2-mercaptoethanol Research Articles

Reaction of Hydrogen Sulphide and β-Mercaptoethanol with Hydrazones of Aromatic Aldehydes in the Presence of Triethylamine

Avec H 2 S obtention de salicylidenehydrazinosulfenylhydrazone du salicylaldehyde, avec le mercapto-2 ethanol, formation du salicylidenehydrazide de l'acide hydroxy-2 ethanesulfenique

Soluble Mn2+-dependent adenylate cyclase activity in the testis of the blue fox (Alopex lagopus).

Soluble Mn2+-dependent adenylate cyclase (MnAC) activity was found in testicular cytosol from blue foxes castrated during the breeding season. The rate of MnAC activity was approximately constant for 30 min at 35 degrees C and for 2 hr after storage at 25 degrees C. Activity was directly proportional to cytosol protein concentration and was optimal in the physiological pH range. Enzyme activity declined in the presence of an alkylating agent (N-ethyl maleimide, NEM) and was eliminated at a concentration of 1 mM NEM. Low concentrations (0.1-10 mM) of a reducing agent (beta-mercapto ethanol, beta ME) did not increase MnAC activity, whereas a high concentration (100 mM) led to a significant reduction (p less than 0.01) in activity. Substitution of Mn2+ in the assay medium with Mg2+ led to a total loss of enzyme activity, which could not be regained by adding hormones or by preincubation of cytosol for 60 min. The Km for Mn2+ was estimated to be 3.5 mM. The affinity of the enzyme for Mn2+ was not altered by varying the concentration of ATP. In contrast, increasing concentrations of Mn2+ appeared to increase the affinity of the enzyme for MnATP2-. The Km for MnATP2- thus varied from 6 to 18 mM.

Alzheimer paired helical filaments: bulk isolation, solubility, and protein composition.

A method has been developed for the bulk isolation of Alzheimer neurofibrillary tangles (ANT) of paired helical filaments (PHF) from histopathologically confirmed cases of Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT). The fresh or frozen autopsied cerebral cortex affected with Alzheimer neurofibrillary changes is dissociated by homogenization and sieving through nylon bolting cloth and the ANT are separated by a combination of sucrose discontinuous density gradient centrifugation, glass bead column chromatography, and sodium dodecyl sulfate (SDS) treatment. The isolated ANT produce red-green birefringence when viewed through polarized light after staining with Congo red. Ultrastructurally, the isolated PHF are well preserved and have the dimensions of the PHF seen in situ. Two major Populations of ANT which exist in different proportions in AD/SDAT brains are identified on the basis of their solubility in SDS. The ANT I and the ANT II are soluble and insoluble respectively on treatment with 2% SDS at room temperature for 5 min. Solubilization of the ANT II requires several repeated extractions with a solution containing 10% each of SDS and beta-mercaptoethanol (BME) at 100 degrees C for 10 min. Sonication of the ANT II greatly facilitates their solubilization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated ANT reveals the presence of two major polypeptides with molecular weights (MW) of 62,000 and 57,000, several minor polypeptides with MW below 57,000, and a significant amount of material not entering the stacking and the resolving gels. Re-electrophoresis of polypeptides extracted from various areas of the resolving gel or of the material which does not enter the gel generates the same polypeptide profile as on the first gel, suggesting that the PHF material which does not enter the gel may result from the reaggregation of the polypeptides that enter the resolving gel. None of the polypeptides that enter the resolving gel. None of the polpeptides observed in the isolated PHF comigrate in the SDS-PAGE with any of the neurofilament polypeptides, tubulin, actin, or myosin.

Experimental Emphysema

The synthesis of nano eight-armed poly{POSS lactide-b-N, N-(dimethylamino)ethyl methacrylate} copolymers is reported in this paper. Thio-click chemistry combined with living/controlled polymerization methods that allow the preparation of well-defined polymeric building block copolymers. This was carried out using Thio-Click reaction of octavinyl POSS with 2-mercapto ethanol to prepare Thiolate-POSS. Thiolate POSS-lactide polymers containing different l-Lactide repeated units (10, 25, 50 and 100) were synthesized by ring-opening polymerization of (l-lactide) in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a catalyst. The resulting polymers were copolymerized with N-hydroxyethyl-2-(dimethylamino) ethyl methacrylate (DMAEMA) via atom transfer radical polymerization. All the prepared polymers and copolymers are characterized by different spectroscopic and analytical techniques, such as infrared spectroscopy, nuclear magnetic resonance (1H NMR and 13C NMR) and by gel permeation chromatography (GPC), where all these analyses have verified the expected structure and compositions. On the other hand, images obtained from Scanning Electronic Microscopy (SEM) revealed the existence of nanostructures in the prepared copolymers due to the presence of the lactide array to give nanofiber within final compositions prepared, and with the presence of POSS in their structures, they enhance the copolymers thermal stability, and they are expected to be suitable for different applications as nano-carriers in the biomedical field.

Intralysosomal hydrolysis of thyroglobulin

Degradations of recently (1 h) iodinated thyroglobulin (Tg) and of Tg iodinated for 48 h (fully iodinated molecule) inside thyroid phagolysosomes have been measured in vitro, at different pH, in basal (lysosomes from normal rats) and stimulated (lysosomes from TSH-treated rats) conditions. In basal conditions, recently iodinated Tg was shown to be preferentially degraded at pH 5 but not at pH 8. Mercaptoethanol (ME) was found to preferentially stimulate the degradation of 48 h-iodinated Tg at pH 5 but not at pH 8. In stimulated conditions and in the presence of ME, no preferential hydrolysis of recently iodinated Tg was observed, whatever the pH. Since the degradation of Tg and its stimulation by ME are known to depend on the mean iodine content of the molecule, it was concluded that: 1) the recently iodinated Tg population contains both poorly and fully iodinated molecules, the degradations of which are faster or similar to that of 48 h-iodinated Tg, respectively, 2) among thyroid phagolysosomes, only those which exhibit a pH-dependent proteolytic activity contain poorly iodinated molecules while others, which are functional at either pH, contain solely fully iodinated molecules (1 and 48 h labelled), 3) reduction of the disulphide bridges of fully iodinated Tg, mediated by ME and probably a lysosomal transhydrogenase, is a prerequisite for its optimal degradation by lysosomal proteinases, 4) in addition, TSH, which preferentially stimulates the degradation of fully iodinated Tg, was concluded to interfere directly upon the intralysosomal hydrolytic process, independently of its effect upon Tg endocytosis. This effect of TSH, similar and additive to that of ME suggests that the hormone might activate a lysosomal transhydrogenase.

Latent and active human polymorphonuclear leukocyte collagenases. Isolation, purification and characterisation.

Latent and active human polymorphonuclear leukocyte collagenase could be purified to apparent homogenei by a rapid and reproducible method involving affinity chromatography on concanavalin‐A–Sepharose, collage Sepharose and activated thiol‐Sepharose 4‐B, gel filtration on Sephacryl S‐300 and ion‐exchange chromatograpl on DEAE‐Sephacel.The latent and active collagenase were shown to be single entities with apparent Mr 91000 and 6400 respectively, as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without mercapto ethanol. Dodecyl sulfate gel electrophoresis of the latent enzyme in the presence of mercaptoethanol showed two bands with apparent Mr of 64000 and 24000 indicating the cleavage of a disulfide bond.Activation of the native latent collagenase could be achieved by_ disulfide‐containing compounds. A concomitant decrease in molecular weight of approximately 25000 was observed on activation of the latent enzyme. The activators proved ineffective after alkylation of the latent enzyme with iodoacetamide. This lends support to the proposed thiol/disulfide interchange mechanism in the activation reaction [Macartney, H. W. and Tschesche, H (1980) FEBS Lett. 119, 327–332]. The active collagenase could be completely inhibited by an inhibitor dissociated from the latent enzyme, i.e. a complex of enzyme and inhibitor. Regulation of the collagenase activity could be achieved by the redox potential of the glutathione cycle [Tschesche, H. and Macartney, H. W. (1981) Eur. J. Biochem. 120, 183–190].The latent and active enzymes were shown to be acidic proteins with isoelectric points at pH 5.5 and 6.4 respectively. Amino acid analyses of both enzymes have been obtained and indicate a difference of about 222–223 amino acid residues between the latent and active collagenases. This corresponds to a difference of approximately Mr25000 between the active and the latent enzyme, the latter being a mixed disulfide of the enzyme and its inhibitor.The optimum pH for collagenolytic activity was observed near neutrality. The enzyme specificity was revealed to be that of a true mammalian collagenase splitting the native type I tropocollagen molecule into three‐quarter and one‐quarter fragments according to dodecyl sulfate/polyacrylamide gel electrophoresis. No other contaminating proteolytic activity was observed in the purified latent and active collagenase preparations. The active enzyme was found to be stable for over one year when stored at ‐ 70°C. The collagenase was capable of reducing the specific viscosity of a native type I collagen solution by approximately 40%.The amount of latent collagenase present in the leukocytes was estimated to 0.65 mg/1010 cells. The overall yield obtained after the purification to homogeneity was 75–80% of the theoretical value.

Off-line liquid chromatographic—mass spectrometric studies of o-phthalaldehyde-primary amine derivatives

The structures of the products formed between several n-aliphatic amines and o-phthalaldehyde (OPT) during routine post-column derivatization after high-performance liquid chromatographic (HPLC) elution, were determined by gas chromatography—mass spectrometry. OPT derivatization studies were carried out separately in the presence of mercaptoethanol (MCE) and in the presence of ethanethiol (ETH). Comparison of the mass spectra of the silylated OPT—MCE derivatized amines with those of the non-silylated OPT—ETH derivatives indicated a consistent fundamental structure with similar fragmentation patterns. It was shown that the structure of the fluorescent product obtained under analytical post-column HPLC derivatization conditions is a 1 -alkylthio-2-alkyl-substituted isoindole. This finding is in agreement with results reported earlier for preparative-scale reactions.

We can make enough Phillips Beta-mercaptoethanol to fill every need in the U.S. and then some.

We can make enough Phillips Beta-mercaptoethanol to fill every need in the U.S. and then some.

We can make enough Phillips Beta-mercaptoethanol to fill every need in the U.S. and then some.

We can make enough Phillips Beta-mercaptoethanol to fill every need in the U.S. and then some.

A mechanism of augmentation of in vitro antibody response by 2-mercaptoethanol in murine lymphocytes

The mechanism of augmentation of antibody response in vitro by 2-mer captoethanol (2 ME) was investigated. By employing cystine-free RPMI-1640 medium, it was demonstrated that cystine was absolutely required for eliciting the in vitro antibody response in murine lymphocytes. The augmentation by 2 ME was corn pletely dependent on the presence of cystine. Maximal response was reached when cysteine or cystine (as half cystine) was added at 2.5 mM where 2ME did not further enhanced the response. The serial feeding of cysteine, but not of cystine, amplified its potentiating activity at lower concentrations (≤1 mM) . The addition of 10-5M 2 ME to the medium shifted the dose-response curve to lower concentrations by about one order of magnitude. It was found that 35S-cystine was incorporated into lymphocytes one fifth more slowly than 35S-cysteine. Cystine uptake was accelerated by about 2.5-fold in the presence of 10-5M 2 ME. A close correlation was observed between doseresponse profiles of 2 ME in augmenting the antibody response and the stimulation of cystine uptake. These data suggest that one of the roles of 2 ME in augmenting antibody response in vitro is to facilitate the uptake of cystine which is contained in RPMI-1640 medium and which is utilized less efficiently than cysteine.

Serodiagnosis of type A hepatitis by detection of immunoglobulin M-type antibody to hepatitis A virus.

Serum specimens from 12 patients with type A hepatitis were analyzed for immunoglobulin M-type antibody to hepatitis A virus (IgM anti-HA). A recently developed solid-phase radioimmunoassay kit for IgM anti-HA (HAVAB-M, Abbott Laboratories) and a competitive binding radioimmunoassay kit (HAVAB, Abbott Laboratories) with or without 2-mercaptoethanol treatment, as modified by Yano et al. (Acta Hepatol. Jpn. 21, 704-712, 1980) were used to obtain an M-index. All specimens obtained within 60 days of the onset of illness and specimens from 2 of 4 patients later than 60 days after the onset were positive with the HAVAB-M test. This test gave negative results to sera which were positive for anti-HA by a standard HAVAB test in the following: 3 patients with type B hepatitis; 5 with non-A, non-B hepatitis; 11 healthy adults; and 10 sera strongly positive for rheumatoid factor. The M-index for type A hepatitis in sera within 30 days of the onset (mean value of the M-index, m, = 1.52; standard deviation, SD, = 0.25) was significantly higher than that for non-A hepatitis (m = 1.05; SD = 0.15) and for healthy adults (m = 1.02; SD = 0.10). The simplicity and usefulness of the HAVAB-M test in diagnosis of acute type A hepatitis over those measuring the M-index by HAVAB tests were shown by direct comparison of the results.

Acid‐extracted nuclear proteins and ultrastructure of human sperm chromatin as revealed by differential extraction with urea, mercaptoethanol, and salt

AbstractBasic proteins were differentially extracted from the purified heads of ejaculated human sperm by successive treatment with (1) 1% Triton X‐100, 1% mercaptoethanol (ME); (2) 8 M urea, 1% ME; (3) 8 M urea, 1% ME, 0.2 M NaCl; and (4) 8 M urea, 1% ME, 0.6 M NaCl. Nonhistones were extracted in the first, second, and third treatments; histones, in the second and third; and most of the protamines, in the fourth, together with DNA. Corresponding electro microscopic studies of the sperm pellets after each treatment suggest that the nucleoprotamines are discrete portions of chromatin which are organized in the form of long thick cords and oval bodies of about 400–550 Å in diameter interlinked with very thin strands of fibers about 20–50 Å thick, which could represent the naked DNA depleted of its constituent histones.

Mechanism of augmentation of antibody response in vitro by 2-mercaptoethanol

The mechanism of augmentation of antibody response in vitro by 2-mer captoethanol (2 ME) was investigated. By employing cystine-free RPMI-1640 medium, it was demonstrated that cystine was absolutely required for eliciting the in vitro antibody response in murine lymphocytes. The augmentation by 2 ME was corn pletely dependent on the presence of cystine. Maximal response was reached when cysteine or cystine (as half cystine) was added at 2.5 mM where 2ME did not further enhanced the response. The serial feeding of cysteine, but not of cystine, amplified its potentiating activity at lower concentrations (≤1 mM) . The addition of 10-5M 2 ME to the medium shifted the dose-response curve to lower concentrations by about one order of magnitude. It was found that 35S-cystine was incorporated into lymphocytes one fifth more slowly than 35S-cysteine. Cystine uptake was accelerated by about 2.5-fold in the presence of 10-5M 2 ME. A close correlation was observed between doseresponse profiles of 2 ME in augmenting the antibody response and the stimulation of cystine uptake. These data suggest that one of the roles of 2 ME in augmenting antibody response in vitro is to facilitate the uptake of cystine which is contained in RPMI-1640 medium and which is utilized less efficiently than cysteine.

Fluorescence stopped-flow study of the o-phthaldialdehyde reaction

The fluorogenic reaction involving three species, namely, a primary amine, o-phthaldialdehyde (OPA), and a thiol compound was studied with the fluorescence stopped-flow technique. The results are consistent with the reaction of the amine with a 1:1 adduct of OPA and the thiol compound. The equilibrium constant for the formation of the adduct, OPAME, from OPA and mercaptoethanol (ME) was determined to be 164 m −1. A survey of the rates of reaction of OPAME with various amino acids demonstrated that with OPA: ME:amine equal to 1:2.4:1 (total OPA concentration 0.5 to 3.0 × 10 −3 m), the reaction followed second-order kinetics, with k = 150 to 450 m −1s −1 at pH 9.O. The differences in rates are discussed in relation to structural differences between the amines. The reaction, when conducted under conditions of excess OPAME yielded pseudo-first-order kinetics, with rates consistent with the second-order rate constants. The rate of reaction of OPAME with alanine was maximal at pH 10.5–11, and a great excess of ME resulted in a slower rate. Slower rates were also observed if ME was replaced by dithiothreitol or 1-propanethiol.

Genetic Analysis of the Humoral Immune Response of White Leghorn Chicks

Total agglutinin antibody titers, 2-mercapto ethanol (2-ME) sensitive and 2-ME resistant antibody titers were determined in 598 White Leghorn chicks after intramuscular injection with sheep red blood cells. Antibody titers were determined on day 0 and on days 3, 7, 10, 13 postinjection. Mean total titer (5.2, log2 value) was highest on day 7. Females showed a significantly higher response to injection with sheep red blood cells than males. Also, significant hatch effects were noted.Heritability estimates generally varied from 0 to .5 for all parameters. In the earlier stages of the immune response the sire estimate of heritability for total and 2-ME sensitive antibody titer was higher than the dam estimate. Additive genetic correlations between 2-ME sensitive (days 3 to 13) and resistant (days 7 to 13) antibody titers were negative, varying from −.30 to −.93. The response to selection for total antibody titer is, therefore, not easily predicted.

The effect of reducing agents on the apparent molecular weight of the myelin P0 protein and the possible identity of the P0 and "Y" proteins.

Abstract:During sodium dodecyl sulfate polyacrylamide gel electrophoresis of sciatic nerve myelin proteins, the P0 protein migrated to two different positions on the gels. The distribution of P0 between the two forms depended on the concentration of mercaptoethanol (ME) or dithiothreitol (DTT) in the sample solvent. At >1% ME or 3.5% DTT, P0 was almost entirely in a form with an apparent molecular weight of 30,000, whereas in the absence of ME or DTT all the P0 migrated with an apparent molecular weight of 25,000. The distribution of the P0 protein between the two bands was independent of whether proteinase inhibitors were used during preparation of myelin. Furthermore, in a sample containing the more rapidly migrating form of P00, the P0 could be converted to the slower‐migrating form by addition of mercaptoethanol. These data show that interconversion of the two forms of P0 does not involve proteolytic degradation, but suggest rather that ME cleaves one or more intramolecular disulfide bonds in P0 unfolding the amino acid chain and thereby converting the form with a lower apparent molecular weight to that with a higher apparent molecular weight. We postulate that the “Y” protein band, which has been observed, in variable quantities, migrating slightly ahead of P0, may represent its oxidized form.

Quantitative SDS–PAGE of total protein from different wheat varieties

AbstractBuffers containing sodium dodecyl sulphate (SDS) and mercaptoethanol (ME) were used to extract total protein from the flour or single seeds of five different wheat (Triticum aestivum L.) varieties (Scout 66, Red River 68, Bankuti 1201, Atlas 66, and Omar). A dye‐binding analysis for protein was adapted to these extracts. The extracted proteins were separated according to molecular weight (mol. wt) by gel electrophoresis with SDS and the patterns were quantitated by densitometry of the gels, after proteins had been stained with Coomassie brilliant blue R250. The patterns were divided into five different areas corresponding to mol. wt ranges of 63 × 103 and greater (A1), 48–63 × 103 (A2), 40–48 × 103 (A3), 31–40 × 103 (A4), and 8–31 × 103 (A5). The proportions of the total area corresponding to these subareas were compared for the different varieties. Dye absorption was assumed to be directly proportional to protein concentration (in the linear range) with the same proportionality holding for the first four areas (largely storage proteins). The proteins of A5 (largely albumins) were assumed to have a three‐fold greater dye‐binding capacity than the other proteins and a correction to this area was based on this assumption. Red River 68, a variety with strong mixing characteristics, had a comparatively large proportion of its total protein in A3 and a notable peak of mol. wt 46 × 103 in this area. Bankuti 1201 and Omar, with weak mixing characteristics, had comparatively small proportions of their protein in A3, but large proportions in A4 (largely gliadins). Atlas 66 had a comparatively large proportion of its protein in A5 (largely albumins) and a smaller proportion in A4, but there were no strong differences in the pattern that could be related clearly to the high‐protein character of Atlas 66. All the varieties had most of their protein (65–69%) in the mol. wt ranges corresponding to A3 and A4.

An immunological approach to the study of protein conformational heterogeneity: its application to growth hormone.

The study of multiple forms of a hormone due to changes in its secondary or tertiary structure (conformational heterogeneity) has been restricted to methods of physical chemistry which require relatively large quantities of purified preparations and has precluded such studies on circulating hormones. The present report describes an immunological approach to the study of GH conformational heterogeneity based on the simultaneous use of two homologous RIAs: one which recognizes porcine (p) GH in its native conformation (pGH RIA) and a second (pB RIA) for a 55-residue S-aminoethylated cyanogen bromide fragment (pB) of pGH (corresponding to residues 126-180 of human GH). In the pGH RIA, pB was nonreactive, whereas pGH displacement of [125I]iodo-pB from anti-pB serum required about 10(3) M excess and was nonparallel to pB. Treatment of pGH with urea plus mercaptoethanol (ME), which would be expected to affect the conformation of the GH molecule, increased its immunoreactivity 68-fold in the pB RIA and its dilution curve became parallel to that of pB. Similar treatment of porcine pituitary extract increased its immunoreactivity 97-fold in pB RIA with a corresponding slope change. In contrast, urea plus ME decreased pGH and pituitary extract immunoreactivity by 84% and 92%, respectively, in the pGH RIA. The pB RIA/pGH RIA ratio of immunoreactivity for pGH and for pituitary extract increased 795-fold and 569-fold, respectively, after combined treatment, whereas lesser increases were observed after urea or ME treatment alone. In conditions where alterations in GH conformation were suspected (highly purified, aged human GH preparations which were no longer satisfactory as radioiodinated tracers for radioimmunoassay), calculation of the pB RIA/hGH RIA ratio of immunoreactivity indicated increased values. Based on these results, a model is proposed in which unfolding a native hormone, with concomitant exposure of the antigenic determinant of pB, hidden within pGH, leads in increased immunoreactivity in the pB RIA and decreased immunoreactivity in the pGH RIA. In this model, the pB RIA/pGH RIA ratio of immunoreactivity would thus serve as an index of the state of hormonal unfolding.

The purification of rabbit liver guanine aminohydrolase by affinity chromatography.

Abstract Rabbit liver guanine aminohydrolase (GAH) was purified 7100 fold to a specific activity of 35.5 units/mg with 60% yield. The three step purification involved homogenization at pH 5.0, 40–80% (NH4)∗S04 fractionation at pH 6.5 and affinity chromatography on 9-(p-aminoethoxyphenyl)guanine-Sepahros.e 48 (AEPGS). GAH was eluted from the AEPGS by decreasing the ionic strength of the eluting solution to below 7 × 10−3. Three closely spacedprotein bands were seen inpolyacrylamide gel after electrophorsis was carried out on the purified GAH. These Coomassie Blue tained bands are believed to represent very similar forms of GAH. Mercaptoethanol (ME) was found to prevnt aggregation of GAH. High molecular weight species of GAH, reported previously, were eliminatd by treatment with 5 × 10−3M Me. The purified GAH had a molecular weight of 110,000 in the presence of ME. The Km with guanine as a substrate for GAH was 3.8 × 10− 6 at pH 7.0. The affinity ligand, 9-(p-aminoethoxyphenyl) guanine (AEPG), inhibited GAH...

Effect of60Co γ Radiation on the Valyl-tRNA Synthetase Isolated from Chick Embryo Brain

The effect of 60Co gamma irradiation on the activity of valyl-tRNA synthetase isolated from chick embryo brain was studied. The enzyme activity exponentially decreased in the dose range 10--200 krad. The first step of the enzyme action, i.e. the amino acid activation, was found to be less sensitive to irradiation than the whole reaction, the formation of valyl-tRNA, 2-Mercapto ethanol and/or glycerol had a significant radioprotective effect. The lesion caused by radiation in the enzyme was also influenced by its concentration during exposure (dilution effect). According to gel-electrophoretic experiments, no chain rupture occurred in the enzyme molecule. Not even a change in Km was observed; however, the maximum velocity of the reaction was found to decrease with increasing radiation dose.