A simple rapid biuret method for the estimation of protein in samples containing thiols

Interference in the Lowry protein determination by thiol compounds is now well known (1–3). We have found that the estimation of protein by the biuret reaction is also subject to interference when the protein sample contains various thiols. We wish to report that this interference can be prevented in most cases by using a biuret reagent which is chelated with ethylenediaminetetraacetate (EDTA). In samples containing dithiothreitol (DTT) it is also necessary to add iodoacetamide prior to the addition of the biuret reagent. The use of iodoacetate to eliminate thiol interference in the Lowry procedure has been reported previously (3).This report details the extent of interference of dithiothreitol, β-mercaptoethanol, β-mercaptoethylamine, and glutathione, and illustrates the extent of neutralization which is attained in each case. We have also introduced modifications which permit the development of a stable color in only 5 min.     

Importance of disulfide bonds in receptors for vasoactive intestinal peptide and secretin in rat pancreatic plasma membranes

(1) Vasoactive intestinal peptide (VIP), secretin, and C-terminal octapeptide of cholecystokinin (CCK-8) receptors were identified in rat pancreatic plasma membranes by the ability of these peptides to stimulate adenylate cyclase activity. The membrane preparation procedure was conducted through a series of steps including discontinuous sucrose density gradient fractionation. 5 mM β-mercaptoethanol was added stepwise. Membrane preparations obtained stepwise were preincubated for 10 min at 25°C in the presence of various concentrations of β-mercaptoethanol or dithiothreitol before assaying adenylate cyclase. The use of the reducing agents exerted no effect on p[NH]ppG-, NaF-, and CCK-8- stimulated activities. By contrast, stimulation of adenylate cyclase by low VIP concentrations was specifically altered when β-mercaptoethanol was used during tissue homogeneization at 5°C. (2) In addition, both VIP and secretin responses were highly sensitive towards a preincubation of 10 min at 25°C in the presence of dithiothreitol. (3) These results were likely to reflect alterations at the receptor level. ¹²⁵I-VIP binding was, indeed, reduced after dithiothreitol preincubation, low concentrations of the thiol reagent decreasing the apparent number of high-affinity VIP receptors and higher dithiothreitol concentrations reducing the affinity of VIP receptors.     

Changes in antigenic properties of the p35 protein of vaccinia virus in various protein fractions of the virion with the use of monospecific antisera

Three monospecific antisera to the major 35 kD (p35) surface protein of vaccinia and ectromelia viruses have been obtained. Two of them are obtained to p35 protein isolated by electrophoresis in the presence of sodium dodecylsulfate from the protein fractions of vaccinia virus, soluble in NP40 and NP40 with dithiothreitol (NP40 and DTT-fractions). The third serum is obtained to NP40-fraction of ectromelia virus, containing practically only p35 protein. The obtained antisera were compared in the reactions with the different fractions of viral proteins in two versions of solid phase radioimmunoassay. The effect of such reagents as sodium dodecylsulfate, NP40, 2-mercaptoethanol, ethanol on the antigenic properties of p35 protein from vaccinia virus is discussed.     

Thiols and the diazo group in photoaffinity labels

The photoactivable carbene precursor, 2-diazo-3,3,3-tri-fluoro-propionyloxy group, has been introduced recently for light-induced covalent crosslinking in studies of protein-phospholipid interactions in biomem-branes. The diazo group in this reagent has now been shown to undergo reduction in the dark by a number of thiols (dithiothreitol, 2-mercaptoethanol, cysteine and reduced glutathione) used commonly as protective agents for proteins. In contrast, thioglycolate did not cause significant reduction and, therefore, can be safely used as a protective agent. In vesicles formed from phospholipids containing the above photolabel in the fatty acyl chain, dithiothreitol and 2-mercaptoethanol caused reduction. However, cysteine and reduced glutathione caused insignificant reduction of the diazo group, presumably because of their non-permeant nature.     

Life history of mouse sperm protein. Intratesticular stages

A basic protein fraction, migrating as a single band in acetic acid-urea gel, distinct from histones, was isolated from mouse sperm collected from vasa deferentia and caudae epididymides and was used to immunize female rabbits. The presence of antibodies to the mouse sperm protein (MSP) in the rabbit antisera was demonstrated by a cytoimmunofluorescence procedure using the cells of origin of the antigenic protein, the mature mouse sperm. The specificity of the antisera was verified by fluid and gel precipitation tests and by crossed immunoelectrophoresis. The latter procedure demonstrated the presence of two antigen-antibody systems, consonant with earlier reports that the basic chromosomal protein of mouse sperm is heterogeneous. MSP antigen in situ was recognized by the specific antibodies of the rabbit antisera only after the smear of mature sperm was treated with either of two reducing agents: 2-mercaptoethanol or dithiothreitol. However, when the immunofluorescence procedure was applied to untreated smears of mouse testicular cells, spermatids of all stages from 1 to 14-15 were positive, while spermatocytes, stage 16 spermatids and spermatozoa were negative. After treatment of testes smears with reducing agent, only spermatocytes remained negative. Those observations indicate the following: (a) MSP is immunogenic in a heterologous species; (b) its antigenic sites are detectable in spermatozoa and spermatids of all stages, but not in primary spermatocytes; (c) those antigenic sites become masked at about stage 15 of spermiogenesis and may be unmasked by treatment with a reducing agent. The interpretation is made, therefore, that one or more components of MSP are assembled at the beginning of spermiogenesis and undergo an alteration in the final intratesticular stage of spermatid maturation. That alteration may be presumed to be the formation of disulfide linkages between the cysteine residues.     

Assay of proteins by Lowry's method in the presence of high concentrations of β-mercaptoethanol

β-Mercaptoethanol interferes in the determination of protein by the Lowry method (1–6). The interference can be overcome by the precipitation of proteins by trichloroacetic acid or acetone or by the use of H₂O₂ which oxidizes the sulfhydryl groups of β-mercaptoethanol (5). Both these methods have inherent disadvantages. Ross and Schatz (5) described a procedure for protein determination in the presence of high concentrations of β-mercaptoethanol where they removed the interference by the addition of iodoacetate. But addition of iodoacetate decreased the sensitivity of the reaction. The removal of interference by β-mercaptoethanol by heating has also been reported (3), but we observed that this procedure is not feasible when a large amount of β-mercaptoethanol is present in the protein samples. In the method reported in this communication, we made use of vacuum drying for the removal of interference by β-merceptoethanol. This method is simple, sensitive, takes less time, and can be used for the determination of protein in the presence of β-mercaptoethanol at levels as high as 10% in a sample volume of 1.0 ml (1.43 mmol) without using any additional chemical steps.     

Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydroquercetin and Flavan-3,4-cis-Diol (Leucocyanidin) to (+)-Catechin by Reductases Extracted from Cell Suspension Cultures of Douglas Fir

Extracts of cell suspension cultures from Douglas fir (Pseudotsuga menziesii) needles catalyzed the production of (+)-catechin (2,3-trans flavan-3-o1) from the 2,3-trans-flavan,3,4-cis-diol (leucocyanidin) in a NADPH-dependent reductase reaction at pH 7.4. Catechin was also produced, along with the 3,4-cis-diol, in a double step reduction from (+)-dihydroquercetin. It was necessary to eliminate any thiol such as 2-mercaptoethanol or dithiothreitol from the extract or assay mixture because these thiols apparently formed thioethers with the 3,4-diols.     

Novikoff hepatoma deoxyribonucleic acid polymerase. Sensitivity of the beta-polymerase to sulfhydryl blocking agents.

Unlike other beta-class eukaryotic DNA polymerases, the enzyme purified from the Novikoff hepatoma is inhibited by both sulfhydryl blocking agents N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pHMB). The degree of sensitivity varies depending on the enzyme purity, pH of the reaction, and the presence of sulfhydryl reducing agents. Novikoff beta-polymerase activity is unaffected by the presence of 2-mercaptoethanol (2-Me) or dithiothreitol (DTT); however, the combination of 2-mercaptoethanol and NEM or pHMB acts to reverse the inhibition of the sulfhydryl blocking agent. The reversal of inhibition involves more than just a titration of NEM with 2-mercaptoethanol since a) the combination of these two reagents actually stimulates the DNA polymerase, and b) dithiothreitol did not reverse the inhibition. Binding of the polymerase to DNA did not affect the enzyme sensitivity to NEM.     

Activation and Inactivation of Alcohol Dehydrogenase in Germinating Pea Cotyledons

Alcohol dehydrogenase (E.C.1.1.1.1.) activity increases markedly in the germinating pea cotyledon in the first 2 days. The activity was not suppressed by the administration of actinomycin D, 6-methylpurine, DL-p-fluorophenylalanine, and D-chloramphenicol. The compounds rather depressed the decrease of alcohol dehydrogenase activity in cotyledons after 3 days of germination. The alcohol dehydrogenase activity in ungerminated pea seeds was activated by treatment with sodium lauryl sulphate, sodium dioctyl sulfosuccinate, dithiothreitol, 2-mercaptoethanol and NADH. The inhibitory effect caused by the extract from 7 day-old cotyledons was diminished markedly in the presence of dithiothreitol and 2-mercaptoethanol, as well as by addition of bovine serum albumin. If dithiothreitol was added to the extraction medium, the enzyme activity from older cotyledons was greatly enhanced.     

A method for repetitive artifact suppression in multichannel EEG recordings

An artifact suppression method based on the assumption of linear independence of EEG and artifact signals is described. This assumption allows the use of principal component analysis for their separation. The method makes it possible to eliminate electrooculogram (EOG) signals from multichannel EEG recordings regardless of the use of special EOG channels in the recording, as well as to eliminate any other repetitive artifacts, including pulse and electrocardiogram artifacts, ones related to mechanical instability of the reference electrode, etc.     

双链DNA模板循环测序的研究

采用末端标记引物进行双链ＤＮＡ循环测序是实验室快速,准确获得双链ＤＮＡ模板序列的有效方法。实验结果表明：该测序法不仅能消除由于模板不纯所导致的引物的非特异性结合等因素而产生的非特异条带,而且能降低ＰＣＲ产物测序的背景问题。     

Fat metabolism in higher plants. Properties of a soluble stearyl-acyl carrier protein desaturase from maturing Carthamus tinctorius

A soluble extract from maturing safflower seeds (Carthamus tinctorius) synthesized [¹⁴C]oleic acid from [¹⁴C]malonate, or [¹⁴C]stearyl-acyl carrier protein. Stearyl-acyl carrier protein was generated from [¹⁴C]malonate by the seed extract. The desaturase had only a trace of activity when stearyl-CoA was the substrate. The stearyl-acyl carrier protein desaturase had a specific requirement for ferredoxin which was only partially replaced by flavodoxin. While NADPH was an effective reductant, NADH was ineffective. However, the most effective reductant was a system composed of ferredoxin, grana lamellae, ascorbic acid, dichlorophenolindophenol, and light. No NADPH requirement was observed when this reducing system was employed. Stearylacyl carrier protein desaturase activity was enhanced by dithiothreitol and reduced glutathione, but was partially inhibited by β-mercaptoethanol. The desaturase activity was inhibited by 1 mm potassium cyanide but insensitive to carbon monoxide. No lipid micelle requirement could be demonstrated.     

Conversion of codeine to morphine in isolated capsules of Papaver somniferum

The biotransformation of codeine to morphine was studied in isolated capsules of Papaver somniferum. Cofactors such as nicotinamide adenine dinucleotide, adenosine 5′-triphosphate, S-acetyl coenzyme A and pyridoxal phosphate were not required in the conversion of codeine to morphine. Reducing agents such as dithiothreitol, glutathione and β-mercaptoethanol strongly promoted codeine and morphine degradation, while morphine formation remained at a constant level. Hydrogen peroxide (concentration > 0.25 mM) caused the conversion of codeine and morphine to N-oxides by non-enzymatic oxidation. Isolated capsules of P. somniferum provide a method of studying the biotransformation of codeine to morphine.     

Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins

Despite little supportive data, differential target protein susceptibility to redox regulation by thioredoxin (Trx) f and Trx m has been invoked to account for two distinct Trxs in chloroplasts. However, this postulate has not been rigorously tested with phosphoribulokinase (PRK), a fulcrum for redox regulation of the Calvin cycle. Prerequisite to Trx studies, the activation of spinach PRK by dithiothreitol, 2-mercaptoethanol, and glutathione was examined. Contrary to prior reports, each activated PRK, but only dithiothreitol supported Trx-dependent activation. Comparative kinetics of activation of PRK showed Trx m to be more efficient than Trx f because of its 40% higher V(max) but similar S(0.5). Activations were insensitive to ribulosebisphosphate carboxylase, which may complex with PRK in vivo. To probe the basis for superiority of Trx m, we characterized site-directed mutants of Trx f, in which unique residues in conserved regions were replaced with Trx m counterparts or deleted. These changes generally resulted in V(max) enhancements, the largest (6-fold) of which occurred with T105I, reflective of substitution in a hydrophobic region that opposes the active site. Inclusive of the present study, activation kinetics of several different Trx-regulated enzymes indicate redundancy in the functions of the chloroplastic Trxs.     

Microeletrophoresis in continuous polyacrylamide gel gradients. IV. The effect of reducing agents on the electrophoresis of proteins in sodium dodecylsulfate buffer systems (author's transl)]

The migration of reducing agents (e.g. 2-mercaptoethanol, dithiothreitol and thioglycolic acid) was analysed in various electrophoretic buffer systems containing sodium dodecylsulfate. It is shown that proteins loaded with dodecylsulfate and previously not reduced can be reduced during the electrophoretic separation.     

Replacing β-mercaptoethanol in RNA extractions

RNA extractions are potentially compromised in terms of both yield and quality by ribonucleases (RNases). The pungent and toxic reducing agent β-mercaptoethanol (β-ME), therefore, is commonly added to the biospecimen’s lysis buffer to aid in RNase deactivation. Using different tissue types (liver tissue, kidney tissue, and cell pellets), extraction kits (RNeasy Mini Kit, Illustra RNA Spin Mini Kit, and PureLink Mini Kit), RNA quality assays (RNA integrity numbers [RINs] and quantitative real-time polymerase chain reaction [qRT–PCR]), yield assessments, and in vitro functional RNase assays (RNaseAlert Kit), we demonstrate that β-ME should be replaced by the less toxic dithiothreitol (DTT) alternative.     

Activation of rat liver pyrimidine nucleoside monophosphate kinase.

The activity of the pyrimidine nucleoside monophosphate kinase (ATP:dCMP phosphotransferase, EC 2.7.4.14) from rat liver is dependent upon the presence of sulfhydryl-reducing agents. Addition to the inactive enzyme of 2-mercaptoethanol (5 mM), a reagent specific for cleavage of disulfide bonds, effects a reduction in molecular weight from approx. 53 000 to 17 000, measured by molecular sieve chromatography. This low molecular weight form is partially active in the presence of 2-mercaptoethanol (f mM). In absence of 2-mercaptoethanol, the low molecular weight form is inactive. Higher concentrations of 2-mercaptoethanol (50 mM) fully reactivate the CMP(ATP) kinase activity followed by dCMP(ATP) and CMP(dCTP) kinase activities in a sequential manner, without further change in moelcular weight. Alkylation by iodoacetamide of the enzyme at different stages of reactivation in dithiothreitol suggests an ordered appearance of the various enzyme activities. Furthermore, iodoacetamide inactivates the fully active enzyme. Thioredoxin was found to activate the enzyme in a manner similar to 2-mercaptoethanol and dithiothreitol. These results are consistent with the interpretation that the mechanism of activation of the enzyme involves cleavage of inter- and intramolecular disulfide bonds.     

Thiol-based photocycle of the blue and teal light-sensing cyanobacteriochrome Tlr1999

Cyanobacteriochromes are a spectrally diverse photoreceptor family that binds a bilin chromophore. For some cyanobacteriochromes, in addition to the widely conserved cysteine to anchor the chromophore, its ligation with a second cysteine is responsible for a remarkable blue shift. Herein, we report a newly discovered cyanobacteriochrome Tlr1999 exhibiting reversible photoconversion between a blue-absorbing form at 418 nm (P418) and a teal-absorbing form at 498 nm (P498). Acidic denaturation suggests that P418 harbors C15-Z phycoviolobilin, whereas P498 harbors C15-E phycoviolobilin. When treated with iodoacetamide, which irreversibly modifies thiol groups, P418 is slowly converted to a green-absorbing photoinactive form denoted P552. The absorption spectrum of P498 appears to be unaffected by iodoacetamide, but when iodoacetamide modified, it is photoconverted to P552. These results suggest that a covalent bond exists between the second Cys and the phycoviolobilin in P418 but not in P498. Subsequent treatment with dithiothreitol converts P552 into P418, whereas dithiothreitol reduces P498 to yield P420, a photoinactive form. Site-directed mutagenesis shows that the second Cys is essential for assembly of the photoactive holoprotein and that the photoactivity of this inert mutant is partially rescued by β-mercaptoethanol. These results suggest that the covalent attachment and detachment of a thiol, although not necessarily that of the second Cys, is critical for the reversible spectral blue shift and the complete photocycle. We propose a thiol-based photocycle, in which the thiol-modified P552 and P420 are intermediate-like forms.     

Purification and properties of benzoate-4-hydroxylase from a soil pseudomonad.

Benzoate-4-hydroxylase from a soil pseudomonad was isolated and purified about 50-fold. Polyacrylamide gel electrophoresis of this enzyme preparation showed one major band and one minor band. The approximate molecular weight of the enzyme was found to be 120,000. Benzoate-4-hydroxylase was most active around pH 7.2. The enzyme showed requirements for tetrahydropteridine as the cofactor and molecular oxygen as the electron acceptor. NADPH, NADH, dithiothreitol, β-mercaptoethanol, and ascorbic acid when added alone to the reaction mixture did not support the hydroxylation reaction to any significant extent. However, when these compounds were added together with tetrahydropteridine, they stimulated the hydroxylation. This stimulation is probably due to the reduction of the oxidized pteridine back to the reduced form. This enzyme was activated by Fe²⁺ and benzoate. It was observed that benzoate-4-hydroxylase could catalyze the oxidation of NADPH in the presence of benzoate,p-aminobenzoate, p-nitrobenzoate, p-chlorobenzoate, and p-methylbenzoate, with only benzoate showing maximum hydroxylation. Inhibition studies with substrate analogs and their kinetic analysis revealed that the carboxyl group is involved in binding the substrate to the enzyme at the active center. The enzyme catalyzed the conversion of 1 mol of benzoate to 1 mol of p-hydroxybenzoate with the consumption of slightly more than 1 mol of NADPH and oxygen.     

The removal of exogenous thiols from proteins by centrifugal column chromatography

Centrifugal column chromatography was shown to provide a rapid, efficient, and useful means of separation of various low molecular weight thiols from proteins. The single chromatographic step procedure employed standard 5 ml plastic syringes containing Sephadex G-25 as the bed matrix and required less than 5 min to produce average dilutions of 5000-, 980-, and 25-fold, respectively, from 5 to 200 mM initial concentrations of 2-mercaptoethanol, dithiothreitol, and reduced glutathione in the sample as measured by titration with 5,5'-dithiobis-(2-nitrobenzoic acid). Dihydrofolate reductase solutions of 0.07-0.08 mM were separated from 50 mM 2-mercaptoethanol, dithiothreitol, or reduced glutathione with a minimum 16,500-fold dilution of the thiol after centrifugal chromatography on two consecutive columns. Thymidylate synthase solutions of 0.06 mM were effectively separated from 50 mM 2-mercaptoethanol or dithiothreitol with a minimum average 5900-fold dilution of the thiol after consecutive column chromatography. There was no change in either the physical or chemical properties of the enzyme throughout the course of the experiments as determined by activity, active site sulfhydryl group titration, and binding assays. Recoveries of protein obtained in the load fraction were usually in excess of 70% of the protein loaded with virtually no dilution from the initial concentration. This method was developed in order to facilitate the study of the active site sulfhydryl groups in enzymes.