RESUMEN
In this study we explore the preparation of core-crosslinked micelles of linear-dendritic methoxy-poly(ethylene glycol) (MPEG)-co-poly(ester-sulfide) (PES) polymers to improve the stability of such polymeric micelle systems against premature disintegration and drug release. A series of MPEG-PES copolymers were synthesised via stepwise reactions of acetylation and thiol-ene photoreaction. Surface tension measurement showed that the copolymers with ethenyl surface groups could self-associate in dilute aqueous solutions to form micelles. Crosslinking within the micelle cores in the presence of dithioerythritol (DTT) linker was initiated under UV radiation. The formation of core-crosslinked micelles was confirmed by HPLC in combination with charged aerosol detection (CAD). The copolymers were found to readily hydrolyse under acidic conditions due to the ester-containing dendrons. Drug solubilisation capacities of the micellar solutions were determined using griseofulvin as a poorly water-soluble model drug. The solubility of griseofulvin showed a 10-fold enhancement in 1% w/v micelle solution and increased with the concentration of the copolymers. Drug release studies indicated that a more sustained release of griseofulvin was achieved for the core-crosslinked micelles compared to the non-crosslinked micelles, attributable to greater stability of the crosslinked core structure. The findings of this study present a new pathway towards developing biodegradable polymeric nanocarriers.
Asunto(s)
Dendrímeros/química , Micelas , Poliésteres/química , Polietilenglicoles/química , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/efectos de la radiación , Dendrímeros/efectos de la radiación , Ditioeritritol/química , Ditioeritritol/efectos de la radiación , Liberación de Fármacos , Griseofulvina/química , Poliésteres/efectos de la radiación , Polietilenglicoles/efectos de la radiación , Propano/análogos & derivados , Propano/química , Propano/efectos de la radiación , Solubilidad , Rayos UltravioletaRESUMEN
The inherently present seventeen disulfide bonds of the circulatory protein, human serum albumin (HSA) provide the necessary structural stability. Various spectroscopic approaches were used to investigate the effect of reduction of these disulfide bonds and its binding with the anionic surfactant, sodium dodecyl sulfate (SDS). Based on several spectroscopic analyses, our investigations highlight the following interesting aspects: (1) HSA on reduction loses not only its tertiary structure but also a significant amount of secondary structure as well. However, the reduced state of the protein is not like the molten-globule, (2) this structural loss of the protein due to reduction is more prominent than that caused by higher SDS concentrations alone and can certainly be attributed to the role of disulfide bonds, (3) lower surfactant concentrations provide marginal structural rigidity to the native state of the protein, whereas, higher concentrations of SDS induces secondary structure to the reduced state of HSA, (4) the binding of SDS with both the native and reduced states of HSA, occurred in three distinct stages which was followed by a saturation stage. However, the nature of such binding is different for both the states as investigated by using the Stern-Volmer equations and estimating the thermodynamic parameters. Besides, in contrast to the native state, the reduced state of HSA shows that the lone tryptophan residue gets more buried. However, there occurs a sudden decrement in the lifetime of the tryptophan and the hydrodynamic diameter increases by twofold.
Asunto(s)
Albúmina Sérica/química , Dodecil Sulfato de Sodio/química , Tensoactivos/química , Dicroismo Circular , Disulfuros/química , Ditioeritritol/química , Dispersión Dinámica de Luz , Humanos , Unión Proteica , Estabilidad Proteica , Espectrometría de Fluorescencia , Reactivos de Sulfhidrilo/químicaRESUMEN
The broad interest of using reversible covalent bonds in chemistry, in particular at its interfaces with biology and materials science, has been recently established through numerous examples in the literature. However, the challenging exchange of peptide fragments using a dynamic covalent peptide bond has not yet been achieved without enzymatic catalysis because of its high thermodynamic stability. Here we show that peptide fragments can be exchanged by a chemoselective and reversible native chemical ligation (NCL) which can take place at N-(methyl)-cysteine residues. This very mild reaction is efficient in aqueous solution, is buffered at physiological pH in the presence of dithiothreitol (DTT), and shows typical half-times of equilibration in the 10 h range.
Asunto(s)
Cisteína/análogos & derivados , Péptidos/síntesis química , Cisteína/síntesis química , Cisteína/química , Ditioeritritol/química , Péptidos/química , TermodinámicaRESUMEN
Biodegradable segmented polyurethanes were prepared with poly(caprolactone) diol as a soft segment, 4,4'-methylene bis(cyclohexyl isocyanate) (HMDI) and either butanediol or dithioerythritol as chain extenders. Platelet adhesion was similar in all segmented polyurethanes studied and not different from Tecoflex® although an early stage of activation was observed on biodegradable segmented polyurethane prepared with dithioerythritol. Relative viability was higher than 80% on human umbilical vein endothelial cells in contact with biodegradable segmented polyurethane extracts after 1, 2 and 7 days. Furthermore, both biodegradable segmented polyurethane materials supported human umbilical vein endothelial cell adhesion, spreading, and viability similar to Tecoflex® medical-grade polyurethane. These biodegradable segmented polyurethanes represent promising materials for cardiovascular applications.
Asunto(s)
Materiales Biocompatibles/metabolismo , Adhesividad Plaquetaria/efectos de los fármacos , Poliuretanos/metabolismo , Venas Umbilicales/citología , Materiales Biocompatibles/química , Plaquetas/citología , Plaquetas/efectos de los fármacos , Butileno Glicoles/química , Butileno Glicoles/metabolismo , Cianatos/química , Cianatos/metabolismo , Ditioeritritol/química , Ditioeritritol/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ensayo de Materiales , Poliésteres/química , Poliésteres/metabolismo , Poliuretanos/química , Venas Umbilicales/efectos de los fármacosRESUMEN
DNA extracted from keratinous and chitinous materials can be a useful source of genetic information. To effectively liberate the DNA from these materials, buffers containing relatively high levels of DTT, proteinase K, and detergent are recommended, followed by purification using either silica-column or organic methods.
Asunto(s)
Quitina/química , ADN/aislamiento & purificación , Fósiles , Queratinas/química , Animales , ADN/metabolismo , Ditioeritritol/química , Endopeptidasa K/metabolismo , Plumas/química , Cabello/química , Dióxido de Silicio/químicaRESUMEN
Phytosterols (PS; ß-sitosterol and campesterol) and cholesterol precursors (CP; desmosterol and lathosterol) have been suggested as important biochemical markers of cholesterol intestinal absorption and liver biosynthesis, respectively. Given that these compounds appear in human blood in low amounts, sensitive and accurate methodology is required, such as gas chromatography-mass spectrometry (GC-MS) the most frequently used. One of the most critical factors of the GC analytical determination is the step of derivatization. Thus, the main objective of the present study was compare and select the better (one out of three) silylation mixtures as follows: N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide/ammonium iodide (MTBSTFA:NH(4)I), N-O-bis-(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane (BSTFA:TMCS), and N-methyl-N-(trimethylsilyl)-trifluoroacetamide/1,4-dithioerythritol/trimethyliodosilane (MSTFA:DTE:TMIS). The results of this study are discussed and accompanied by a brief review on the importance and principles of derivatization process, specifically in silylation reactions in GC-MS sterols analyses. Furthermore, a scrutiny of some published results is presented, along with additional information about mass spectral data of these potentially useful compounds. Interestingly, the results of the study showed that from the three validated methodologies, the selected one, based on the best relation specificity/sensibility, is MSTFA:DTE:TMIS. With this silylation procedure for simultaneous determination of PS and CP in human serum by GC-MS in selected ion monitoring (SIM) mode, good linearity (r(2)≥0.931), precision (repeatability ranging from 0.92 to 3.91 CV and intermediate precision ranging from 5.12 to 6.33) and recoveries (≥93.6%) were obtained. Thus, it proved to be a helpful methodology in the quantification of predominant serum cholesterol origin in each patient.
Asunto(s)
Colesterol/sangre , Desmosterol/sangre , Cromatografía de Gases y Espectrometría de Masas/métodos , Indicadores y Reactivos/química , Fitosteroles/sangre , Acetamidas , Colesterol/metabolismo , Ditioeritritol/química , Fluoroacetatos , Humanos , Modelos Lineales , Compuestos de Organosilicio , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Ácido Trifluoroacético/química , Compuestos de Trimetilsililo/químicaRESUMEN
A ternary surface monolayer, consisting of coassembled thiolated capture probe, mercaptohexanol and dithiothreitol, is shown to offer dramatic improvements in the signal-to-noise characteristics of electrochemical DNA hybridization biosensors based on common self-assembled monolayers. Remarkably low detection limits down to 40 zmol (in 4 µL samples) as well as only 1 CFU Escherichia coli per sensor are thus obtained without any additional amplification step in connection to the commonly used horseradish peroxidase/3,3',5,5'-tetramethylbenzidine system. Such dramatic improvements in the detection limits (compared to those of common binary alkanethiol interfaces and to those of most electrochemical DNA sensing strategies without target or signal amplification) are attributed primarily to the remarkably higher resistance to nonspecific adsorption. This reflects the highly compact layer (with lower pinhole density) produced by the coupling of the cyclic- and linear-configuration "backfillers" that leads to a remarkably low background noise even in the presence of complex sample matrixes. A wide range of surface compositions have been investigated, and the ternary mixed monolayer has been systematically optimized. Detailed impedance spectroscopy and cyclic voltammetric studies shed useful insights into the surface coverage. The impressive sensitivity and high specificity of the simple developed methodology indicate great promise for a wide range of nucleic acid testing, including clinical diagnostics, biothreat detection, food safety, and forensic analysis.
Asunto(s)
Técnicas Biosensibles/métodos , ADN/análisis , Ditioeritritol/química , Hibridación de Ácido Nucleico/métodos , Compuestos de Sulfhidrilo/química , Bencidinas/química , Técnicas Electroquímicas/métodos , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/diagnóstico , Humanos , Límite de Detección , Masculino , ARN Bacteriano/análisisRESUMEN
The phthalodinitrile derivative 3 was prepared by the reaction of 1,4-dithioerythritol 1 and 4-nitrophthalonitrile 2 in dry DMF as the solvent in the presence of K(2)CO(3) as the base by the method of nucleophilic substitution of an activated nitro group in an aromatic ring. The template reaction of compound 3 with the corresponding metal salts gave the novel binuclear MPcs of ball-type (M = Zn 4, Co 5, Cu 6) and their water soluble phthalocyanines 7-9 were obtained from refluxing a suspension of the compounds bearing eight OH side groups, in aqueous NaOH (%30) solution. Newly synthesized compounds were characterized by elemental analysis, UV/VIS, IR, MALDI TOF mass and (1)H-NMR spectroscopy techniques. The electronic spectra exhibit an intense πâπ* transition of characteristic Q and B bands of the phthalocyanine core. The electrochemical measurements showed the formation of various mixed-valence oxidation and reduction species of 4 and 6 due to weak intramolecular interactions between the two MPc units. Complex 5 displayed a much higher catalytic activity than those of 4 and 6. It was found that oxygen reduction on the 5-based catalyst occurs through a direct 4-electron transfer pathway with a high water selectivity. However, the overpotential for oxygen reduction is high, probably due to a long distance between the two CoPc units in 5. A.c. and d.c. conductivity measurements were performed as a function of temperature (300-543 K) and frequency (40-10(5) Hz). It was found from d.c. measurements that the values of the pre-exponential factor σ(0) for the investigated samples are in the interval from 1.36 × 10(-3) to 6.20 × 10(2)Ω(-1) cm(-1), inferring that the conduction occurs most probably by hopping between the localized states in band tails. Based on the existing theory of a.c. conduction, it has been concluded that for the low frequency region the dominant conduction mechanism is multihopping at high temperatures (>390 K) whereas for the high frequency region the correlated barrier hopping model is the dominant mechanism. The sensing properties of the films for CO(2) gas were also investigated.
Asunto(s)
Dióxido de Carbono/análisis , Técnicas de Química Analítica/instrumentación , Ditioeritritol/química , Electricidad , Indoles/química , Compuestos Organometálicos/química , Agua/química , Catálisis , Electroquímica , Polímeros de Fluorocarbono/química , Isoindoles , Compuestos Organometálicos/síntesis química , Oxidación-Reducción , Oxígeno/química , Protones , Solubilidad , Propiedades de SuperficieRESUMEN
Comparison of the effectiveness of antioxidant activity of three thiol compounds, D-penicillamine, reduced L-glutathione, and 1,4-dithioerythritol, expressed as a radical-scavenging capacity based on the two independent methods, namely a decolorization 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] assay and a rotational viscometry, is reported. Particular concern was focused on the testing of potential free-radical scavenging effects of thiols against hyaluronan degradation, induced by hydroxyl radicals. A promising, solvent-independent, antioxidative function of 1,4-dithioerythritol, comparable to that of a standard compound, Trolox(®), was confirmed by the 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] assay. The new potential antioxidant 1,4-dithioerythritol exhibited very good solubility in a variety of solvents (e.g., H(2)O, EtOH, and DMSO) and could be widely accepted and used as an effective antioxidant standard instead of a routinely used Trolox(®) on 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] assay.
Asunto(s)
Antioxidantes/química , Cromanos/química , Evaluación Preclínica de Medicamentos/métodos , Ácido Hialurónico/química , Compuestos de Sulfhidrilo/química , Ácidos Sulfónicos/química , Tiazoles/química , Antioxidantes/farmacología , Benzotiazoles , Cromanos/farmacología , Ditioeritritol/química , Depuradores de Radicales Libres/química , Glutatión/química , Ácido Hialurónico/farmacología , Radical Hidroxilo/química , Oxidación-Reducción , Penicilamina/química , Solubilidad , Solventes , Espectrofotometría , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/farmacología , ViscosidadRESUMEN
A strategy for the rational design of a novel colorimetric sensor based on dithioerythritol-modified gold nanoparticles for the selective recognition of Hg2+ in aqueous media is presented. This approach relies on the combination of gold nanoparticles with Hg2+ through sulfur-Hg2+-sulfur interaction. The gold nanoparticles showed high selectivity toward Hg2+ with binding-induced red shift in the absorption spectra, with no response to major interfering cations such as Pb2+, Cd2+, and Cu2+ in the presence of ethylenediamine tetraacetic acid. The system responds to Hg2+ with a detection limit of 100 nM and might open a new avenue for the development of Hg2+ sensing probes.
Asunto(s)
Ditioeritritol/química , Oro/química , Mercurio/análisis , Nanopartículas del Metal/química , Calorimetría/métodos , Sensibilidad y EspecificidadRESUMEN
In this study, we describe the use of Tween 20-capped gold nanoparticles (AuNPs) as selective probes for the extraction of aminothiols from an aqueous solution. Tween 20 molecules noncovalently attached to the surface of AuNPs to form Tween 20-AuNPs were used for the selective extraction of aminothiols through the formation of Au-S bonds. After extraction and centrifugation, the aminothiols were detached from the surface of the AuNPs by adding DTT in a high concentration. We used this probe in combination with CE and UV absorption detection. On-line concentration and separation of the released aminothiols were performed by using 1.6% v/v poly(diallyldimethylammonium chloride) as an additive in CE. Under optimal extraction and stacking conditions, the LOD at a S/N of 3 were 28, 554, and 456 nM for glutathione (GSH), cysteine (Cys), and homocysteine (HCys), respectively. In comparison with the normal injection without the extraction procedure, approximately 2280-, 998-, and 904-fold improvements in the sensitivity were observed for GSH, Cys, and HCys, respectively. We have validated the application of our method on the basis of the analysis of GSH and HCys in human urine samples. It is believed that this approach has significant potential to be extended to clinical diagnosis.
Asunto(s)
Aminas/química , Electroforesis Capilar/métodos , Nanopartículas/química , Compuestos de Sulfhidrilo/química , Aminas/orina , Cisteína/orina , Ditioeritritol/química , Glutatión/orina , Oro/química , Homocisteína/orina , Humanos , Masculino , Polisorbatos/química , Sensibilidad y Especificidad , Espectrofotometría Ultravioleta , Compuestos de Sulfhidrilo/orinaRESUMEN
The aqueous solutions of arsenous acid with the meso and racemic forms of 1,4-dithiol-butane-2,3-diol, namely, dithioerythritol (dte) and dithiothreitol (dtt), respectively, were titrated pH-metrically in different molar ratios. The p K a values determined for As(OH) 3, and dtt were in good accordance with the literature data, and we determined for the first time the p K a value of dte. The deprotonation steps of both M (As(OH) 3 considered as a central metal ion) and H 2L components dte and dtt (considered as ligands) appeared at a higher pH in the titration curves of the ternary systems (M, H 2L, H (+)) than in the individual component. This unusual observation is explained by the condensation reactions between the reagents taking place in the pH < 8 range. In the solutions of c As(III) > 5.10 (-3) M, the precipitate formed upon mixing the arsenous acid and H 2L solutions in neutral medium, and the formation of the precipitate shifted toward acidic pH on the increase of the total concentrations. This indicated that pH-metry can follow the reactions only in an indirect way. Useful, but not satisfactory, information can be obtained by means of this method alone. Combined with NMR and UV spectroscopic measurements it is revealed that depending on the As(III)/H 2L molar ratio, different complexes form in the solutions. In the species with 1:2 composition, one of the ligands is strongly bound to the arsenic(III) probably via its two thiolate, while the second one is attached only weakly. The crystal structure of an As(III)-dte crystal of 1:1 composition, grown from ethanolic solution, shows that As(III) binds the ligand through its three p-orbitals in a manner similar to that expected in aqueous solution. While the uptake of the second ligand cannot be detected by pH-metry, the decomposition of thioether bonds above pH approximately 10 is confirmed by the change in UV spectra at approximately 265 nm to be a base-consuming process. In such alkaline solutions, most probably, rearrangement of the bonding scheme occurs, resulting in ligands being bound to the arsenic(III) through the oxygen donor atoms.
Asunto(s)
Arsenitos/química , Ditioeritritol/química , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Cinética , Resonancia Magnética Nuclear Biomolecular , Espectrofotometría UltravioletaRESUMEN
Plasma protein adsorption patterns on surfaces may give vital information to evaluate biocompatibility of biomaterials designed for direct blood-contacting applications or tissue integration. Adsorption of human serum proteins on four different types of biomaterials (glass, aminosilanized glass, hyaluronan and sulfated hyaluronan) was analyzed by two-dimensional electrophoresis. Desorption of proteins from the surfaces was first classically achieved by sodium dodecyl sulfate (SDS) elution. We introduced a second elution step (by use of isoelectric focusing (IEF) sample buffer consisting of urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate, and dithioerythritol) which allows more stringent elution conditions and is a tool to evaluate the protein adsorption strength to biomaterials. Moreover, the two-step elution may discriminate between irreversible and reversible adsorption of plasma proteins for biomaterials, thus helping to elucidate the structure of protein multilayers which form a complex system at the surfaces. The IEF sample buffer proved not to alter the biomaterial structure and integrity. Hydrophobic bonds resulted to be the main strength driving protein adsorption onto our biomaterials. Apolipoproteins were the most important proteins interacting with the surfaces suggesting that high-density lipoprotein (HDL) particles could play a role in biocompatibility due to their beneficial effects on endothelial cells.
Asunto(s)
Apolipoproteínas/química , Materiales Biocompatibles/química , Proteínas Sanguíneas/análisis , Ácido Hialurónico/química , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Sanguíneas/química , Ditioeritritol/química , Electroforesis en Gel Bidimensional , Vidrio/química , Humanos , Dodecil Sulfato de Sodio/química , Espectroscopía Infrarroja por Transformada de Fourier , Urea/químicaRESUMEN
Bacterial phytochromes (Bphs) are ancestors of the well characterized plant photoreceptors. Whereas plant phytochromes perform their photoisomerization exclusively via a covalently bound bilin chromophore, Bphs are variable in their chromophore selection. This is demonstrated in the cyanobacterium Calothrix PCC7601 that expresses two Bphs, CphA and CphB. CphA binds phycocyanobilin (PCB) covalently, whereas CphB, lacking the covalently binding cysteine of the plant phytochromes, carries biliverdin IXalpha (BV) as the chromophore. Our experiments elucidate the different modes of chromophore-protein interaction in CphA and CphB and offer a rationale for their chromophore selectivity. The tight binding of BV by CphB prevents PCB from competing for the binding cavity. Even when the chromophore-binding cysteine has been inserted (CphB-mutant L266C), PCB replaces BV very slowly, indicating the tight, but not irreversible binding of BV. The mutant CphB L266C showed a redox-sensitivity with respect to its PCB binding mode: under reducing conditions, the chromoprotein assembly leads to spectra indicative for a covalent binding, whereas absence of dithiothreitol or its removal prior to assembly causes spectra indicative for noncovalent binding. Regarding the CphB-type Bphs lacking the covalently binding cysteine, our results support the involvement of the succeeding histidine residue in chromophore fixation via a Schiff base-like bond between the bilin A-ring carbonyl and the histidine imidazole group. The assembly process and the stability of the holo-proteins were strongly influenced by the concentration of added imidazole (mimicking the histidine side-chain), making the attachment of the chromophore via the histidine more likely than via another cysteine of the protein.
Asunto(s)
Cianobacterias/metabolismo , Fitocromo/metabolismo , Sustitución de Aminoácidos , Biliverdina/metabolismo , Sitios de Unión , Unión Competitiva , Cianobacterias/química , Cianobacterias/genética , Cisteína/genética , Cisteína/metabolismo , Oscuridad , Ditioeritritol/química , Histidina/genética , Histidina/metabolismo , Cinética , Oxidación-Reducción , Ficobilinas , Ficocianina/metabolismo , Fitocromo/genética , Unión Proteica , Pirroles/metabolismo , Espectrofotometría/métodos , TetrapirrolesRESUMEN
The oxidative folding pathway(s) of single-domain proteins can be characterized by the existence, stability, and structural nature of the intermediates that populate the regeneration pathway. Structured intermediates can be disulfide-secure in that they are able to protect their existing (native) disulfide bonds from SH/SS reshuffling and reduction reactions, and thereby form the native protein directly, i.e., by oxidation of their exposed (or locally exposable) thiols. Alternatively, they can be disulfide-insecure, usually requiring global unfolding to expose their free thiols. However, such an unfolding event also exposes the existing native disulfide bonds. Thus, the subsequent oxidation reaction to form the native protein in a disulfide-insecure intermediate competes with the intramolecular attack by the thiols of the macromolecule on its own native disulfide bonds, resulting in a large population of intermediates that are reshuffled instead of being oxidized. Under stabilizing conditions, disulfide-insecure species become long-lived kinetically trapped intermediates that slowly and only indirectly convert to the native protein through reshuffling reactions. In this study, trans-[Pt(en)(2)Cl(2)](2+), a strong oxidizing agent which has not traditionally been used in oxidative folding, was applied to shift the competition between reshuffling and oxidation reactions in des [58-110] and des [26-84], two long-lived disulfide-insecure intermediates of RNase A, and oxidize them directly under stable conditions to form the native protein. Such a successful direct conversion of kinetically trapped intermediates to the native molecule by trans-[Pt(en)(2)Cl(2)](2+) may be helpful in facilitating the oxidative folding processes of multi-disulfide-containing proteins in general.
Asunto(s)
Disulfuros/química , Metanosulfonato de Etilo/análogos & derivados , Ribonucleasa Pancreática/química , Compuestos de Sulfhidrilo/química , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Ditioeritritol/química , Metanosulfonato de Etilo/química , Guanidina/química , Cinética , Compuestos Organoplatinos/química , Oxidación-Reducción , Pliegue de ProteínaRESUMEN
The more biologically active thyroid hormone 3,5,3'-triiodothyronine (T(3)), is primarily derived from peripheral deiodination of thyroxine (T(4)). We characterized hepatic deiodination for a commercially important, warm water teleost fish, the red drum (Sciaenops ocellatus). Low K(m) outer-ring deiodination (ORD) activity was determined by production of free iodide ((125)I) upon incubation of hepatic microsomes with radiolabeled T(4). HPLC analysis demonstrated that (125)I, and T(3) were produced in equal amounts, thereby validating 125I as a measure of T(3) production. A small amount of 3,3',5'-triiodothyronine (reverse T(3)) was also produced by inner-ring deiodination. Production of (125)I was linear over a range of 0--100 microg protein/ml and for incubations of 30 min--4 h. Maximal ORD activity was measured at pH 6.6, 50 mM dithiothreitol (DTT) and an incubation temperature of 20 degrees C. Double reciprocal plots demonstrated that the average apparent K(m) was 5.1 nM and the average V(max) was 3.7 pmol T(4) converted/h per mg protein. ORD was not inhibited by propylthiouracil but was 50% inhibited by 90 microM of iodoacetic acid and 7 microM of gold thioglucose. The substrate analog preference was T(4) = tetraiodoacetic acid = reverse T(3) > triiodoacetic acid >> T(3). In relation to other tissues, ORD for liver>gill>intestine>kidney. Similar hepatic deiodination activity was present in adult wild, aquacultured and laboratory-reared red drum, but in adult wild red drum the optimum temperature was higher. Red drum hepatic low-K(m) deiodination activity appears to most closely resemble rainbow trout hepatic and mammalian Type II deiodination. Evidence of inner-ring T(4) deiodination suggests a more active hepatic iodothyronine catabolic pathway than in other teleost species.
Asunto(s)
Yoduro Peroxidasa/metabolismo , Microsomas Hepáticos/enzimología , Perciformes/metabolismo , Tiroxina/metabolismo , Triyodotironina/metabolismo , Animales , Antirreumáticos/farmacología , Antitiroideos/farmacología , Aurotioglucosa/farmacología , Cromatografía Líquida de Alta Presión , Ditioeritritol/química , Inhibidores Enzimáticos/farmacología , Radioisótopos de Yodo , Ácido Yodoacético/farmacología , Cinética , Microsomas Hepáticos/efectos de los fármacos , Propiltiouracilo/farmacología , Triyodotironina Inversa/metabolismoRESUMEN
At pH 4.0 to 4.5, 5,10-methenyltetrahydrofolate is hydrolyzed to only 5-formyltetrahydrofolate if reducing agents are present or iron-redox cycling is suppressed. At pH 4.0, the equilibrium position for this hydrolysis is approximately equal concentrations of both folates. If no reducing agents are used or iron-redox cycling is promoted, considerable amounts of 10-formyldihydrofolate are also formed. It is likely that 10-formyldihydrofolate has been misidentified as 5,10-hydroxymethylenetetrahydrofolate, which was reported to accumulate during the hydrolysis of 5, 10-methenyltetrahydrofolate to 5-formyltetrahydrofolate [Stover, P. and Schirch, V. (1992) Biochemistry 31, 2148-2155 and 2155-2164; (1990) J. Biol. Chem. 265, 14227-14233]. Since 5, 10-hydroxymethylenetetrahydrofolate is reported to be the viable in vivo substrate for serine hydroxymethyltransferase-catalyzed formation of 5-formyltetrahydrofolate, and 5, 10-hydroxymethylenetetrahydrofolate probably does not accumulate, the above folate metabolism is now doubtful. It is hypothesized that mildly acidic subcellular organelles provide an environment for the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate in vivo, and there is no requirement for enzyme catalysis. Finally, 10-formyltetrahydrofolate is susceptible to iron-catalyzed oxidation to 10-formyldihydrofolate at pH 4 to 4.5.
Asunto(s)
Formiltetrahidrofolatos/química , Tetrahidrofolatos/química , Ácido Ascórbico/química , Tampones (Química) , Cromatografía en Gel , Citratos/química , Ditioeritritol/química , Ácido Fólico/química , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Fosfatos/química , Soluciones , Espectrofotometría UltravioletaRESUMEN
Trypsin-subtilisin inhibitor from marine turtle eggwhite refolded quantitatively from its fully reduced state at pH 8.5 in the presence of reduced and oxidized glutathione. The refolding process was studied by following the accompanying changes in inhibitory activity, fluorescence, sulfhydryl group titer, and hydrodynamic volume. The refolding process followed second-order kinetics with rate constants of 4.80 x 10(2) M-1 sec-1 for trypsin-inhibiting domain and 0.77 x 10(2) M-1 sec-1 for subtilisin-inhibiting domain of the inhibitor at 30 degrees C and their respective activation energies of the refolding process were 15.9 and 21.6 kcal/mol. Fluorescence intensity of the reduced inhibitor decreased with time of refolding until it corresponded to the intensity of the native inhibitor. The inhibitor contained 1-2% alpha-helix, 40-42% beta-sheet, and 57-58% random coil structure. Refolded inhibitor gave a circular dichroic spectrum identical to that of the native inhibitor. A number of principal intermediates were detected as a function of the refolding time. Size-exclusion chromatography separated the intermediates differing in hydrodynamic volume (Stokes radius). The Stokes radius ranged from 23 A (fully reduced inhibitor) to 18.8 A (native inhibitor). Results indicated the independent refolding of two domains of the inhibitor and multiple pathways of folding were followed rather than an ordered sequential pathway.
Asunto(s)
Pliegue de Proteína , Subtilisinas/antagonistas & inhibidores , Subtilisinas/química , Inhibidores de Tripsina/química , Animales , Bovinos , Cromatografía Líquida de Alta Presión/métodos , Dicroismo Circular , Disulfuros/química , Ditioeritritol/química , Proteínas del Huevo/química , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta/métodos , Subtilisinas/metabolismo , Reactivos de Sulfhidrilo/química , Temperatura , Factores de Tiempo , Inhibidores de Tripsina/metabolismo , TortugasRESUMEN
The interactions of arsenate and arsenite with meso-2,3-dimercaptosuccinic acid (DMSA) have been characterized using carbon-13 nuclear magnetic resonance. These studies show that DMSA reduces arsenate to arsenite and complexes arsenite. Monitoring the carbon-13 signals of complexed DMSA and liberated glutathione shows that DMSA readily extracts arsenite from a (glutathione)3-arsenite complex, proving the affinity of arsenite for dithiols is greater than that for monothiols. Competition between DMSA (vicinal thiols) and dithioerythritol (1,4-dimercapto-2,3-butanediol) for binding of arsenite indicates that the binding affinity is inversely related to the distance between the two thiol groups. On the basis of these findings, a model for the interaction of arsenic with mono- and dithiol-containing molecules is proposed.
Asunto(s)
Arsenitos/química , Glutatión/química , Compuestos de Sulfhidrilo/química , Arseniatos/química , Ditioeritritol/química , Espectroscopía de Resonancia Magnética , Modelos Químicos , Oxidación-Reducción , Succímero/químicaRESUMEN
A recombinant chimeric plasminogen activator, MA-15C5Hu/scu-PA-32k, composed of a humanized fibrin fragment-D-dimer-specific monoclonal antibody (MA-15C5Hu) and a recombinant low-molecular-mass single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scu-PA-32k), was produced by cotransfecting Chinese hamster ovary (CHO) cells with the cDNA encoding the MA-15C5Hu light-chain sequence and the cDNA encoding the MA-15C5Hu heavy-chain sequence fused with the cDNA encoding scu-PA-32k. Purified MA-15C5Hu/scu-PA-32k migrated as a 215-kDa band on non-reducing SDS/PAGE, which is consistent with a molecule composed of one antibody and two scu-PA-32k moieties. However, the chimera was obtained as a mixture of single-chain u-PA-32k (37%) and amidolytically inactive (50%) and active (13%) two-chain u-PA-32k, the latter of which was removed by immunoadsorption on a monoclonal antibody specific for two-chain urokinase. The fragment-D-dimer affinity and enzymatic properties of MA-15CHu/scu-PA-32k were similar to those of MA-15C5Hu or of scu-PA-32k. In an in vitro system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated human plasma, MA-15C5Hu/scu-PA-32k had a 12-fold higher fibrinolytic potency than scu-PA-32k: 50% lysis in 2 h required 0.43 +/- 0.12 micrograms u-PA-32k equivalent of the chimera/ml versus 5.4 +/- 0.3 micrograms/ml of scu-PA-32k (mean +/- SEM, n = 4). Addition of purified fibrin fragment-D dimer reduced the fibrinolytic potency of MA-15C5Hu/scu-PA-32k in a concentration-dependent way, indicating that the increased potency is the result of antibody targeting. Thus, a recombinant humanized antifibrin antibody/u-PA chimera has been obtained in which only the variable domains of the antibody moiety are of non-human origin. The chimera has intact antigen-binding capacity, u-PA enzymatic activity and a significantly increased fibrinolytic potency in a plasma medium in vitro.