase gel

Immobilized glucoamylase, invertase, and β-galactosidase were prepared by using N-vinylpyrrolidone monomer (VP) under γ-ray irradiation. The enzyme-VP solutions were gelled by irradiation with 2.9 Mrad and the added enzymes were almost completely entrapped. Activity losses on entrapping were 55% for the VP-glucoamylase gel, and more than 90% in the case of VP-invertase and VP-β-galactosidase gels. No leakage of enzyme from these gels could be detected within 1 hr. The VP-glucoamylase gel was capable of hydrolyzing dextrin (mol wt 10,400) to glucose and the glucose equivalent was equal to that obtain able with native enzyme. The optimum temperature, heat stability, pH activity curve, and pH stability of VP-glucoamylase gel were slightly inferior to those of native enzyme, while Km was a little larger than that of native enzyme.     

THE PROTEIN ASSOCIATED WITH HEMOLYSIN IN RABBIT SERUM AND PLASMA

1. The water-insoluble globulin with which hemolysin is associated, may be separated from immune serum or plasma by dilution and simple dialysis at optimal pH. 2. This optimum in plasma is influenced by the presence of the fibrinogen. 3. Fibrinogen carries no immune body, or only an insignificant amount; when present in immune body solutions in other form than fibrin gel, it depresses the hemolytic activity. The conditions for the formation of fibrin gel are similar to those for the formation of a gel by banana protein. 4. The hemolytic activity is a more labile property of the immune protein than the agglutinating activity; hemolysin is destroyed, hemagglutinin shows an apparent increase, as a result of alcohol extraction.     

Studies on chitosan: 2. Solution stability and reactivity of partially N-acetylated chitosan derivatives in aqueous media

The stability of the solutions of partially N-acetylated chitosans was studied by two methods: (1) 1% solutions of the chitosan derivatives in 0.1 M aqueous acetic acid were added dropwise to buffer solutions with pH from 8.6 to 12 and to a 0.1 M NaOH solution; (2) to each 0.5% solution of the derivatives in 0.1 M acetic acid was added the desired amount of a 1 M NaOH solution. The stability data obtained were summarized with respect to the degree of N-acetylation. It was found that the solutions of the derivatives with more than 50% acetyl content were stable even in alkaline conditions and the gelation and precipitation of the solutions did not occur. The reactivity of the derivatives with the degree of N-acetylation of more than 50% was studied using methyl 4-azidobenzoimidate (MABI) and ethylene glycol diglycidyl ether in homogeneous states. It was found that MABI reacted with amino groups of the chitosans only at neutral pH and glycidyl groups reacted at neutral and alkaline pH. It seems that these unique properties of chitosans with a degree of N-acetylation of more than 50% will enable us to prepare new chitosan derivatives.     

Effect of Physicochemical Parameters on the Formation of Chitosan-Based Gels

The possibility for forming physical gels based on Pchelozan (bee chitosan with a molecular weight of 230 kDa and an acetylation degree of 26–65%) has been demonstrated. Conditions for obtaining the gels (1% solution in 1% glycolic acid, 25°C, pH 5.5–7.5) were selected. The effects of (1) the concentration of the original solution, (2) the degree of acetylation of Pchelozan, and (3) the value of pH on the process of gel formation, were studied. The gels obtained may be classified with reversible toxotropic systems. These gels are stable for a long time when stored within a temperature range of 18–55°C. The gel with a degree of acetylation of 34% was characterized by irreversible syneresis.     

Preparation and characterization of physical gels and beads from chitin solutions

A detailed account of physical bulk gel and bead formation from various chitin solutions and nonsolvents is given. Instant gel formation occurs upon contact of chitin solutions in dimethylacetamide (DMAc)/lithium chloride (LiCl) or N-methyl-pyrrolidinone (NMP)/LiCl solvents and nonsolvents such as water, ethanol, or acetone. Ethanol was found to be the optimal nonsolvent for homogeneous spherical bead formation from chitin solutions. Similarly, DMAc-based chitin solutions proved to yield higher quality beads compared to NMP-based solutions. The differences in bead morphology, crystallinity, and thermal degradation are explained in light of the attainment of a balance between attractive hydrogen bonding in the chitin gel network and segment–nonsolvent interactions. The dependence of swelling of chitin gels on pH indicated a maximum of swelling ratio value of 4.3 at pH 11 in aqueous solutions while the equilibrium swelling ratio value of chitin beads formed with ethanol reached a maximum of 2.4. Bulk gels formed under favorable conditions were demonstrated to be recyclable after solvent separation and drying.     

Whey Protein/Arabic Gum Gels Formed by Chemical or Physical Gelation Process

Whey proteins (WP) gelation process with addition of Arabic gum (AG) was studied. Two different driving processes were employed to induce gelation: (1) heating of 12% whey protein isolate (WPI) solutions (w/w) or (2) acidification of previous thermal denatured WPI solutions (5% w/w) with glucono-δ-lactone (GDL). Protein concentrations were different because they were minimal to form gel in these two processes, but denaturation conditions were the same (90 °C/30 min). Water-holding capacity and mechanical properties of the gels were evaluated. The BST equation was used to evaluate the nonlinear part of the stress–strain data. Cold-set gels were weaker than heat-set gels at the pH range near the isoelectric point (pI) of the main whey proteins, but heated gels were more deformable (did not exhibit rupture point) and showed greater elasticity modulus. However, gels formed by heating far from the pI (pH 6.7 or 3.5) showed more fragile structure, indicating that, in these mixed gels, there are prevailing biopolymers interactions. Cold-set and heat-set gels at pH near or below the WP pI showed strain-weakening behavior, but heated gels at neutral pH showed strong strain-hardening behavior. Such results suggest that differences in stress–strain curve at the nonlinear part of the data could be correlated to structure particularities obtained from different gelation processes.     

Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole), PVIm, gels were prepared by γ-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption–desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.