Reducción de proteínas y glucosa por reacción de Maillard en leche con lactosa hidrolisada / Protein and glucose reduction by Maillard reaction in milk with hydrolyzed lactose

Se evaluó el efecto de la temperatura sobre la desnaturalización de proteínas y la reacción de Maillard en leche entera y descremada con lactosa hidrolizada. Las leches hidrolizadas se trataron térmicamente a 100, 110, 120 y 130 °C durante un período de 1 hora y se midió la concentración de glucosa, el grado de pardeamiento y la desnaturalización de proteínas. El grado de dorado en la leche entera varió de 14.4 (100 °C) a 42.6 (130 °C). Para la leche descremada fue de 20.2 (100 °C) a 38.0 (130 °C). La concentración de glucosa en leche entera (47% p/v) y en leche descremada (41% p/v) después del tratamiento térmico (130 °C) mostró una reducción significativa en relación con el control (25 °C). El efecto de la temperatura en la desnaturalización de proteínas en leche entera y descremada en relación con el control (25 °C) fue del 100%. La leche tratada térmicamente con lactosa hidrolizada promovió la desnaturalización de proteínas con un aumento del pardeamiento característico de la reacción de Maillard, lo que afectó la calidad nutricional.

The effect of temperature in protein denaturation and Maillard reaction in whole and skim milk with hydrolyzed lactose was evaluated. Hydrolyzed milk was thermally treated at 100, 110, 120 and 130 °C over a period of 1 hour and glucose concentration, browning degree and protein denaturation were measured. The browning degree in whole milk varied from 14.42 (100 °C) to 42.63 (130 °C) and 20.21 (100 °C) to 38.03 (130 °C) in skim milk. Glucose concentration in whole milk (47% - w/v) and skim milk (41% - w/v) after heat treatment (130 °C) showed a significant reduction in relation to the control (25 °C). The temperature effect in protein denaturation in whole and skim milk in relation to the control (25 °C) was 100%. Thermally treated milk with hydrolyzed lactose promoted protein denaturation with increasing browning characteristic of the Maillard reaction, thus affecting the nutritional quality.

Antipsicóticos para la esquizofrenia: paradigma de los medicamentos psiquiátricos / Antipsychotics for schizophrenia: the paradigm of psychiatric drugs

Los antipsicóticos no parecen revertir las causas de la esquizofrenia y, aunque son fármacos que pueden aliviar los síntomas a corto y mediano plazo, a largo plazo pueden no ser beneficiosos e incluso ser contraproducentes. Su empleo debería limitarse a situaciones agudas con agitación y tensión incapacitante. Presentan considerables efectos adversos y, ante la negativa de una persona a seguir tomándolos, adoptar una estrategia de reducción de daños apoyando y supervisando la retirada puede ser preferible a la coerción. Existen alternativas a los neurolépticos. Los prescriptores deberían estar más atentos y considerar las valoraciones que los usuarios hacen de sus efectos. El apego a las guías de tratamiento es escaso, seguramente por basarse en ensayos clinicos de calidad deficente, que deben mejorar y prolongarse en el tiempo. La raíz del problema probablemente se encuentra en la tautología sobre la etiología y naturaleza biológica de lo que llaman esquizofrenia, que realmente no parece ser más que un constructo ideológico-comercial.

Antipsychotic drugs do not appear to reverse the causes of schizophrenia, and although they can relieve symptoms in the short to medium term, in the long term they may not be beneficial and could even be counterproductive. Their use should be limited to acute situations in which agitation and tension is disabling. The drugs have significant adverse effects, and given the refusal of a person to continue taking them, a harm reduction strategy to support and monitor the withdrawal may be preferable to coercion. There are alternatives to neuroleptics. Prescribers should be more vigilant and consider the assessments of users regarding the drugs' effects. Adherence to treatment guidelines is low, probably because the guidelines are based on clinical trials of deficient quality which consequently should be improved and extended over a greater period of time. The root of the problem is likely the tautology on the etiology and biological nature of what is known as schizophrenia, which in fact does not seem to be more than a commercial and ideological construct.

Riesgo potencial de parásitos zoonóticos presentes en heces caninas en Puerto Escondido, Oaxaca / Potential hazard of zoonotic parasites present in canine feces in Puerto Escondido, Oaxaca

Objetivo. Estimar la prevalencia de parásitos potencialmente zoonóticos en heces caninas de Puerto Escondido. Material y métodos. La ciudad se dividió en diez zonas de estudio y éstas se categorizaron en hábitats natural, urbano y suburbano. Se colectaron muestras fecales caninas del piso. Se recuperaron los parásitos por medio de técnicas coproparasitológicas de flotación y frotis directo para su observación microscópica y posterior identificación. Se estimó la prevalencia parasitaria en las heces caninas. Resultados. Todas las zonas presentaron fecalismo canino. La prevalencia parasitaria fue de 73.33%. Los parásitos con mayor prevalencia fueron Toxocara canis (47.78%), Ancylostoma caninum (17.88%) y Dipylidium caninum (13.89%). Conclusión. El fecalismo canino proviene de perros errantes y con dueño. Del total de parásitos encontrados, 66.66% son zoonóticos. Los factores que favorecen la problemática son el hábitat suburbano, el manejo indeseable de la basura y la tenencia irresponsable de los cánidos.

Objective. To estimate the zoonotic parasites prevalence in feral dog feces in Puerto Escondido. Material and methods. The fecalism frecuency was estimated in ten zones. To identify the parasites parasitological flotation and direct smear methods were used. The parasitic prevalence was estimated in the canine feces. Results. All the zones presented canine fecalism. The parasitic prevalence in the feces was 73.33%. The parasites with the highest prevalence were Toxocara canis (47.78%), Ancylostoma caninum (17.88%), and Dipylidium caninum (13.89%). Conclusion. Canine fecalism comes from strayed and owned dogs. 66.66% of the parasites found in the dog feces are zoonotics. The factors associated to this problem are the suburban habitat, waste mishandling and nil tenure of stray dogs.

Residual ordered structure in denatured proteins and the problem of protein folding.

Structural characteristics of numerous globular proteins in the denatured state have been reviewed using literature data. Recent more precise experiments show that in contrast to the conventional standpoint, proteins under strongly denaturing conditions do not unfold completely and adopt a random coil state, but contain significant residual ordered structure. These results cast doubt on the basis of the conventional approach representing the process of protein folding as a spontaneous transition of a polypeptide chain from the random coil state to the unique globular structure. The denaturation of proteins is explained in terms of the physical properties of proteins such as stability, conformational change, elasticity, irreversible denaturation, etc. The spontaneous renaturation of some denatured proteins most probably is merely the manifestation of the physical properties (e.g., the elasticity) of the proteins per se, caused by the residual structure present in the denatured state. The pieces of the ordered structure might be the centers of the initiation of renaturation, where the restoration of the initial native conformation of denatured proteins begins. Studies on the denaturation of proteins hardly clarify how the proteins fold into the native conformation during the successive residue-by-residue elongation of the polypeptide chain on the ribosome.

Study on the chaperone properties of conserved GTPases

As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.

In silico evaluation of cross linking effects on denaturant meq values and delta Cp upon protein unfolding

Important thermodynamic parameters including denaturant equilibrium m values [meq] and heat capacity changes [delta Cp] can be predicted based on changes in Solvent Accessible Surface Area [SASA] upon unfolding. Cross links such as disulfide bonds influence the stability of the proteins by decreasing the entropy gain as well as reduction of SASA of unfolded state. The aim of the study was to develop mathematical models to predict the effect of cross links on delta SASA and ultimately on meq and delta Cp based on in silico methods. Changes of SASA upon computationally simulated unfolding were calculated for a set of 45 proteins with known meq and delta Cp values and the effect of cross links on delta SASA of unfolding was investigated. The results were used to predict the meq of denaturation for guanidine hydrochloride and urea, as well as delta Cp for the studied proteins with overall error of 20%, 31% and 17%, respectively. The results of the current study were in close agreement with those obtained from the previous studies

In vitro refolding process of bovine allergen β-lactoglobulin by Multispectroscopic method / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To characterize the relationship between the refolding process of recombinant bovine β-lactoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation.</p><p><b>METHODS</b>The refolding process of recombinant bovine β-lactoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro.</p><p><b>RESULTS</b>Renaturation of recombinant bovine β-lactoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity.</p><p><b>CONCLUSION</b>The degree of protein renaturation correlated with the IgE-binding capacity of the protein. Results from this study may be of help for food allergy therapy and development of vaccination in the future.</p>

Nutrient digestibility in Arctic fox (Vulpes lagopus) fed diets containing animal meals / Digestibilidade dos nutrientes em raposa polar (Vulpes lagopus) alimentada com dietas que continham farinhas de origem animal

Three digestibility experiments on Arctic foxes were carried out. Control groups were fed standard diets (C1 and C2) composed of fresh or frozen animal by-products and steamed ground grain. Dry experimental diets (E1 and E2) contained animal meals, extracted meals and fat, were mixed with water prior to administration. In a preliminary experiment, the digestibility of dry diet E1 moistened with water for 15min and 24h was compared to determine the optimum moistening time during the experimental period proper. The preliminary experiment showed that moistening time had no significant effect on digestibility. In the main experiment, two independent digestibility trials were performed to compare the digestibility of diets fed to foxes during growth (C1 vs. E1) and fur development (C2 vs. E2). Better nutrient digestibility was noted for control diets, compared to experimental. The greatest differences were reported for total protein digestibility. Protein contained in meals undergoes denaturation during heat treatment, which reduces digestibility. It was found that the retention of nitrogen in relation to nitrogen digestion was higher in foxes fed experimental diets (E1 and E2).

Realizaram-se três ensaios de digestibilidade em raposas polares. Os grupos controles receberam dietas-padrão (C1 e C2) compostas por subprodutos de origem animal frescos ou congelados e sementes de cereais em grão. As dietas secas (E1, E2) usadas nos ensaios que continham farinha de origem animal e sementes em grãos extrudadas eram hidratadas antes de administradas. Em ensaio preliminar, avaliou-se a digestibilidade da dieta seca E1, submetida a 15 minutos e a 24 horas de hidratação. Verificou-se que o tempo de hidratação não influenciou a digestão. No experimento principal, foram realizados dois ensaios para comparar a digestibilidade das dietas fornecidas às raposas no período de crescimento (C1 vs E1) e no desenvolvimento de pelo (C2 vs E2). Melhor digestibilidade dos nutrientes foi observada para as dietas controle. As maiores diferenças foram relatadas para a proteína total. A desnaturação das proteínas, durante o tratamento térmico, reduz o índice de digestibilidade das dietas. Observou-se alto teor de retenção de hidrogênio em relação ao hidrogênio digerido nas raposas alimentadas com as dietas E1 e E2.

Effects of solvent environment on the structure of hepatitis B surface antigen (HBsAg) / 生物工程学报

As a virus-like particle, hepatitis B surface antigen (HBsAg) was the primary component of hepatitis B vaccine. HBsAg was maintained by the non-covalent interaction of proteins and lipids. The intact structure of HBsAg particle was vital to its function. However, there was no report about the effects of solvent environment on HBsAg structure. In this paper, we studied the effects of temperature, pH, ionic type and salt concentration on HBsAg structure. The results showed that HBsAg was stable at normal temperature, but began to denature above 60 degrees C. The aggregation of HBsAg at pH 3.0 and 4.0 was nearly irreversible, but partly reversible at pH 5.0. The influence of ionic type on HBsAg was generally in accordance with Hofmeister sequence, except that SO4(2-) caused more aggregation than F-. HBsAg aggregates started to be visible in 0.4 mol/L (NH4)2SO4, and the extent of aggregation increased with the salt concentration. Therefore, caution must be taken when using (NH4)2SO4 in the hydrophobic chromatography purification of HBsAg.

comparison of milk protein status of healthy and mastitic cows under denaturing conditions

Mastitis is one of the most important diseases of dairy cattle in the world. The identification and characterization of the constituent proteins in milk can be useful for studying the biochemistry and pathogenesis of mastitis. In this study, the electrophoretic patterns of milk from 10 healthy and 30 mastitic cows were studied. All of the latter milk samples were California Mastitis Test [CMT] positive, and these were cultured to isolate the infective agents. The electrophoretic patterns of these samples and those of healthy cows [negative CMT and cultures] were studied with the SDS-PAGE technique. The approximate molecular weight of protein bands were categorized by their different flow rates [Rf], and these ranged between 18.5 - 220 KDa in mastitis samples of milk. The electrophoretogram showed that higher molecular weight bands appeared in the milk of mastitic cows60-220KDa and many were in the range of 176-208 kDa.The major band for the healthy samples was 220 KDa. In this respect, the mastitis samples had a minimum of two bands and a maximum of five bands, while milks from healthy cows did not show any bands in this range. On the basis of the different result between the electrophoretic patterns of milk from healthy and mastitic cows, it can be concluded that SDS-PAGE is a suitable method for the diagnosis of cows withsub sub clincal mastitis

Anti-calcification Effects in Decellularized and Variously Fixed Bovine Pericardium / 대한흉부외과학회지

BACKGROUND: Our goal was to evaluate anti-calcification effects of decellularization and diverse fixing methods including preincubation of the bovine pericardium with ethanol. We also assessed changes in mechanical properties. MATERIAL AND METHOD: Harvested bovine pericardium was decellularized with 0.25% sodim dodecysulfate and then treated with 5 methods of fixation: (1) 0.5% glutaraldehyde (GA) for 14 days, (2) 0.5% GA for 5 days, 2% GA for 2 days and 0.25% GA for 7 days, (3) 0.5% GA for 5 days, 2% GA for 2 days, 0.25% GA for 7 days, and then 70% ethanol for 2 days, (4) 0.5% GA for 5 days, a mixture of 2% GA and 70% ethanol for 2 days, and 0.25% GA for 7 days, (5) 0.5% GA for 5 days, a mixture of 2% GA, 65% ethanol, and 5% octanediol for 2 days and then 0.25% GA for 7 days. All treated bovine pericardia were tested for histological variables, lipid content, and mechanical properties including tensile strength and thermal stability. A total 10 kinds of differently treated bovine pericardia were implanted into rat subdermis and harvested 8 weeks later. Harvested pericardia were evaluated for calcium content. RESULT: No protein denaturation was observed microscopically after decellularization. There was a 32% mean decrease in tensile strength index after decellularization in the bovine pericardium group fixed. Octanediol preincubation attenuated the decrease in tensile strength and maintained thermal stability. TG and cholesterol were not affected by decellularization but were decreased by organic solvent. Calcium content was decreased after decellularization, and organic solvent preincubation decreased calcification in the non-decellularized bovine pericardium group. CONCLUSION: Decellularization and organic solvent preincubation have anti-calcification effects but decellularization may cause mechanical instability. A method of decellularization and fixation that does not cause damage to matrices will be needed for evaluation of the next step in using tissue-engineering for replacement of cardiac valves.

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

The in vitro anti-denaturation effects induced by natural products and non-steroidal compounds in heat treated (immunogenic) bovine serum albumin is proposed as a screening assay for the detection of anti-inflammatory compounds, without the use of animals, in the early stages of the drug discovery process / Los efectos de la anti-desnaturalización in vitro inducida por productos naturales y compuestos no esteroidales en la albúmina sérica bovina (inmunogénica) tratada con calor, se proponen aquí como ensayo de pesquizaje para la detección de compuestos inflamatorios sin el uso de animales en las etapas tempranas del proceso de descubrimiento del medicamento

There are emerging ethical issues with regards to the use of animals in the early stages of drug discovery for anti-inflammatory and degenerative diseases from natural products using the activity-directed isolation pathways when many compounds (eg > 100) are present in the crude extract or fraction and are to be tested. The above-mentioned is the main reason for proposing the use of the in vitro anti-denaturation (stabilization) effects of heat treated (immunogenic) bovine serum albumin (BSA) as an assay. Current methods used for detecting and isolating a wide range of anti-inflammatory compounds in the early stages of the drug discovery process utilize a large number of animals. When BSA is heated and is undergoing denaturation, it expresses antigens associated to Type III hyper-sensitive reaction and which are related to diseases such as serum sickness, glomerulonephritis, rheumatoid arthritis and systemic lupus erythematosus. Thus, the assay that is being proposed should be applicable to the discovery of drugs for treating the above mentioned diseases and others, once the compounds stabilize the denaturation process.

Actualmente surgen problemas éticos en relación con el uso de animales en las etapas tempranas del descubrimiento de medicamentos anti-inflamatorios y contra enfermedades degenerativas, a partir de productos naturales, usando vías de aislamiento dirigido por actividad, cuando muchos compuestos están presentes (p.ej. > 100) en la fracción o extracto crudo, y deben ser probados. Lo anterior representa la razón principal para proponer el uso de los efectos de la anti-desnaturalización (estabilización) in vitro de la albúmina sérica bovina (inmunogénica) tratada con calor (ASB) como ensayo. Los métodos corrientes usados para detectar y aislar una amplia gama de compuestos anti-inflamatorios en las etapas tempranas del proceso de descubrimiento del medicamento, utilizan un gran número de animales. Cuando la ASB es calentada y sometida a un proceso de desnaturalización, expresa antígenos en relación con la reacción hipersensitiva de tipo III, relacionada a su vez con enfermedades tales como la enfermedad del suero, la glomerulonefritis, la artritis reumatoide, y el lupus sistémico y eritematoso. De este modo, el ensayo que aquí proponemos debe ser aplicable al descubrimiento de medicamentos para el tratamiento de las enfermedades anteriormente mencionadas y otras, una vez que los compuestos estabilicen el proceso de desnaturalización.

Protein content in irrigating solutions in contact with pulp tissue

Las soluciones de irrigación endodóntica pueden tener distintas accionesLas soluciones de irrigación endodóntica pueden tener distintas acciones. Una de ellas es la de disolver el tejido pulpar. Seevaluó in vitro la capacidad de diferentes soluciones de irrigaciónpara disolver tejido pulpar vital y necrótico mediante un estudio cuanti y cualitativo de proteínas pulpares totales solubles. Se utilizaron pulpas de dientes de bovinos jóvenes, vitales y con necrosis inducida. Se seccionaron en trozos pequeños, que se pesaron y colocaron en 1 ml de las siguientes soluciones: hipoclorito de sodio al 1por ciento y 2,5 por ciento, hidróxido de calcio al 1 por ciento y 5 por ciento, gluconato de clorhexidina al 0,2 por ciento, té al 1 por ciento y aguadestilada (control). Se colocaron a 37°C y se extrajeron muestras de 20 μl a luego de 30 min, 90 min y 20 hs. Se dosaron proteínas totales utilizando el método de Lowry y por electroforesis(SDS-Page 12 por ciento). Se determinaron bandas de proteínassolubles. Los resultados se analizaron por Anova En el analisis químico, de las corridas electroforéticas de proteínaspulpares bovinas se evidenció que tanto el hipoclorito de sodio en ambas concentraciones como el hidróxido de calcio producen desnaturalización de proteínas. No se demuestra acción solvente con clorhexidina, té y agua destilada.

Optimization of induction and purification of HIV-1 Gag protein in Escherichia coli expression system / 生物工程学报

To investigate the effects of induction temperature on the expression product and the impact of urea concentration on the purification, HIV-1 Gag inclusion bodies from E. coli induced at 30 degrees C (IB30) and 37 degrees C (IB37) were dissolved with urea of different concentrations. The solubility and yield of refolding were compared. IB30 were dissolved with 2 mol/L and 8 mol/L urea, and then purified with chromatography. IB30 were found easier to be solubilized in low concentration of urea and easier to be refolded than IB37. Furthermore, compared to the IB30 dissolved in 8 mol/L urea, Gag protein solubilized in 2 mol/L urea was purified to higher purity with gel filtration (GF) and ion exchange (IEX) chromatography. Gag inclusion body induced at lower temperature may contain more protein with native-like or reversibly-denatured structures, and solubilization in the presence of low concentrations of urea can help to retain these structures. This study has provided new insights into the purification of proteins from inclusion bodies.

Dynamic light scattering study of peanut agglutinin: size, shape and urea denaturation.

Peanut agglutinin (PNA)is a homotetrameric protein with a unique open quaternary structure. PNA shows non-two state profile in chaotrope induced denaturation. It passes through a monomeric molten globule like state before complete denaturation (Reddy et al 1999). This denaturation profile is associated with the change in hydrodynamic radius of the native protein. Though the molten globule-like state is monomeric in nature it expands in size due to partial denaturation. The size and shape of the native PNA as well as the change in hydrodynamic radius of the protein during denaturation has been studied by dynamic light scattering (DLS). The generation of two species is evident from the profile of hydrodynamic radii. This study also reveals the extent of compactness of the intermediate state.

The effect of some osmolytes on the activity and stability of mushroom tyrosinase.

The thermodynamical stability and remained activity of mushroom tyrosinase (MT) from Agaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40 degrees C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obey the first order law. Inactivation rate constant (kinact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of kinact (0.7x10(-4) s-1) in comparison with their absence (2.5x10(-4) s-1). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodynamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.

Effect of denaturants on the structure and activity of 3-hydroxybenzoate-6-hydroxylase.

The effect of denaturants such as urea, sodium dodecylsulphate (SDS), guanidinium hydrochloride (Gu.HCl) on the structure of enzyme 3-hydroxybenzoate-6-hydroxylase was studied using intrinsic fluorescence and far and near-UV-CD spectroscopic techniques. Also, activity profiles of the enzyme, as a function of increasing concentrations of denaturants were studied. The far-UV CD spectrum of the enzyme did not show appreciable alterations in the presence of urea, SDS or Gu.HCl, thereby suggesting that the protein does not undergo gross conformational changes in its alpha-helical secondary structure. The treatment of enzyme with 2 M urea resulted in almost complete loss of catalytic activity, accompanied by the reduction of emission fluorescence of enzyme. Similarly, treatment with 0.01% SDS also caused almost complete loss of activity and quenching of enzyme fluorescence as well as a red shift in the emission peak. In addition, reduction in the intensity of near-UV-CD spectrum, especially at 280 nm was observed. About 70% of the activity was lost by treatment with 20 mM Gu.HCl, accompanied by quenching of intrinsic fluorescence of the enzyme. The change in intrinsic fluorescence of the enzyme in the presence of 5 mM-100 mM Gu.HCI could be correlated to progressive loss of catalytic activity. Thus, intrinsic fluorescence (due to tryptophan residues) could be used as an effective probe to provide an insight into the relation between the activity and subtle conformational changes of the enzyme. The results suggested that denaturants caused very slight conformational changes in the enzyme that perturbed the microenvironment of aromatic amino acid residues such as tryptophan accompanied by reduction or loss of catalytic activity.

Effect of some cryoprotectant agents on physiochemical properties of frozen stored bolti [Tilapia Nilotica] fish fillets

The effectiveness of four cryoprotectant agents i.e. mixture of sucrose and sorbitol 8% [w/v] [S/S], sodium lactate 8% [w/v] [SL], sodium tripolyphosphate 11.8% [w/v] [STPP], and polydextrose 8% [w/v] [PDT] on protein denaturation of bolti fish fillets during freezing and frozen storage for 6 months were studied. The parameters of extractable proteins [salt and water-soluble factions], water holding capacity, total sulfhydryl content, and expressible moisture [drip under pressure] content were used to measure the protein denaturation. The used cryoprotectant agents were significantly [p<0.5] retarded protein denaturation during freezing storage [6 months]. Among all cryoprotectants used, polydextrose exhibited the greatest protective effect on protein denaturation as shown by the effectiveness in maintaining protein solubility. Sucrose/sorbitol provided more effective cryoprotection than sodium lactate in preservation of protein structure. Polydextrose and sodium lactate appeared to be promising alternative cryoportectants for fish muscle and its products owing to their low sweetness and caloric value

Estabilização de proteínas por osmólitos / Protein stabilization by osmolytes

Os osmólitos constituem um grupo de solutos de baixo peso molecular envolvidos na estabilização protéica, em resposta a condições ambientais de estresse. Esses solutos, encontrados mesmo em organismos bastante distantes dentro da escala evolutiva, forçam as proteínas a se enovelarem e a resistirem a desnaturação. Evidências sugerem que os osmólitos atuariam modificando as propriedades do solvente em que a proteína encontra-se imersa, afetando desta forma as interações que a superfície da proteína estabelece com o solvente.