Fast events in protein folding: structural volume changes accompanying the early events in the N-->I transition of apomyoglobin induced by ultrafast pH jump

Ultrafast, laser-induced pH jump with time-resolved photoacoustic detection has been used to investigate the early protonation steps leading to the formation of the compact acid intermediate (I) of apomyoglobin (ApoMb). When ApoMb is in its native state (N) at pH 7.0, rapid acidification induced by a laser pulse leads to two parallel protonation processes. One reaction can be attributed to the binding of protons to the imidazole rings of His24 and His119. Reaction with imidazole leads to an unusually large contraction of -82 +/- 3 ml/mol, an enthalpy change of 8 +/- 1 kcal/mol, and an apparent bimolecular rate constant of (0.77 +/- 0.03) x 10(10) M(-1) s(-1). Our experiments evidence a rate-limiting step for this process at high ApoMb concentrations, characterized by a value of (0. 60 +/- 0.07) x 10(6) s(-1). The second protonation reaction at pH 7. 0 can be attributed to neutralization of carboxylate groups and is accompanied by an apparent expansion of 3.4 +/- 0.2 ml/mol, occurring with an apparent bimolecular rate constant of (1.25 +/- 0.02) x 10(11) M(-1) s(-1), and a reaction enthalpy of about 2 kcal/mol. The activation energy for the processes associated with the protonation of His24 and His119 is 16.2 +/- 0.9 kcal/mol, whereas that for the neutralization of carboxylates is 9.2 +/- 0.9 kcal/mol. At pH 4.5 ApoMb is in a partially unfolded state (I) and rapid acidification experiments evidence only the process assigned to carboxylate protonation. The unusually large contraction and the high energetic barrier observed at pH 7.0 for the protonation of the His residues suggests that the formation of the compact acid intermediate involves a rate-limiting step after protonation.     

pH stability of HLA-DR4 complexes with antigenic peptides

Complexes between antigenic peptides and class II proteins of the major histocompatibility complex (MHC) trigger cellular immune responses. These complexes usually dissociate more rapidly at mildly acidic pH, where they are formed intracellularly, as compared to neutral pH, where they function at the cell surface. This paper describes the pH dependence of the dissociation kinetics of complexes between MHC proteins and antigenic peptides containing aspartic and glutamic acid residues. Some of these complexes show an unusual pH dependence, dissociating much more rapidly at pH 7 than at pH 5.3. This occurs when the carboxylate group of the aspartic or glutamic acid residue is located in a neutral pocket of the protein. In contrast, solvent-exposed carboxylate groups or carboxylate groups buried in pockets where they form salt bridges with the protein do not show this unusual pH dependence. The kinetic data having the unusual pH dependence conform closely to a model in which there is a rapid reversible equilibration between a less stable deprotonated complex and a more stable protonated complex. In this model, the pK(a) of the protonation reaction for the partially buried peptide carboxylate group ranges from 7.7 to 8.3, reflecting the strongly basic conditions required for deprotonation. One of the few peptide/MHC complexes demonstrated to play a role in autoimmunity in humans contains a buried peptide carboxylate and shows this unusual pH dependence. The relevance of this finding to understanding the chemical basis of autoimmunity is briefly discussed.     

Photolytic depolymerization of alginate

A photochemical reaction has been developed for the partial de-polymerization of sodium alginate, a polysaccharide utilized in medicine, pharmacy, basic sciences and foods. An aqueous solution of sodium alginate was photochemically depolymerized to ∼40% of its average molecular weight using ultraviolet light in the presence of titanium dioxide catalyst at pH 7 over a period of 3 h. The products were separated giving four fractions all having an average molecular weight that was smaller than that of the starting material. Characterization of the guluronate (G) and mannuronate (M) contents, and determination of the M/G ratio of photochemically depolymerized alginate, were accomplished using ¹H NMR spectroscopy. The resulting M/G ratio was compared to that obtained for alginate fractions produced by acid hydrolysis. The M and G content, of each alginate fraction, was also assigned with regards to their occurrence in G-rich, M-rich or M/G heteropolymeric domains. This new depolymerization method might also be applicable in the preparation of alginate oligosaccharides for use in the food and pharmaceutical industries.     

Sequential immobilization of urease to glycidyl methacrylate grafted sodium alginate

Objective of this study is to realize appropriate enzyme immobilization onto a suitable support material and to develop a model which enables reactions catalyzed with different enzymes arranged in order. Thence, this model was potential for developing a multi-enzyme system. The reactions need more than one enzyme can be realized using immobilized form of them and the enzymes will be in one support at wanted activities. In this study, sodium alginate was used as immobilization material and glycidyl methacrylate was grafted onto sodium alginate. Thus reactive epoxy groups were added to sodium alginate which also has carboxyl groups. Average molecular weight of sodium alginate was determined using Ubbelohde viscosimetri. The molecular mass of sodium alginate was calculated as 15,900 Da. Graft polymerization was made in two steps. Firstly, sodium alginate was activated with benzophenone using UV-light at 254 nm. Secondly, glycidyl methacrylate was grafted under UV-light at 365 nm onto activated sodium alginate. Grafted glycidyl methacrylate was determined gravimetric and titrimetric. Additional groups after grafting were showed with FT-IR spectrum. 1-Ethyl-3-(3-dimetylaminopropyl)-carbodiimide was used for immobilization urease from carboxyl groups at pH 5.0. Suitable 1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide/–COOH ratio was found 1/10 and immobilized product activity was 197 U/g support. Reaction medium pH was 8.0 for immobilization from epoxy group. Optimum immobilization reaction time was found as 2 h and immobilized product activity was 285 U/g support. Sequential immobilization of urease to glycidyl methacrylate grafted sodium alginate was made from –COOH and epoxy groups, respectively.     

On the structure-function relationship of acyl carrier protein of Escherichia coli.

The conformations of Escherichia coli acyl carrier protein (ACP) and acetylated ACP have been studied as a function of pH and salt concentration by circular dichroism measurements. The results show that the amino groups of ACP in their protonated form are important for maintaining the native conformation of the protein at physiological pH. However, externally added cations (divalent more effectively than monovalent ones) can substitute for the ammonium groups in maintaining the ordered structure pf ACP. It is suggested that both the ammonium groups of ACP and externally added cations reduce the repulsion between carboxylate groups of ACP and thereby prevent the unfolding of the protein. A reduction of the number of negatively charged carboxylate groups by either protonation or chemical modification abolished the requirement for either ammonium groups or other cations. A qualitative agreement between the effect of salt on the conformation and on the biological activity of acetylated ACP has been observed. The single arginine residue of acetylated ACP has been modified by treatment with a trimer of 2,3-butanedione with the resulting derivative of ACP retaining most of its biological activity.     

海藻酸钠包埋法制备固定化菠萝蛋白酶

以海藻酸钠为载体,包埋法固定菠萝蛋白酶,对固定化奈件进行优化,同时探讨固定化菠萝蛋白酶的部分酶学性能。结果表明：固定化菠萝蛋白酶的质量受海藻酸钠质量分数、固定化酶量、固定化时间以及CaCl2质量分数的影响,其最佳固定化条件为：海藻酸钠质量分数1．0％,CaCl2质量分数3％,固定化酶液量与海藻酸钠体积之比1：2,固定化时间60min,在此条件下,制备的固定化菠萝蛋白酶的比活力为211．8U／g（湿质量载体）,由此制得的固定化酶的最适pH为7．6,与游离酶相比,升高了0．8个pH单位,同时显示固定化菠萝蛋白酶能耐受较高的碱性环境,固定化酶最适温度与游离酶相同,均为50℃,固定化酶在较高温度范围内,仍能保持较高的相对活力。     

Immobilization of a thermostable α‐amylase by covalent binding to an alginate matrix increases high temperature usability

Thermostable α‐amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95°C. 1‐ethyl‐3‐(3‐dimethylamino‐propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized α‐amylase were investigated. Covalently bound thermostable α‐amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95°C in a stirred batch reactor. The free and covalently bound α‐amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis‐Menten constant (K_m) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (V_max) of immobilized and free enzyme were determined to be 10.4‐mg starch degraded/mL min mg bound protein and 25.7‐mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable α‐amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Biosorption of reactive dye by waste biomass of Nostoc linckia

Potential of spent biomass of a cyanobacterium, Nostoc linckia HA 46, from a hydrogen bioreactor was studied for biosorption of a textile dye, reactive red 198. The waste biomass was immobilized in calcium alginate and used for biosorption of the dye from aqueous solution using response surface methodology (RSM). Kinetics of the dye in aqueous solution was studied in batch mode. Interactive effects of initial dye concentration (100-500 mg/L), pH (2-6) and temperature (25-45 °C) on dye removal were examined using Box-Behnken design. Maximum adsorption capacity of the immobilized biomass was 93.5 mg/g at pH 2.0, initial concentration of 100 mg/L and 35 °C temperature, when 94% of the dye was removed. Fourier transform infrared (FT-IR) studies revealed that biosorption was mainly mediated by functional groups like hydroxyl, amide, carboxylate, methyl and methylene groups present on the cell surface.     

N-(2-Carboxyethyl)chitosans: regioselective synthesis,characterisation and protolytic equilibria

N-(2-Carboxyethyl)chitosans were obtained by reaction of low molecular weight chitosan with a low degree of acetylation and 3-halopropionic acids under mild alkaline media (pH 8-9, NaHCO3) at 60 degrees C. The chemical structure of the derivatives obtained was determined by 1H and 13C NMR spectroscopies. It was found that alkylation of chitosan by 3-halopropionic acids proceeds exclusively at the amino groups. The products obtained are described in terms of their degrees of carboxyethylation and ratio of mono-, di-substitution and free amine content. The protonation constants of amino and carboxylate groups of a series of N-(2-carboxyethyl)chitosans were determined by pH-titration at ionic strength 0.1 M KNO3 and 25 degrees C.     

Pilot plant scale extraction of alginates from Macrocystis pyrifera 4. Conversion of alginic acid to sodium alginate, drying and milling

The last three steps of the alginate production process were studied:conversion of alginic acid to sodium alginate, drying, and milling. Threemethods were used to follow the conversion reaction: measuring the pH (a) intheethanol-water liquid of the reaction mixture, (b) after dissolving a sample ofthe fiber taken from the reaction mixture, (c) after dissolving the driedsodiumalginate obtained from the reaction. To obtain a neutral dried sodium alginate,in the first method the pH should be adjusted to 9, and in the second the pHshould be adjusted to 8. The best method to control the reaction was todissolvea sample of the fiber and adjust the pH to 8. The best proportion to reach thecritical point, where pH just begins to rise, was 0.25 parts of sodiumcarbonateto 1 part of alginate in the initial dry algae. A pH above 7 may produce abreakdown of the molecule, reducing significantly the viscosity of the finalalginate. Four different temperatures were used to dry the alginate: 50, 60,70,and 80 °C. Drying time to reach 12% moisture ranged from 1.5h at 80 °C to 3 h at 50°C. The best drying temperature was 60 °C for2.5 h. The effect of drying temperature on alginate viscosity wasdependent on the alginate type. Low and medium viscosity alginates were notsignificantly affected, but alginate with high viscosity was reduced by 40 to54% using the temperature range of 60 to 80 °C. A fixed hammermill was used to reduce the particle size of the dried sodium alginate.Particlesize measurements showed that after a first milling the product contained 76%large particles (20–60 mesh) and 24% fine particles (80–120 mesh).After a third milling the product still contained 42.9% large particles. Nosignificant effect was found on alginate viscosity because of the millingsteps.     

Hydrophobic modification of sodium alginate and its application in drug controlled release

Sodium alginate was hydrophobically modified by coupling of polybutyl methacrylate onto the alginate. The polybutyl methacrylate was previously prepared through polymerization of butyl methacrylate in the presence of 2-amino-ethanethiol as a chain transfer agent. The structure of the product was characterized by Fourier-transformed infrared spectrometry, nuclear magnetic resonance (¹HNMR) and thermogravimetry. The result of fluorescence analysis showed that the hydrophobicity of the modified alginate was obviously increased. The modified alginate conjugate was used for immobilization of bovine serum albumin in the presence of calcium chloride. In addition, the release behavior of the drug-loaded alginate in deionized water and Tris–HCl buffer solution (pH 7.2) was investigated. It was found that the modified sodium alginate possessed prolonged release behavior compared to unmodified sodium alginate, and it had potential application in controlled release as a drug carrier.     

Selective oxidation of primary alcohol groups of beta-cyclodextrin mediated by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)

Beta-cyclodextrin (beta-CD) was reacted with catalytic amounts of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO), sodium hypochlorite and sodium bromide at 2 degrees C and a pH value of 10 in water. The primary alcohol groups were selectively oxidized into carboxylate groups within a few minutes, and mono- and dicarboxy-beta-cyclodextrin sodium salts were isolated and characterized by 1H, 13C NMR and mass spectroscopy. With this reaction system, the degradation of the cyclodextrin was limited, provided the oxidation was performed at 2 degrees C, at constant pH value of 10, with catalytic amounts of TEMPO and controlled quantities of sodium hypochlorite and sodium bromide for the continuous regeneration of the oxoammonium salt.     

Utilization of an exopolysaccharide produced by Chryseomonas luteola TEM05 in alginate beads for adsorption of cadmium and cobalt ions

Cadmium and cobalt adsorption from aqueous solution onto calcium alginate, sodium alginate with an extracellular polysaccharide (EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and immobilized C. luteola TEM05 was studied. In addition, solutions containing both of these ions were prepared and partial competitive adsorption of these mixtures was investigated. Metal adsorption onto gel beads was carried out at pH 6.0 and 25 degrees C. The maximum adsorption capacities determined by fitting Langmuir isotherms to the data for calcium alginate, calcium alginate+EPS, calcium alginate + C. luteola TEM05 and calcium alginate + EPS + C. luteola TEM05 were 45.87, 55.25, 49.26, 51.81 mg g(-1) for Co(II) and 52.91, 64.10, 62.5, 61.73 mg g(-1) for Cd(II), respectively. The biosorption capacity of the carrier for both metal ions together in competition was lower than those obtained when each was present alone.     

Viscosity study of interactions between sodium alginate and CTAB in dilute solutions at different pH values

Interactions between anionic polyelectrolyte sodium alginate and the cationic surfactant cetytrimethylammonium bromide (CTAB) have been investigated by viscosity measurement techniques. The polymer–surfactant interactions are observed between alginate and CTAB at different pH in dilute solution. The results show that the rheological response of alginate dilute solutions is sensitive to a change of pH in the low pH range. The steady shear and intrinsic viscosity measurements reveal that the strong association between alginate and CTAB by electrostatic attraction above pH 5.0. However, as the pH value of solution decrease from 5.0 to 3.0, the strong association between alginate and CTAB is affected by not only electrostatic attraction but also hydrophobic interaction.     

Facile preparation of well-defined near-monodisperse chitosan/sodium alginate polyelectrolyte complex nanoparticles (CS/SAL NPs) via ionotropic gelification: A suitable technique for drug delivery systems

Polymeric nanoparticles have emerged as a promising approach for drug delivery systems. We prepared chitosan (CS)/sodium alginate (SAL) polyelectrolyte complex nanoparticles (CS/SAL NPs) via a simple and mild ionic gelation method by adding a CS solution to a SAL solution, and investigated the effects of molecular weight of the added CS, and the SAL:CS mass ratio on the formation of the polyelectrolyte complex nanoparticles. The well-defined CS/SAL NPs with near-monodisperse particle size of about 160 nm exhibited a pH stable structure, and pH responsive properties with a negatively or positively charged surface. The so-called “electrostatic sponge” structure of the polyelectrolyte complex nanoparticles enhanced their drug-loading capacity towards the differently charged model drug molecules, and favored controlled release. We also found that the drug-loading capacity was influenced by the nature of the drugs and the drug-loading media, while drug release was affected by the solubility of the drugs in the drug-releasing media. The biocompatibility and biodegradability of the polyelectrolytes in the polyelectrolyte complex nanoparticles were maintained by ionic interactions. These results indicate that CS/SAL NPs can represent a useful technique for pH-responsive drug delivery systems.     

Calorimetric studies of the interaction between sodium alginate and sodium dodecyl sulfate in dilute solutions at different pH values

Interactions between the polyelectrolyte sodium alginate (NaAlg) and the anionic surfactant sodium dodecyl sulfate (SDS) have been investigated by microcalorimetric techniques. The polymer-surfactant interactions were observed between NaAlg and SDS at different pH values in dilute solution. The thermodynamic parameters for their interaction process are evaluated from the results of the observed dilution enthalpy curves. As the pH value of the solution decreases from 7 to 6, NaAlg polymers have an obvious effect on the cmc of SDS as a simple salt, which indicates no association between SDS and NaAlg owing to electrostatic repulsion. With the progressive decrease of pH value from 5 to 3, the hydrophobic segments in the alginate chains are increasing and the hydrophilic segments decreasing, and the aggregation between SDS and alginate due to hydrophobic interactions is observed.     

Structural studies with the uveopathogenic peptide M derived from retinal S-antigen

The 18-residue fragment of bovine S-antigen, corresponding to amino acid positions 303-320, is highly immunogenic and is known to induce experimental autoimmune uveitis. The solution conformation of this immunogenic peptide, known as peptide M, was studied by Fourier-transform infrared spectroscopy and by circular dichroism. In the pH range between approximately 4 and 9.5, peptide M has a strong tendency to form macromolecular assemblies in which it adopts an intermolecular beta-sheet structure. The intermolecular beta-sheets are stabilized by ionic interactions ("salt bridges") between the carboxylate groups and basic residues of the neighboring peptide molecules. These interactions can be disrupted by neutralization of either acidic (pH range below 4) or basic residues (pH range above 9.5) or by elevated hydrostatic pressure. The secondary structure of the peptide under conditions favoring the monomeric state appears to be a mixture of unordered structure and beta-sheets. The present data are consistent with a recently proposed model [Sette, A., Buns, S., Colon, S., Smith, J. A., Miles, C., & Grey, H. M. (1987) Nature 328, 395-399], which assumes that certain immunogenic peptides adopt an extended beta-type conformation in which they are "sandwiched" between the major histocompatibility complex and the T-cell receptor.     

Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base.

The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.     

Fabrication of curcumin-loaded zein nanoparticles stabilized by sodium caseinate/sodium alginate: Curcumin solubility,thermal properties,rheology, and stability

Curcumin is a polyphenol with multiple biological activities, but its extremely poor water solubility severely limits its application in the food industry. The purposes of this work were to study the effect of nano-encapsulation on the water solubility of curcumin (C), the interaction of curcumin with zein (Z), the thermal properties, rheological properties, and the stability under different environmental pressures of the nanoparticles. The results of particle size, zeta potential, and surface hydrophobicity (H₀) indicated that the combination of coating materials including sodium caseinate (SC) and sodium alginate (SA) with zein nanoparticles by electrostatic interaction led to a gradual increase in the particle size of composite nanoparticles and a decrease in surface hydrophobicity. The nano-encapsulation significantly improved the water solubility of curcumin and causing its crystal structure to change to an amorphous state. Fourier transform infrared spectroscopy confirmed that curcumin bound to zein through hydrogen bonding. Rheological test results showed that the coating materials combined with zein led to an increase in the apparent viscosity of the nanoparticles. The stability analysis results indicated that the composite nanoparticles with a sodium alginate coating have excellent stability of pH, salt solution and storage, and excellent anti-gastrointestinal fluids digestion characteristics when compared to pure protein nanoparticles.