The role of hydration on the mechanism of allosteric regulation: In situ measurements of the oxygen-linked kinetics of water binding to hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98%, relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.

Water regulation of actinomycin-D binding to DNA: the interplay among drug affinity, DNA long-range conformation, and hydration

Actiaomycin-D (actD) binds to natural DNA at two different classes of binding sites, weak and strong. The affinity for these sites is highly dependent on DNA se(sequence and solution conditions, and the interaction appears to be purely entropic driven Although the entropic character of this reaction has been attributed to the release of water molecules upon drug to DNA complex formation, the mechanism by which hydration regulates actD binding and discrimination between different classes of binding sites on natural DNA is still unknown. In this work, we investigate the role of hydration on this reaction using the osmotic stress method. We skew that the decrease of solution water activity, due to the addition of sucrose, glycerol ethylene glycol, and betaine, favors drug binding to the strong binding sites on DNA by increasing both the apparent binding affinity Delta G, and the number of DNA base pairs apparently occupied by the bound drug n(bp/actD). These binding parameters vary linearly with the logarithm of the molar fraction of water in solution log(X-w), which indicates the contribution of water binding to the energetic of the reaction. It is demonstrated that the hydration change measured upon binding increases proportionally to the apparent size of the binding site n(bp/uctD). This indicates that n(bp/actD) measured from the Scatchard plod is a measure of the size of the DNA molecule changing conformation due to ligand binding. We also find that the contribution of DNA deformation, gauged by n(bp/act) to the total free energy of binding Delta G, is given by Delta G = Delta G(local) + n(bp/actD) x delta G(DNA), where Delta G(local), = -8020 +/- 51 cal/mol of actD bound and delta G(DNa) = -24.1 +/- 1.7cal/mol of base pair at 25 degrees C. We interpret Delta G(local), as the energetic contribution due to the direct interactions of actD with the actual tetranucleotide binding site, and it n(bp/actB) X delta G(DNA) as that due to change inconformation, induced by binding, of it n(bp/actD) DNA base pairs flanking the local site. This interpretation is supported by the agreement found between the value of delta G(DNA) and the torsional free energy change measured independently. We conclude suggesting an allosteric model for ligand binding to DNA, such that the increase in binding affinity is achieved by increasing the relaxation of the unfavorable free energy of binding storage at the local site through a larger number of DNA base pairs. The new aspect on this model is that the size of the complex is not fixed but determined by solutions conditions, such as water activity, which modulate the energetic barrier to change helix conformation. These results may suggest that long-range allosteric transitions of duplex DNA are involved in the inhibition of RNA synthesis by actD, and more generally, in the regulation of transcription. (C) 2000 John Wiley & Sons, Inc.

The water effect on allosteric regulation of hemoglobin probed in water glucose and water glycine solutions

We have previously proposed a role of hydration in the allosteric control of hemoglobin based on the effect of varying concentrations of polyols and polyethers on the human hemoglobin oxygen affinity and on the solution water activity (Colombo, M. F., Rau, D. C., and Parsegian, V. A. (1992) Science 256, 655-659). Here, the original analyses are extended to test the possibility of concomitant solute and water allosteric binding and by introducing the bulk dielectric constant as a variable in our experiments. We present data which indicate that glycine and glucose influence HbA oxygen affinity to the same extent, despite the fact that glycine increases and glucose decreases the bulk dielectric constant of the solution. Furthermore, we derive an equation linking changes in oxygen affinity to changes in differential solute and water binding to test critically the possibility of neutral solute heterotropic binding. Applied to the data, these analyses support our original interpretation that neutral solutes act indirectly on the regulation of allosteric behavior of hemoglobin by varying the chemical potential of water in solution. This leads to a displacement of the equilibrium between Hb conformational states in proportion to their differential hydration.

Significance of bound water to local chain conformations in protein crystals.

We examine how the polypeptide chain in protein crystal structures exploits the multivalent hydrogen-bonding potential of bound water molecules. This shows that multiple interactions with a single water molecule tend to occur locally along the chain. A distinctive internal-coordinate representation of the local water-binding segments reveals several consensus conformations. The fractional water occupancy of each was found by comparison of the total number of conformations in the database regardless of the presence or absence of bound water. The water molecule appears particularly frequently in type II beta-turn geometries and an N-terminal helix feature. This work constitutes a first step into assessing not only the generality but also the significance of specific water binding in globular proteins.

Penetration of hydrolysed soy protein-added brine and its effect on yield and pH of beef steaks from the biceps femoris muscle

This study was aimed to evaluate the penetration behaviour of different brines with tumbled beef steaks from the biceps femoris muscle, specifically their interactions with pH and effects on yield. Six muscles from different animals, divided into origin (OP) and insertion (IP) portions, were cut into 60 steaks of 2.5 cm thickness and tumbled for 30 or 60 min. The steaks were tumbled with two brines, with (WTB/HSP) or without (WTB) hydrolysed soy protein (HSP), and steaks that were not tumbled with brine or water were used as controls. Brine penetration was verified by measuring the amount of dye-containing brine (absorbance at 627 nm) recovered from homogenates of four thin (2 mm) slices from the surface of the beef steaks after tumbling. The WTB/HSP steaks exhibited greater (P < 0.05) brine penetration when tumbled for 60 min than for 30 min. The OP steaks showed greater yield and lower pH (P < 0.05) than IP steaks. HSP-added brine increased the water absorption and retention in the first slices of the steaks, and its efficiency was increased with a longer tumbling time. The portion of the biceps femoris muscle used influenced brine absorption and retention, impacting meat yield. (C) 2010 Elsevier Ltd. All rights reserved.

Caracterização físico-química do amido e da farinha da fruta-pão (Artocarpus altilis) e aplicação em pães de forma

A fruta-pão é um fruto rico em vitaminas e minerais do qual pode ser extraído o amido ou processado à forma de farinha aumentando sua vida de prateleira. Essa farinha pode substituir parcialmente a farinha de trigo em produtos panificáveis. O objetivo do presente estudo foi extrair e analisar algumas características físicas, químicas e tecnológicas do amido nativo de fruta-pão, além de realizar a caracterização físico-química da farinha obtida desse fruto, de suas misturas com a farinha de trigo e verificar a viabilidade destas na elaboração de pão de forma. O amido de fruta-pão foi submetido às análises de umidade, capacidade de ligação de água a frio (CLAF), poder de inchamento (PI), índice de solubilidade (IS), sinérese, microscopia eletrônica de varredura (MEV) e difração de raio X. O amido de milho foi analisado para efeito de comparação. O amido de fruta-pão apresentou maior umidade (18,32%), CLAF (80,33%), PI (198,71 g/g a 90 0C), IS (7,22% a 90 0C) e sinérese (2,69 mL) quando comparado com o amido de milho. Pela análise de MEV, verificou-se que o amido de fruta-pão apresentou grânulos menores que os do amido de milho, mas ambos com formato poliédrico. A difração de raio X permitiu classificar o amido de milho como tipo A e o de fruta-pão como tipo B. As amostras de farinha analisadas continham um percentual de farinha de fruta-pão de 0% (controle), 5%, 10%, 15%, 20% e 100% e foram submetidas às análises de umidade, cinzas, extrato etéreo, carboidratos, proteína, energia, fibra total, solúvel e insolúvel, pH, cor e granulometria. Os mesmos percentuais de substituição foram utilizados para a elaboração dos pães, analisados em relação a umidade, peso, volume, volume específico, altura, perfil de textura, aceitação sensorial e intenção de compra. A substituição da farinha de trigo pela farinha de fruta-pão influenciou na granulometria das misturas, reduziu o teor de proteína de 10,8% para 3,99%, elevou o percentual de carboidratos, cinzas e pH de 73,84%, 0,52% e 5,94 para 83,10%, 2,19% e 6,34 , respectivamente. No entanto, para a umidade, teor de lipídeos e diferença global de cor das amostras não foi verificada influência significativa do percentual de substituição. A farinha de fruta-pão apresentou um maior percentual de fibras totais (21,17%), solúveis (10,39%) e insolúveis (10,77%). Para os pães de forma, o aumento da substituição não influenciou na umidade e no peso dos pães, exceto para a amostra com 100% de farinha de fruta-pão, que apresentou 41,28% de umidade e peso de 588,65 g. Para as demais análises, os maiores percentuais de substituição influenciaram negativamente as características do produto. Em relação a caracterização físico-química das amostras de farinha, o aumento do percentual de substituição influenciou na maioria dos parâmetros avaliados. Para os pães, as formulações com maiores notas hedônicas e com melhor intenção de compra foram as com substituição de 5% e de 10%.

High pressure processing of swede (Brassica napus): Impact on quality properties

The effects of combined pressure/temperature treatments (200, 400 and 600 MPa, at 20 and 40 °C) on the physical and nutritional properties of swede roots (Brassica napus var. napobrassica) were assessed. Changes induced by high pressure processing (HPP) on the original properties of swede samples were compared with those produced by thermal treatment (blanching). All studied treatments altered the physical properties of swede, resulting in a loss of hardness and water binding capacity. The strongest alteration of texture was observed after HPP at 400 MPa, while 600 MPa was the treatment that better preserved the texture properties of swede. Blanching caused less total colour changes (ΔE) than HPP. Antioxidant properties of swede were measured as total antioxidant capacity, ascorbic acid and total phenol content. All treatments caused a loss of antioxidant capacity, which was less pronounced after HPP at 600 MPa and 20 °C and blanching. Four glucosinolates were detected in swede roots, glucoraphanin, progoitrin, glucobrassicanapin and glucobrassicin. Glucobrassicanapin and glucobrassicin contents were reduced with all studied treatments. Progoitrin content was not affected by blanching and HPP at 200 MPa. HPP at higher pressure levels (400 and 600 MPa), though, induced an increase of progoitrin levels. The results indicated that blanching and HPP at 600 MPa and 20 °C were the treatments that better preserved the original quality properties of swede.

Modificação química e física do amido de quirera de arroz para aproveitamento na indústria de alimentos

The study evaluated the efficiency of chemical (phosphorylation) and physical (extrusion) modifications of the starch of broken rice. Results demonstrated a reduction in the moisture content of extruded and phosphorylated broken rice and an increase in the ash content of phosphorylated broken rice. Both phosphorylation and extrusion increased cold water binding capacity, swelling power, and solubility. Extruded and phosphorylated pastes were stable under refrigeration, but only extruded paste was stable when submitted to freezing. Phosphorylated paste had the lowest viscosity and the highest stability during heating, while the extruded one gelatinized without heating, but had higher losses during heating.

Impacts of morphological, physical and chemical properties of sludge flocs on dewaterability of activated sludge

This study examined how the floc characteristics affect dewaterability of activated sludge. The floc properties were characterized by morphological parameters (floc size distribution, fractal dimension and filament index), physical properties (flocculating ability, surface charge, relative hydrophobicity and viscosity), and chemical constituents in sludge and extracted extracellular polymeric substances (EPS), including the polymeric compounds protein, humic substances, carbohydrates and the ions Ca2+, Mg2+, Fe3+ and Al3+. The dewaterability was defined in terms of the bound water content and capillary suction time (CST). The bound water and CST corresponded to a similar indication with respect to dewaterability of activated sludge. The floc physical parameters were the most important factors which effect significantly on the water binding ability of the sludge flocs. The morphological characteristics had relatively weak impact on the dewaterability. The polymeric components protein and carbohydrate had a significant contribution to enhance the water binding ability of the sludge flocs. The effect of humic substances in the sludge on the dewaterability was, however, insignificant. The CST had good statistical correlations with the polymeric constituents measured in both sludge and the extracted EPS, and the bound water was only correlated well with the individual polymers measured in the sludge. High concentration of Ca2+, Mg2+, Fe3+ and Al3+ had significant improvement for dewaterability. (C) 2003 Elsevier B.V. All rights reserved.

Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein

The aim of this work was to construct short analogues of the repetitive water-binding domain of the Pseudomonas syringae ice nucleation protein, InaZ. Structural analysis of these analogues might provide data pertaining to the protein-water contacts that underlie ice nucleation. An artificial gene coding for a 48-mer repeat sequence from InaZ was synthesized from four oligodeoxyribonucleotides and ligated into the expression vector, pGEX2T. The recombinant vector was cloned in Escherichia coli and a glutathione S-transferase fusion protein obtained. This fusion protein displayed a low level of ice-nucleating activity when tested by a droplet freezing assay. The fusion protein could be cleaved with thrombin, providing a means for future recovery of the 48-mer peptide in amounts suitable for structural analysis by nuclear magnetic resonance spectroscopy.

Novel hydrogel copolymers and semi-interpenetraing polymer networks

Interpenetrating polymer networks (lPN's), have been defined as a combination of two polymers each in network form, at least one of which has been synthesised and / or crosslinked in the presence of the other. A semi-lPN, is formed when only one of the polymers in the system is crosslinked, the other being linear. lPN's have potential advantages over homogeneous materials presently used in biomedical applications, in that their composite nature gives them a useful combination of properties. Such materials have potential uses in the biomedical field, specifically for use in hard tissue replacements, rigid gas permeable contact lenses and dental materials. Work on simply two or three component systems in both low water containing lPN's supplemented by the study of hydrogels (water swollen hydrophilic polymers) can provide information useful in the future development of more complex systems. A range of copolymers have been synthesised using a variety of methacrylates and acrylates. Hydrogels were obtained by the addition of N-vinyl pyrrolidone to these copolymers. A selection of interpenetrants were incorporated into the samples and their effect on the copolymer properties was investigated. By studying glass transition temperatures, mechanical, surface, water binding and oxygen permeability properties samples were assessed for their suitability for use as biomaterials. In addition copolymers containing tris-(trimethylsiloxy)-y-methacryloxypropyl silane, commonly abbreviated to 'TRlS', have been investigated. This material has been shown to enhance oxygen permeability, a desirable property when considering the design of contact lenses. However, 'TRIS' has a low polar component of surface free energy and hence low wettability. Copolymerisation with a range of methacrylates has shown that significant increases in surface wettability can be obtained without a detrimental effect on oxygen permeability. To further enhance to surface wettability 4-methacryloxyethyl trimellitic anhydride was incorporated into a range of promising samples. This study has shown that by careful choice of monomers it is possible to synthesise polymers that possess a range of properties desirable in biomedical applications.

Novel cationic polymers for use at biological interfaces

One of the main problems with the use of synthetic polymers as biomaterials is the invasion of micro-organisms causing infection. A study of the properties of polymeric antibacterial agents, in particular polyhexamethylene biguanide, has revealed that the essential components for the design of a novel polymeric antibacterial are a balance between hydrophilicity and hydrophobicity coupled with sites of cationicity. The effect of cation incorporation on the physical properties of hydrogels has been investigated. Hydrogel systems copolymerised with either N-vinyl imidazole or dimethylaminoethyl methacrylate have been characterised in terms of their water binding, mechanical and surface properties. It has been concluded that the incorporation of these monomers does not adversely affect the properties of such hydrogels and that these materials are potential candidates for further development for use in biomedical applications. It has been reported that hydro gels with ionic character may increase the deposition of biological material onto the hydrogel surface when it is in contact with body fluids. An investigation into the deposition characteristics of hydrogels containing the potentially cationic monomers has been carried out, using specific protein adsorption and in vitro spoilation techniques. The results suggest that at low levels of cationicity, the deposition of positively charged proteins is reduced without adversely affecting the uptake of the other proteins. The gross deposition characteristics were found to be comparable to some commercially available contact lens materials. A preliminary investigation into the development of novel antibacterial polymers has been completed and some novel methods of bacterial inhibition discussed. These methods include development of an hydrogel whose potential application is as a catheter coating.

Novel hydrogel polymers

Hydrogels are a unique class of polymers which swell, but do not dissolve in water. A range of 2-hydroxyethyl methacrylate based copolymer hydrogels have been synthesised and are described in this thesis. Initially, hydrogels were synthesised containing acryloylmorpholine, N,N-dimethyl acrylamide and N-vinyl pyrrolidone. Variations in structure and composition have been correlated with the sequence distribution, equilibrium water content (EWC) , mechanical and surface properties of the hydrogels. The sequence distribution was found to be dependant on the structure and reactivity of the monomers. The EWC was found to be dependant on the water structuring groups present in the hydrogel, although the water binding abilities were modified by steric effects. The mechanical properties were also investigated and were found to be dependant on the monomer structure, sequence distribution and the amount and nature of water in the hydrogel. The macroscopic surface properties of the hydrogels were probed using surface energy determinations and were found to be a function of the water content and the hydrogel composition. At a molecular level, surface properties were investigated using an in vitro ocular spoilation model and single protein adhesion studies. The results indicate that the sequence distribution and the polarity of the surface affect the adhesion of biological species. Finally, a range of 2-hydroxyethyl methacrylate based copolymer hydrogels containing both charged monomer groups and linear polyethers have been synthesised and described. Although variations in the EWC are observed with the structure of the monomers, it was observed that the EWC increased due to the polar character of the charged monomers and the chain length and hydrophilicity of the polyethers. Investigation of these hydrogel surfaces revealed subtle changes. The molecular surface properties indicate the significance of the effect of charge and molecular mobility of the groups expressed at the hydrogel surface.