Analysis of the interaction between human plasma fibronectin and gelatin by affinity electrophoresis

The interaction between human plasma fibronectin and gelatin was analyzed by affinity electrophoresis, in which the fibronectin was subjected to electrophoresis in a 4% polyacrylamide gel in the presence and absence of gelatin, as an affinity ligand, and the fibronectin band was stained by an immunoblotting method. The apparent dissociation constants (Kd) of fibronectin for gelatin were calculated from affinity plots based on the original affinity equation at different pHs, urea concentrations, and temperatures. The fibronectin exhibited much lower affinity in the presence of urea. The Kds at 37 degrees C were 1.49 X 10(-7) M, 2.50 X 10(-6) M, and 3.58 X 10(-6) M with 2 M, 3 M, and 4 M urea, respectively. The van't Hoff plots of Kd values against absolute temperature (T) showed that the value of log Kd decreased in proportion to the increase in the value of 1/T within the range of 15-50 degrees C. The standard enthalpy, the standard free energy change at 37 degrees C, and the entropy change at 37 degrees C for association were calculated to be -124.7 kJ/mol, -33.23 kJ/mol, and -295.1 J/mol/deg, respectively. These results suggest that a hydrophilic interaction, such as hydrogen bond or van der Waals interaction, plays an important role in the binding of plasma fibronectin to gelatin.     

Inhibition of blood coagulation factor XIIIa-mediated cross-linking between fibronectin and collagen by polyamines

Soluble fibronectin is found in body fluids and media of cultured adherent cells. Insoluble fibronectin is found in tissue stroma and in extracellular matrices of cultured cells. Fibronectin is a substrate for factor XIIIa (plasma transglutaminase) and can be cross-linked to collagen and to the alpha chain of fibrin. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to investigate the possibility that factor XIIIa-mediated cross-linking is influenced by polyamines. Spermidine inhibited cross-linking between fibronectin and type I collagen, isolated alpha 1 (I) collagen chains, or iodinated cyanogen bromide fragment 7 of alpha 1 (I) chains (125I-alpha 1 (I)-CB7). Half-maximal inhibition of cross-linking between 125I-alpha (I)-CB7 and fibronectin was observed when 0.1 mM spermine or spermidine was present. Spermidine, 0.7 mM, partially inhibited cross-linking between fibronectin and the alpha chain of fibrin but failed to inhibit cross-linking between the fibrin monomers of a fibrin clot. Spermidine also failed to inhibit cross-linking between fibronectin molecules when aggregation of fibronectin was induced with dithiothreitol. In contrast, 0.7 mM monodanyslcadaverine inhibited fibronectin-collagen, fibronectin-fibrin, fibronectin-fibronectin, and fibrin-fibrin cross-linking. Spermidine or spermine, 0.7 mM, enhanced the cross-linking between molecules of partially amidinated fibronectin, suggesting that N1,8-(di-gamma-glutamyl)-polyamine cross-linkages were formed. Spermidine and spermine failed to enhance cross-linking between monomers of amidinated fibrin. These results indicate that physiologic concentrations of polyamines specifically disturb transglutaminase-catalyzed cross-linking between fibronectin and collagen.     

Interactions between collagen chains and fiber formation.

The temperature-dependent dissociation of neutral salt-soluble collagen into its component chains was measured in 0.6-1.6 M urea solutions at pH 7.3. The temperature-dependent association of the same radioactively labeled collagen into fibers was measured in 0-0.4 M urea solutions, pH 7.3. The effect of urea on the temperature, Tm(G), for half dissociation into chains was small, and the value extrapolated to zero urea concentration was 39 degrees C. In contrast, the effect of urea on the temperature, Tm(F), for half association into fibers was large, and the value at zero urea concentration was 30 degrees C. We conclude that while body temperature provides excellent conditions for the matching of collagen chains to form molecules, the conditions are not optimal for the formation of highly ordered fibers. The large effects of 0.1 M urea suggest that other factors in vivo may help to destabilize mismatched molecular association during fiber growth. Alternately this might be facilitated by parts of the extension peptides of procollagen.     

The effect of a functional group in penicillin derivatives on the interaction with bile salt micelles studied by micellar electrokinetic chromatography.

A capillary electrophoresis method is developed and validated for the characterization of the affinity and the interaction between aminopenicillanic acid (APS-H) and its derivatives with bile salt micelles. The micellar systems studied contained sodium taurocholate (NaTC) and sodium deoxycholate (NaDC). Using the retention factor k', functional group selectivity, gammaG, is defined as the ratio of the retention factor of a substituted aminopenicillanic acid (APS-R) over the capacity factor of APS-H and the difference in free energy (deltadeltaG degrees) can be used for the characterization of the affinity and interaction between drugs and micelles. The functional group selectivity is a direct measure of the interaction of the functional group with micelles. The calculated deltadeltaG degrees value gives information on the partition equilibrium and interaction between drugs and micelles. A positive deltadeltaG degrees value means that the interaction of a functional group gammaG to the APS-H leads to a decrease in the interaction with the micelles. A negative deltadeltaG degrees value, on the other hand, has the opposite meaning. The results obtained in this study exhibited that these APS-R compounds have smaller affinity to the micelles compared to unsubstituted APS-H. Furthermore, the log k' of the drugs in these systems were correlated with the log Pow in the n-octanol/water system and with log P(G) (permeation coefficient).     

Preparative isoelectric focusing of reduced wheat gluten proteins

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.     

Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretin

Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70-80 degrees C in neutral to mild alkaline buffers or at 37 degrees C and slightly acidic pH (6-7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis.     

Study on interaction of alpha-amylase from Bacillus subtilis with cetyl trimethylammonium bromide

The interaction of cetyl trimethylammonium bromide (CTAB) with alpha-amylase from Bacillus subtilis was investigated at 25 degrees C and various experimental conditions, such as pH, ionic strength and urea concentration. The binding data were measured using CTAB-membrane selective electrodes as a simple, fast, cheap and accurate method. The obtained binding isotherms were analyzed using Wyman binding potential concept. The results represent the highest binding affinity at 10(-3) M of NaBr respect to other salt concentrations. The less binding affinity at pH 9.7 with respect to pH 6.5 is related to increasing of protein self aggregation with pH. The binding data analysis at various urea concentrations also shows that the predominate unfolding of alpha-amylase occurred in the urea concentration range of 3-5 M.     

Affinity Study of Novel Gelatin Cell Carriers for Fibronectin

In the present work, the gelatin/fibronectin affinity was evaluated using SPR, QCM and radiolabelling. The results indicate that type A gelatin films possess a higher affinity for Fn compared to type B gelatin. This is due to a combined hydrophobic and electrostatic interaction between gelatin type A and Fn. In a second part, the affinity of Fn for porous gelatin scaffolds was evaluated. The scaffolds were prepared by a cryogenic treatment and subsequent freeze‐drying yielding type I and type II scaffolds which possess different pore geometries/sizes. The results indicate that the Fn density on the scaffolds can be fine‐tuned by varying the Fn concentration, the gelatin type (A vs. B), the pore size/geometry (type I vs. type II scaffolds).

     

Preparation of stimuli-responsive protein nanogel by quantum-ray irradiation

To prepare the functional nanoparticle with biological affinity, we tried to control the particle diameter and volume phase transition point of protein nanogel by quantum-ray irradiation. We succeeded in controlling the particle diameter of gelatin nanogel in the range of 20–70 nm by gamma-ray irradiation. It was also found that the prepared gelatin nanogel reversibly swelled and shrunk by pH and temperature change. Volume phase transition point and swelling ratio were found to change, depending on the absorbed dose and gelatin concentration.     

Multiple use of slab gels in sequencing apparatus for separation of polymerase chain reaction products

Attempting to assess whether a decrease of the electrophoresis temperature could prevent or reduce the extent of gel well deformations, and whether the utilization of native polyacrylamide gels (without urea) could speed up the separation of polymerase chain reaction (PCR)-amplified products with an automated 377 DNA sequencer, denatured PCR products were subjected to electrophoresis in 6% native gels under 45 degrees C. Results show that a decrease of the electrophoresis temperature from 51 degrees C (recommended by the User's Manual) to 45 degrees C substantially facilitates the preservation of gel wells, and that all PCR products tested migrate significantly faster in native than in denatured (with urea) gels of the same concentration. The combination of a 6% native gel and a lower (45 degrees C) electrophoresis temperature permits multiple uses of a given gel with consistent results, consequently reducing the electrophoresis time and reagent costs.     

The effects of urea and n-propanol on collagen denaturation: using DSC, circular dicroism and viscosity

The effect of urea and n-propanol on circular dichroism (CD) and viscosity of purified type1 collagen solution at various temperatures and differential scanning calorimetry (DSC) of rat-tail tendon (RTT) collagen fibre have been studied. CD reveals a spectrum with a positive peak at around 220 nm and a negative peak at 200 nm characteristics of collagen triple helix. The molar ellipticity decreases as the concentration of urea increases up to particular concentration (collagen solution treated with 265 μM of urea) and after that it increases (collagen solution treated with 500 μM of urea). There is a linear decrease in molar ellipticity as the concentration of n-propanol increases. Denaturation temperature of urea and n-propanol treated with purified collagen solution has been studied using viscosity method. Additives such as urea and n-propanol decrease the thermal stability of collagen triple helix in solution and in RTT collagen fibre. Thermal helix to coil transition of urea and n-propanol treated collagen depends on the degree of hydration and the concentration of these additives. Thermodynamic parameters such as the peak temperature, enthalpy of activation, and energy of activation for collagen-gelatin transition for native, urea and n-propanol treated RTT collagen fibre has been calculated using DSC. The change in the thermodynamic parameters has been observed for native, urea and n-propanol treated RTT collagen fibres. The experimental results show that the change in the water structure, dehydration and desolvation induced by different additives such as urea and n-propanol on RTT may vary with the type of denaturation.     

Influence of decavanadate clusters on the rheological properties of gelatin

The influence of polyoxovanadate clusters ([H(2)V(10)O(28)](4-)) on the thermo-reversible gelation of porcine skin gelatin solution (type A, M w approximately 40 000 g.mol (-1), pH = 3.4 < isoelectric point (IEP) approximately 8) has been investigated as a function of temperature and vanadate concentration by combining rheology and microcalorimetry. This work shows that the rheological properties of the system depend on electrostatic interactions between [H(2)V(10)O(28)](4-) and positively charged gelatin chains. In a first stage, we describe the renaturation of the gelatin triple helices in the presence of decavanadate clusters. We reveal that, when gelatin chains are in coil conformation (30 degrees C < T < 50 degrees C), the inorganic clusters act as physical cross-linkers that govern the visco-elastic properties of the mixture with an exponential dependence of the (G', G') modulus with the vanadate concentration. Below 30 degrees C, we show that gelatin triple helix nucleation is slightly favored by the presence of vanadate, but above a helix concentration of 0.012 g.cm (-3), G' is fully governed by the helix concentration. During the melting process, we reveal the non-fully reversible behavior of the vanadate/gelatin rheological properties and the stabilization of gelatin triple helices due to vanadate species until 50 degrees C. This non-reversible character has also been observed in the same experimental conditions with collagen/vanadate solutions. This is the first time that such a stabilization of triple helices has been reported in the case of gelatin hydrogels chemically cross-linked or not. We propose to analyze these results by considering that triple helix aggregates should persist because of decavanadate bridging, that the nucleation of an extended triple helix network may induce a strong modification of the vanadate cross-linker distribution in the system, or both, thus promoting the formation of thermally stable vanadate/gelatin micro-gels in the dangling end of the triple helices.     

Use of biospecific interactions of collagen, fibronectin and their fragments in affinity chromatography.

Various aspects of the application of fibronectin-collagen biospecific interactions in affinity chromatography are described. A new biospecific method for one-stage isolation of collagen peptides containing fibronectin-binding sites is proposed. The alpha 1 CB7-peptide of type-I collagen cyanogen bromide cleavage was isolated by means of affinity chromatography on adsorbents containing an immobilized gelatin-binding domain (45,000 relative molecular mass) of fibronectin. The method gives highly purified preparations of alpha 1 CB7-peptide. This peptide, as well as some other collagen molecular fragments (alpha-chains, beta-components, alpha 1 CB8-peptide), were immobilized on Sepharose, and the properties of such affinity adsorbents obtained were studied. Adsorbents with immobilized alpha-chains and alpha 1 CB7-peptide had a fibronectin-binding capacity 1.5-2.0 times higher than commercial gelatin-Sepharose. Large-scale production of highly purified fibronectin from human plasma, using affinity chromatography on immobilized individual alpha-chains of collagen, was developed.     

UCST‐type phase transition driven by protein‐derived polypeptide employing gelatin and chitosan

Here, we describe the thermosensitive reversible phase transition behaviors of polyelectrolyte complex composed of gelatin and chitosan (G/C complex). An aqueous dispersion of the G/C complexes showed a clear upper critical solution temperature (UCST) at around 30°C. The thermosensitive phase transition behavior showed excellent reversibility and large thermal hysteresis as a usual phenomenon based on the intra‐ and inter‐molecular interaction change. A high correlation was observed between the UCST of the G/C complex and the helix‐melting temperature of gelatin by circular dichroism, which suggested that the phase transition of the G/C complex corresponded to the secondary structure (helix‐coil) transition of gelatin. Notably, the UCST of the G/C complex shifted to lower temperatures in the presence of urea, which is well known to destabilize gelatin, whereas the addition of salt led to the dissolution of the G/C complex. It is envisaged that the results of this study will have a significant impact on the fabrication of UCST‐type thermosensitive materials, which can be utilized under aqueous physiological conditions using well‐known biopolymers. This protein‐derived functional material, which responds to the secondary structure transition, could also be used for the development of novel UCST‐type thermosensitive biomaterials. Copyright © 2017 John Wiley & Sons, Ltd.     

Thermodynamic characterization of the binding of tetrahydropterins to phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is the key enzyme in the catabolism of L-Phe. The natural cofactor of PAH, 6R-tetrahydrobiopterin (BH4), negatively regulates the enzyme activity in addition to being an essential cosubstrate for catalysis. The analogue 6-methyltetrahydropterin (6M-PH4) is effective in catalysis but does not regulate PAH. Here, the thermodynamics of binding of BH4 and 6M-PH4 to human PAH have been studied by isothermal titration calorimetry. At neutral pH and 25 degrees C, BH4 binds to PAH with higher affinity (Kd = 0.75 +/- 0.18 microM) than 6M-PH4 (Kd = 16.5 +/- 2.7 microM). While BH4 binding is a strongly exothermic process (DeltaH = -11.8 +/- 0.4 kcal/mol) accompanied by an entropic penalty (-TDeltaS = 3.4 +/- 0.4 kcal/mol), 6M-PH4 binding is both enthalpically (DeltaH = -3.3 +/- 0.3 kcal/mol) and entropically (-TDeltaS = -3.2 kcal/mol) driven. No significant changes in binding affinity were observed in the 5-35 degrees C temperature range for both pterins at neutral pH, but the enthalpic contribution increased with temperature rendering a heat capacity change (DeltaCp) of -357 +/- 26 cal/mol/K for BH4 and -63 +/- 12 cal/mol/K for 6M-PH4. Protons do not seem to be taken up or released upon pterin binding. Structure-based energetics calculations applied on the molecular dynamics simulated structures of the complexes suggest that in the case of BH4 binding, the conformational rearrangement of the N-terminal tail of PAH contribute with favorable enthalpic and unfavorable entropic contributions to the intrinsic thermodynamic parameters of binding. The entropic penalty is most probably associated to the reduction of conformational flexibility at the protein level and disappears for the L-Phe activated enzyme. The calculated energetic parameters aid to elucidate the molecular mechanism for cofactor recognition and the regulation of PAH by the dihydroxypropyl side chain of BH4.     

Two-dimensional electrophoresis as a tool for structural and genetic studies of seed proteins from Poaceae and Fagaceae

The application of two-dimensional electrophoretic procedures to structural and genetic studies of seed proteins from Poaceae (including the cultivated cereals) and Fagaceae is described. The following related problems have been considered: covalent and non-covalent association of protein subunits in multiple oligomeric structures; chromosomal locations of genes encoding seed proteins; quantitation of gene products in relation to gene expression and regulation; purification of protein components to study their homology relationships and in vitro activities; evolutionary and phylogenetic relationships; identification of genetic stocks. Isoelectric focusing, pore-gradient electrophoresis, electrophoresis at different pH's, are among the separation procedures used in the first dimension, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis and starch-gel electrophoresis at acid pH have been the preferred second-dimensional methods. Dissociating conditions (sodium dodecyl sulfate, Nonidet P-40, or urea) and reducing conditions (2-mercaptoethanol) have been used when required.     

Metal-metal oxide enzyme electrodes for the determination of urea

Enzyme electrodes for urea assay based on metal-metal oxide (Sb, Bi, W, Ti + RuO₂) with urease immobilized in gelatin gel were examined. It was shown that the best electrodes were obtained for tungsten. The urea response of the electrodes was influenced by the pH and concentration of the buffer used. Increasing additions of inert salt (potassium chloride) change the pH characteristic of the tungsten electrode and buffer capacity, thus influencing the urea response of the electrode.     

Studies on Mg/Fe hydrotalcite-like-compound (HTlc) I. Removal of inorganic selenite (SeO3(2-)) from aqueous medium

A Mg/Fe hydrotalcite-like-compound (HTlc) was prepared and its affinity toward the removal of SeO(3)(2-) from an aqueous medium was studied as a function of pH, time, temperature, particle dose, and SeO(3)(2-) concentration. The fraction of SeO(3)(2-) removal increases with decrease in both pH and temperature. The adsorption data are fitted to the Langmuir adsorption isotherm in the temperature range 303-333 K, and the thermodynamic parameters viz. standard Gibbs' free energy change (DeltaG degrees ), enthalpy change (DeltaH degrees ), and entropy change (DeltaS degrees ) are calculated. The negative value of DeltaH degrees indicates that the adsorption process is exothermic. The apparent equilibrium constants (K(a)) are also calculated and found to decrease with increase in temperature.     

Matériaux réactifs à base de collagène

Studies on the functionalisation of collagen by the introduction of polymerisable unsaturated groups, by analogy with the transformation of -OH or -NH₂ groups in proteins using acryloyl chloride as reagent, are presented. These unsaturated groups participate in copolymerisation reactions with vinyl monomers to produce interpenetrating networks. The influence of the parameters temperature, amount of acryloyl chloride and pH on the degree of transformation of the conjunctive protein (evaluated by IR spectroscopy and by dosage of free amine functions) is presented. It was established that pH has a significant influence on the nature of formed bonds (amine or ester type) and on the degree of transformation. Electron microscopy to analyse the products of copolymerisation between functionalised collagen and water-soluble monomers (acrylamide) was used. Interpenetrating networks with complex morphology were observed.     

Analysis of the interaction between an alpha (1----6)dextran-specific mouse hybridoma antibody and dextran B512 by affinity electrophoresis.

Carbohydrates are common environmental antigens. As dextran B512 is composed of a repeating structure of simple antigenic determinants, it is widely used to study the immunochemical properties of immunoglobulins. Two-dimensional affinity electrophoresis patterns of a mouse monoclonal antidextran antibody (35.8.2H; IgG1, BALB/c) were produced to obtain insights into the microheterogeneity of the monoclonal antibody. The monoclonal antibody was separated into about six spots which had an identical affinity to dextran B512, but differed in their isoelectric points (pI). In addition, the pH dependence of the binding affinity of this antidextran to dextran B512 was examined. By comparing affinities obtained by affinity electrophoresis between weakly basic (pH 9.5) and weakly acidic (pH 3.8) discontinuous buffer systems, the latter showed an affinity about 500 times lower than the former. The change in the affinity was investigated with a continuous pH gradient by an affinity titration curve and was seen to change markedly at about pH 6. This suggests that the histidine at residue 34 in the light-chain CDR1 is largely responsible for the dextran binding.