RESUMEN

BACKGROUND: Generally, proteases in medicinal plants had different therapeutic effects such as anti-inflammatory effect; modulate the immune response and inhibitory effect toward tumor growth. In this study, protease was purified and characterized from miswak roots, as medicinal plant and natural toothbrush. RESULTS: Physical and chemical characterization of cysteine protease P1 were studied such as pH optimum (6.5), optimum temperature (50 °C), thermal stability (50 °C) and Km (3.3 mg azocasein/ml). The enzyme digested some proteins in the order of caseine > haemoglobin > egg albumin >gelatin > bovine serum albumin. Hg2+ had strong inhibitory effect on enzyme activity compared with other metal ions. Kinetic of inhibition for determination the type of protease was studied. Iodoactamide and p-Hydroximercuribenzaoic acid (p-HMB) caused strong inhibitory effect on enzyme activity indicating the enzyme is cysteine protease. CONCLUSIONS: The biochemical characterization of this enzyme will be display the suitable conditions for using of this enzyme in toothpaste in the future and the enzyme may be used in other applications.

Asunto(s)

Proteasas de Cisteína/metabolismo , Proteínas de Plantas/metabolismo , Salvadoraceae/enzimología , Proteasas de Cisteína/química , Proteasas de Cisteína/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Estabilidad de Enzimas , Hidroximercuribenzoatos/química , Hidroximercuribenzoatos/metabolismo , Yodoacetamida/química , Yodoacetamida/metabolismo , Cinética , Mercurio/química , Mercurio/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Raíces de Plantas/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Temperatura

RESUMEN

The aim of our study was to investigate how denaturing agents commonly used in protein analysis influence the labeling between a reactive molecule and proteins. For this reason, we investigated the labeling of ovalbumin (OVA) as a globular model protein with p-hydroxymercurybenzoate (pHMB) in its native state (phosphate buffer solution) and in different denaturing conditions (8 molL(-1) urea, 3 molL(-1) guanidinium thiocyanate, 6 molL(-1) guanidinium chloride, 0.2% sodium dodecyl sulfate, and 20% methanol). In addition to chemical denaturation, thermal denaturation was also tested. The protein was pre-column simultaneously denatured and derivatized, and the pHMB-labeled denatured OVA complexes were analyzed by size exclusion chromatography (SEC) coupled online with chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS). The number of -SH groups titrated greatly depends on the protein structure in solution. Indeed, we found that, depending on the adopted denaturing conditions, OVA gave different aggregate species that influence the complexation process. The results were compared with those obtained by a common alternative procedure for the titration of -SH groups that employs monobromobimane (mBBr) as tagging molecule and molecular fluorescence spectroscopy as detection technique. We also investigated the labeling kinetics for denatured OVA and pHMB, finding that the 4 thiolic groups of OVA have a very different reactivity toward mercury labeling, in agreement with previous studies.

Asunto(s)

Hidroximercuribenzoatos/metabolismo , Mercurio/metabolismo , Ovalbúmina/metabolismo , Desnaturalización Proteica , Coloración y Etiquetado , Animales , Pollos , Cromatografía en Gel , Hidroximercuribenzoatos/química , Cinética , Mercurio/química , Dodecil Sulfato de Sodio/química

RESUMEN

Low molecular mass (LMM) thiols is a diverse group of compounds, which play several important roles in aquatic ecosystems, even though they typically occur at low concentrations. Comprehensive studies of LMM thiols in natural waters have so far been hampered by selectivity and limit of detection constraints of previous analytical methods. Here, we describe a selective and robust method for the quantification of 16 LMM thiols in natural waters. Thiols were derivatized with 4-(hydroxymercuri)benzoate (PHMB) and preconcentrated online by solid-phase extraction (SPE) before separation by liquid chromatography and determination by electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Their quantification was performed by selective reaction monitoring (SRM), while the presence of a product ion at m/z 355, specific for thiols and common for the investigated compounds, also allows to screen samples for unknown thiols by a precursor ion scan approach. The robustness of the method was validated for aqueous matrices with different pH, sulfide, and dissolved organic carbon (DOC) concentrations. The limits of detection for the thiols were in the sub-nanomolar range (0.06-0.5 nM) and the methodology allowed determination of both reduced and total thiol concentrations (using tris(2-carboxyethyl)phosphine (TCEP) as reducing agent). Six thiols (mercaptoacetic acid, cysteine, homocysteine, N-acetyl-cysteine, mercaptoethane-sulfonate, and glutathione) were detected with total concentrations of 7-153 nM in boreal lake or wetland pore waters while four thiols (mercaptoacetic acid, cysteine, homocysteine, and N-acetyl-cysteine) were detected in their reduced form at concentrations of 5-80 nM.

Asunto(s)

Cromatografía Liquida/métodos , Hidroximercuribenzoatos/química , Extracción en Fase Sólida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Compuestos de Sulfhidrilo/análisis , Espectrometría de Masas en Tándem/métodos , Agua/química , Sistemas en Línea , Contaminantes Químicos del Agua/análisis

RESUMEN

In this work we have studied the derivatization of protein disulfide bonds with p-Hydroxymercurybenzoate (pHMB) in strong alkaline medium without any preliminary reduction. The reaction has been followed by the determination of the protein-pHMB complex using size exclusion chromatography coupled to a microwave/UV mercury oxidation system for the on-line oxidation of free and protein-complexed pHMB and atomic fluorescence spectrometry (SEC-CVG-AFS) detection. The reaction has been optimized by an experimental design using lysozyme as a model protein and applied to several thiolic proteins. The proposed method reports, for the first time, that it is possible to label 75-100% cysteines of proteins and, thus, to determine thiolic proteins without the need of any reducing step to obtain reduced SH groups before mercury labelling. We obtained a detection limit of 100 nmol L(-1) based on a signal-to-noise ratio of 3 for unbound and complexed pHMB, corresponding to a detection limit of proteins ranged between 3 and 360 nmol L(-1), depending on the number of cysteines in the protein sequence.

Asunto(s)

Cromatografía en Gel/métodos , Disulfuros/análisis , Hidroximercuribenzoatos/química , Muramidasa/análisis , Sustancias Reductoras , Cromatografía Liquida/métodos

RESUMEN

A novel method is presented for the characterization and determination of thiolic proteins. After the labeling with p-hydroxymercurybenzoate, the pHMB-labeled proteins underwent on-line oxidation with a novel microwave (MW)/UV photochemical reactor, followed by cold vapor generation-atomic fluorescence spectrometry (CVG-AFS) detection. The MW/UV process led to the conversion of pHMB to Hg(II) with a yield of 89.0 ± 0.5% without using chemical oxidizing reagents and avoiding the use of toxic carcinogenic compounds. Hg(II) was reduced to Hg(0) in a knotted reaction coil with NaBH4 solution, stripped from the solution by an argon flow and detected. The chromatographic method for labeled thiolic peptides was linear in the 0.2-100 µmol L(-1) range, with a LOD as mercury of 57 nmol L(-1). This system has proven to be a useful interface for liquid chromatography coupled with CVG-AFS in the determination and characterization of thiolic proteins. This method has been applied to the determination of thiolic peptides after tryptic digestion of serum albumins from different species (human, bovine, rat, horse, and sheep).

Asunto(s)

Frío , Hidroximercuribenzoatos/química , Microondas , Procesos Fotoquímicos , Proteínas/análisis , Proteínas/química , Espectrometría de Fluorescencia/métodos , Animales , Bovinos , Cromatografía de Fase Inversa , Análisis de Inyección de Flujo , Humanos , Oxidación-Reducción , Proteínas/metabolismo , Ratas , Tripsina/metabolismo , Rayos Ultravioleta , Volatilización

RESUMEN

Cysteine residues play a unique role in human hemoglobin (Hb) by affecting its cooperative oxygen binding behavior and the stability of its tetrameric structure. However, how these cysteine residues fulfill their biophysical functions from the molecular level is yet unclear. Here we study the subunit disassembly pathway of human hemoglobin using the sulfhydryl reagent, p-hydroxymercuribenzoate (PMB) and investigate the functional roles of cysteine residues in human hemoglobin. We show evidence from the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry that all three types of cysteine residues, including the surface-exposed ßCys93 and the shielded αCys104 and ßCys112 are reactive to PMB, resolving an issue long under debate. It is demonstrated that all three types of cysteine residues must be blocked by PMB to accomplish the subunit disassembly, and the PMB-cysteine reactions proceed in a stepwise manner with an order of ßCys93, αCys104, and ßCys112. The PMB reactions with the three different cysteine residues demonstrate strong site-specificity. The possible influence of PMB-cysteine reactions to the stability of various intersubunit salt bridges has been discussed based on the crystallographic structure of hemoglobin, providing insights in understanding the hemoglobin subunit disassembly pathway and the site-specific functional role of each cysteine residue in hemoglobin.

Asunto(s)

Cisteína/química , Hemoglobinas/química , Hemoglobinas/metabolismo , Humanos , Hidroximercuribenzoatos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

A method was developed for the precise and accurate determination of ovalbumin labelled with p-hydroxy-mercuribenzoic acid (pHMB) using polyacrylamide gel electrophoresis with ns-laser ablation-inductively coupled plasma mass spectrometry. Following systematic optimisation of the ablation process in terms of detection sensitivity, two different quantification strategies were applied: external calibration using standards of the derivatized protein after (13)C(+) normalization and, as a proof of concept, label-specific isotope dilution analysis (IDA) using pHMB enriched in the isotope (199)Hg. Due to the inhomogeneous distribution of the protein within the gel bands, it could be demonstrated that the IDA approach was superior in terms of precision and accuracy. Furthermore, it permits a reliable quantification, if more complex separation protocols are applied, as typically occurring analyte loss and degradation can be compensated for as soon as complete mixture of spike and sample is achieved. The estimated limit of detection was 160 fmol in the case of ovalbumin. In contrast to earlier studies using metals naturally present in proteins, no loss of mercury was observed during separation under denaturing conditions and other sample preparation steps. Using label-specific IDA, the measured isotope ratios in the gel corresponded to recoveries between 95% and 103%.

Asunto(s)

Isótopos de Mercurio/análisis , Ovalbúmina/análisis , Técnica de Dilución de Radioisótopos , Espectrofotometría Atómica/métodos , Análisis Espectral/métodos , Coloración y Etiquetado/métodos , Animales , Calibración , Pollos , Electroforesis en Gel de Poliacrilamida , Hidroximercuribenzoatos/química , Límite de Detección , Isótopos de Mercurio/química , Ovalbúmina/química , Estándares de Referencia , Reproducibilidad de los Resultados

RESUMEN

Chromatographic determination of glutathione disulfide (GSSG) without any preliminary reduction has been presented using GSSG derivatization by p-hydroxymercuribenzoate (pHMB) in strong alkaline medium followed by the determination of GS-pHMB complex by reversed phase chromatography coupled to chemical vapour generation and atomic fluorescence detector (RPC-CVGAFS). A detection limit of 35 nM for GSSG (corresponding to 1.8 pmol) detected as GS-pHMB species was achieved based on a signal-to-noise ratio of 3 in buffer and in blood. The proposed method was applied to the determination of GSSG in whole blood and validated by the classical determination of GSSG by derivatization after reduction with dithiothreitol (DTT).

Asunto(s)

Disulfuro de Glutatión/sangre , Disulfuro de Glutatión/química , Hidroximercuribenzoatos/química , Álcalis/química , Humanos , Concentración de Iones de Hidrógeno , Límite de Detección , Oxidación-Reducción , Espectrometría de Masa por Ionización de Electrospray

RESUMEN

A liquid chromatography method with mass spectrometric detection has been developed for the simultaneous determination of six thiols in the sulfur metabolic pathway, including cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), gamma-glutamyl-cysteine (Glu-Cys), and S-adenosyl-homocysteine (AdoHcy). Tris(2-carboxyethyl)phosphine (TCEP) was used as the reducing reagent and p-(hydroxymercuri)benzoate (PHMB) as the derivatization reagent. Thiols were extracted from 3 mg of yeast using water in an ultrasonic bath. The absolute detection limits for the compounds studied were in the subpicomole range. It was found that AdoHcy, Cys, GSH, Cys-Gly, Glu-Cys, and very little HCys were present in the selenium-enriched yeast sample studied, and GSH, Glu-Cys, very little AdoHcy, Cys, and Cys-Gly were present in three bakery yeasts.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Hidroximercuribenzoatos/química , Espectrometría de Masas/métodos , Compuestos de Sulfhidrilo/química , Levaduras/química , Compuestos de Sulfhidrilo/metabolismo , Levaduras/metabolismo

RESUMEN

Chemical labeling with subsequent mass spectrometric detection represents a common approach for protein quantification. Whereas most methods make use of stable isotope labels from natural elements such as (2)D, (13)C, (15)N, or (18)O, artificially introduced metals have gained interest as alternative markers. In this work we present the application of p-hydroxymercuribenzoic acid (pHMB) as a labeling reagent for cysteine-containing proteins. As a proof of concept, insulin was chosen as the model protein, and two different workflows were developed to its absolute and relative quantification with the use of complementary MALDI-MS and ICPMS. On the basis of the synthesis of isotopically labeled [(199)Hg]pHMB, and thus on the basis of the label-specific isotope dilution concept, a differential labeling procedure can be applied either to the comparative study of two different samples (relative quantification) or to the absolute quantification of insulin. In both cases, final detection by MALDI-MS followed by isotope pattern deconvolution was applied to extract the quantitative data from the mass spectra. Good agreement with the expected values was obtained for the relative insulin quantification, and the recovery for insulin applying the absolute quantification workflow was between 90% and 110% with an RSD of better than 5% in the low picomole range.

Asunto(s)

Hidroximercuribenzoatos/química , Insulina/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Bovinos , Humanos , Insulina/química , Marcaje Isotópico , Isótopos de Mercurio/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación

RESUMEN

An alternative analytical method was established for simultaneous determination of main urinary low-molecular-mass (LMM) thiols including cysteine (Cys), cysteinylglycine (Cys-Gly), homocysteine (HCys), gamma-glutamyl cysteine (gamma-Glu-Cys) and glutathione (GSH) as well as N-acetylcysteine (NAC) using RPLC coupled on line with UV/HCOOH-induced cold vapor generation atomic fluorescence spectrometry (UV/HCOOH-CVG-AFS) with 4-(hydroxymercuric)benzoic acid (PHMB) as a tag. The LMM thiols were stabilized and labeled by PHMB allowing the determination of reduced form thiols (R-thiols) and total thiols (T-thiols) without and with Tris-(2-carboxyethyl)-phosphine reduction. UV/HCOOH-induced Hg cold vapor generation was used instead of K(2)SO(8)-KBH(4)/NaOH-HCl and/or KBrO(3)/KBr-KBH(4)/NaOH-HCl systems as an effective interface between RPLC and CVG-AFS. The limits of detection (3sigma) of RPLC-(UV/HCOOH)-CVG-AFS with PHMB labeling for Cys, HCys, Cys-Gly, gamma-Glu-Cys and GSH as well as NAC were 4.6, 5.9, 5.9, 8.1, 7.3 and 5.9nM with the RSD of 4.4, 5.1, 3.6, 7.5 4.2 and 3.7% (n=6 at 2microM), respectively, satisfying the simultaneous determination of the main urinary LMM thiols. This developed method was applied successfully to determine the LMM R-thiols and T-thiols in 10 urine samples contributed by 10 healthy volunteers.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Fluorescencia/métodos , Compuestos de Sulfhidrilo/química , Cromatografía Líquida de Alta Presión/instrumentación , Formiatos/química , Hidroximercuribenzoatos/química , Peso Molecular , Oxidación-Reducción , Espectrometría de Fluorescencia/instrumentación

RESUMEN

Conformation and microenvironment at the active site of 1,4-beta-D-glucan glucanohydrolase was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde. OPTA has been known to form a fluorescent isoindole derivative by cross-linking the proximal thiol and amino groups of cysteine and lysine. Modification of lysine of the enzyme by TNBS and of cysteine residue by PHMB abolished the ability of the enzyme to form an isoindole derivative with OPTA. Kinetic analysis of the TNBS and PHMB-modified enzyme suggested the presence of essential lysine and cysteine residues, respectively, at the active site of the enzyme. The substrate protection of the enzyme with carboxymethylcellulose (CMC) confirmed the involvement of lysine and cysteine residues in the active site of the enzyme. Multiple sequence alignment of peptides obtained by tryptic digestion of the enzyme showed cysteine is one of the conserved amino acids corroborating the chemical modification studies.

Asunto(s)

Actinomycetales/enzimología , Glucano 1,4-beta-Glucosidasa/química , Sitios de Unión/genética , Carboximetilcelulosa de Sodio/metabolismo , Carboximetilcelulosa de Sodio/farmacología , Dicroismo Circular , Cisteína/química , Cisteína/genética , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Glucano 1,4-beta-Glucosidasa/genética , Glucano 1,4-beta-Glucosidasa/metabolismo , Hidroximercuribenzoatos/química , Hidroximercuribenzoatos/farmacología , Cinética , Lisina/química , Lisina/genética , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica/efectos de los fármacos , Alineación de Secuencia , Especificidad por Sustrato , Ácido Trinitrobencenosulfónico/química , Ácido Trinitrobencenosulfónico/farmacología

RESUMEN

The endo-beta-1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine.

Asunto(s)

Trichosporon/enzimología , Xilosidasas/metabolismo , Aminoácidos/química , Ácidos Carboxílicos/química , Hidroximercuribenzoatos/química , Imidazoles/química , Cinética , Tetranitrometano/química , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/química

RESUMEN

Neutral cholesterol ester hydrolase from rat liver microsomes was inactivated in a dose and time-dependent manner by classical sulphydryl-reacting reagents such as p-hydroxymercuribenzoic acid, 5,5'-dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide, or iodoacetate. The concentrations at which half-maximal inhibition of the native microsomal cholesterol ester hydrolase occurred (IC50) were 15, 68, and 370 mumol/l and 68 mmol/l, respectively. Only partial reactivation of the enzyme was observed under excess dithiothreitol or mercaptoethanol treatment. The stimulation of cholesterol ester hydrolase by the metal ions Ca2+ and Mg2+ was dependent on the integrity of the thiol groups. Solubilization of cholesterol ester hydrolase from membranes preserved its sensitivity towards sulphydryl reagents and thiols, as well as its ability to be activated by Ca2+ and Mg2+. Dithiothreitol, mercaptoethanol, and Ca2+ and Mg2+ provided total protection of the enzyme against inactivation by thiol-reacting reagents. The results indicate that one or more thiol groups are either at the active centre of the native and solubilized forms of rat liver microsomal cholesterol ester hydrolase or are sufficiently near, to interfere with the catalysis when they are reacted.

Asunto(s)

Proteínas de la Membrana/metabolismo , Microsomas Hepáticos/metabolismo , Esterol Esterasa/metabolismo , Reactivos de Sulfhidrilo/química , Animales , Calcio/química , Cationes Bivalentes/química , Ácido Ditionitrobenzoico/química , Ditiotreitol/química , Relación Dosis-Respuesta a Droga , Activación Enzimática , Etilmaleimida/química , Femenino , Hidroximercuribenzoatos/química , Yodoacetatos/química , Ácido Yodoacético , Hígado/enzimología , Hígado/metabolismo , Magnesio/química , Proteínas de la Membrana/química , Mercaptoetanol/química , Microsomas Hepáticos/química , Microsomas Hepáticos/enzimología , Concentración Osmolar , Ratas , Ratas Sprague-Dawley , Solubilidad , Esterol Esterasa/química , Factores de Tiempo

RESUMEN

Crystals of a complex of chicken gizzard G-actin and DNase I were soaked in a solution of radioactive 4-hydroxymercuribenzoate (MB). The soaked crystals, which contained 0.93 mol of MB per mol of G-actin, were dissolved in "G-buffer" and digested with trypsin, and the resulting peptides were fractionated by thin-layer chromatography. The MB is exchangeable between peptides that contain cysteine residues, but the data obtained here suggested that MB attached to the cysteine residue at the 373rd position of the G-actin molecule.

Asunto(s)

Actinas/química , Desoxirribonucleasa I/química , Hidroximercuribenzoatos/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Pollos , Cristalización , Molleja de las Aves , Datos de Secuencia Molecular , Músculo Liso , Fragmentos de Péptidos/aislamiento & purificación , Tripsina