A simplified method of quantitating protein using the biuret and phenol reagents.

A new modification of the Lowry method of quantitating protein is introduced, whereby the protein sample is mixed first with a diluted biuret reagent and later with 2 n phenol reagent (undiluted) for color development. The method is superior to the original in (i) extremely stable color development (0.3% change from 20 min to 2 hr), (ii) good reproducibility (±2% for 50–600 μg/ml of protein), (iii) elimination of the need to mix reagents for each assay, (iv) good storability (the diluted biuret reagent is storable for months), (v) simplicity (both reagents are available commercially), and (vi) the biuret method can be immediately converted to the Lowry method if the former does not yield a sufficient absorbance. It was found that the relationship between absorbance and protein concentration is expressed by a straight line with a slope of 1 in the Hill plot.     

Measurement of protein using bicinchoninic acid

Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.     

Estimation of labile sulfide in iron-sulfur proteins.

Lowry's method (1) for protein determination is subject to interference from the nonionic detergent Triton X-100 (2,3) which is used in high concentrations (1–5%) to solubilize membrane proteins or enzymes (4–6) and structural acidic proteins (7). Hartree (3) could reduce the errors caused by 0.1% Triton X-100 by a modification of Lowry's method. However, when protein solutions containing 0.2% or more of the detergent are mixed with the Folin-Ciocalteu reagent (1) a precipitate forms that interferes with the assay. We could reduce this interference to an insignificant level either by centrifuging the precipitate and incorporating Triton X-100 in both the reagent blank and standards, or by removing the detergent prior to the assay. This report presents two simple procedures for the Lowry assay of dilute protein samples containing 1–5% Triton X-100.     

An evaluation of protein assays for quantitative determination of drugs

We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red-Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble vitamins (ascorbic acid, niacinamide, pantothenic acid and pyridoxine). The biuret, Lowry and BCA assays responded strongly to most of the drugs tested. The PRM assay gave a sensitive response to the aminoglycoside antibiotics (gentamicin and neomycin) and the antipsychotic drugs. In contrast, the CBB assay showed little response to the aminoglycosides and gave a relatively poor response with the antipsychotics. The BEC assay did not respond significantly to the drugs tested. The response of the protein assays to the drugs was further evaluated by investigating the linearity of the response and the combined response of drug plus protein. The results are discussed with reference to drug interference in protein assays and the development of new methods for the quantification of drugs in protein-free solution.     

Inorganic phosphate estimation in the presence of dithiothreitol

Dithiothreitol (Cleland's reagent) is widely used as a sulfhydryl protective reagent in biochemical systems in vitro. For example, dithiothreitol has been used to achieve maximal rates of enzyme activity for protein phosphokinase reactions (1–4) as well as for phosphoprotein phosphatese assays (1,5). Meisler and Langan (5) have utilized ³²P-labeled histone phosphoprotein as a substrate to examine the protein phosphatase activity of a rat liver cytosol enzyme preparation. However, if one is dealing with a phosphoprotein substrate which may not be labeled with ³²P, it would be desirable to measure the phosphatase activity using a sensitive chemical analysis, e.g., the method of Berenblum and Chain (6) as modified by Martin and Doty (7). We have been interested in examining the protein phosphatase activity associated with prostatic chromatin and the androgenic influences thereupon, using nonhistone and histone phosphoproteins and phosvitin as substrates (Ahmed and Davis, unpublished data). In designing these experiments, 1–3 mm dithiothreitol was added in the reaction medium; this subsequently resulted in interference of P_i analysis using the Berenblum and Chain procedure (6,7). We have, therefore, systematically examined the conditions which may be used to assay P_i when dithiothreitol is present in the sample. The following report deseribes these observations.     

Artificial reductant enhancement of the Lowry method for protein determination

Addition of dithiothreitol in the Lowry procedure 3 min after adding the Folin-Ciocalteau reagent produces immediate color development, with 35 to 60% greater absorbance per mass of protein used.     

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.     

Interference of melanin in protein determination

Using several assay methods, synthetic eumelanin prepared by autooxidation of L-beta-(3,4-dihydroxyphenyl)alanine and a natural melanin isolated from dog hair melanosomes were tested in model experiments to assess their possible interference in protein determination. The degree of interference was assessed by comparing the data obtained with the melanin samples with those derived from measurements of bovine serum albumin. In the common biuret and Lowry methods melanin interferes by falsely increasing the values obtained; the addition of Folin reagent only after melanin removal, as suggested by Doezema, decreased but did not eliminate melanin interference. Methods working at acid pH such as those according to Salo and Honkavaara with Ponceau S or Sedmak and Grossberg or Spector using Coomassie blue G-250 proved much better. Although melanins adsorbed a small amount of dye from the reaction systems in these procedures, their sensitivity to proteins makes the melanin interference negligible. Such procedures can therefore be recommended for protein determination in the presence of melanin.     

Variable sensitivity in the microbiuret assay of protein

Microbiuret methods have been introduced which have sensitivity similar to that of the method of Lowry et al. (1) and are claimed to be less subject to interference (2–5). Each method has three steps: (I) formation of the protein-copper biuret complex in alkaline solution, (II) separation of excess copper reagent from protein-bound copper, and (III) colorimetric assay of the latter with diethyldithiocarbamate. They differ chiefly in steps I and II. However, the concentration of dry bovine serum albumin (BSA) reported to give absorbance 1.0 in these methods ranges from 64 to 99 μg/ml of final coloured solution. In preliminary studies in this laboratory these variations were confirmed and in one case [the method of Westley and Lambeth (3)] linearity and sensitivity were significantly improved by addition of detergents in step III. This was surprising since albumin is added in step III of that procedure to prevent precipitation of the poorly soluble copper complex. This report shows that absorbance and stability of the copper-diethyldithiocarbamate complex is markedly affected both by the concentration of alkali and by the presence or absence of detergents. The method of Klungsöyr (4), in which excess alkaline copper tartrate reagent is removed by adsorption on a Sephadex column, is probably the simplest for multiple assays and a modified procedure giving a reproducible high sensitivity of absorbance 1.0 for 51–53 μg of dry BSA/ml of colour is described.     

Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine

Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.     

The use of high-pressure liquid chromatography in the simultaneous assay of 11beta-hydroxylase and 18-hydroxylase in zona fasciculata-reticularis tissue of the rat adrenal cortex.

Certain reagents utilized in the formation of polyacrylamide gels are shown to interfere in the Lowry assay for protein. Acrylamide (3–30%) and potassium ferrocyanide (0.0015–0.0105%) produced a linear response in color formation. Both compounds are capable of reducing the phenol reagent in the absence of copper and the interference can be compensated for by employing the appropriate blank. An extract of polymerized and electrophoresed gels also interferes in the Lowry assay, however, this increased color formation cannot be corrected by using a gel extract blank. Under the conditions studied, filtration, centrifugation, and dialysis did not sufficiently remove the acrylamide fines responsible for the interference.     

Synthesis of a radioactive thiol reagent, 1-S-[3H]carboxymethyl-dithiothreitol: identification of the phosphorylation sites by N-terminal peptide sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A novel radioactive thiol reagent, 1-S-[3H]carboxymethyl-dithiothreitol (DTT-S-C[3H(2)]CO(2)H, [3H]CM-DTT), was designed and synthesized at the micromole level by reaction of dithiothreitol with tritiated iodoacetic acid (I-C[3H(2)].CO(2)H). The reaction progress was followed by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The usefulness of the synthesized reagent was evaluated in a series of experimental approaches. (i) The synthetic phosphopeptide, NSVS(P)EEGRGDSV, was derivatized by [3H]CM-DTT separated from excess reagent by RP-HPLC. The extent of derivatization was quantitated in terms of the mol of P-Ser/mol of peptide by 3H counting, and the location of the phosphoserine was defined by the N-terminal Edman degradation sequence analysis as being the fourth amino acid residue from the N terminus. (ii) A sample of trypsin-digested alpha-casein was derivatized with [3H]CM-DTT, peptides were separated by RP-HPLC, and aliquots of each fraction were counted for 3H label within the peptide map which rapidly pinpointed the original four phosphoserine-containing peptides. This demonstrated the utility of the synthesized radioactive thiol agent in rapid purification and identification of phosphopeptides from HPLC peptide mapping of proteolytic digests of phosphoproteins. (iii) The [3H]CM-DTT was also used to determine the extent of phosphorylation of phosphoproteins both qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography and quantitatively by 3H counting. The synthesized radioactive thiol reagent [3H]CM-DTT proved to be very efficient and sensitive and should be adaptable to a wide range of routinely utilized laboratory approaches in many fields of the biological sciences.     

Interference of Good's buffers and other biological buffers with protein determination

Interference of low concentrations of Hepes and other buffers commonly used in protein determination was studied. The data show that some of these buffers interfere to differing degrees with protein determination according to the Lowry method. A study of the structure-interference relationship suggests that the group ethanolamine is involved in this interference. No interference was observed when protein was measured using bicinchonic acid at the same concentration as the Lowry reagent.     

Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent.

A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).     

Peptide uptake in germinating barley embryos involves a dithiol-dependent transport protein

An active transport system for small peptides occurs in the scutellar membrane of germinating barley and serves to move the products of partial hydrolysis of storage proteins from the endosperm into the growing embryo. Transport of peptides, but not amino acids or glucose, is inhibited by the thiol reagents, N-ethylmaleimide and p-chloromercuribenzene sulphonic acid (PCMBS). Peptide substrates protect against PCMBS inactivation. The dithiol-specific reagent, phenylarsine oxide (PAO) also inhibits. The reducing agent, dithiothreitol, reverses the inactivation caused by PCMBS and PAO. We conclude that the peptide transport system contains a redox-sensitive, dithiol-dependent protein.     

Postcolumn detection of serum proteins with the biuret and Lowry reactions

The Lowry and biuret reactions have been adapted for the selective detection of chromatographically resolved proteins, specifically proteins separated by high-performance liquid chromatography. The protein reagents are continuously added to the column effluent and produce the characteristic chromophores with both proteins and peptides. The reaction chemistries are compatible with ion-exchange, steric exclusion, and reverse-phase chromatography. Detection limits for proteins resolved by ion-exchange are about 5 to 10 micrograms with the Lowry reaction. Peptides containing tyrosine can be detected at the 100-ng level when chromatographed on reverse-phase columns. The biuret reaction is about 8 times less sensitive for proteins and not very effective for peptides. Reaction detection can be combined with direct absorbance detection in the uv to distinguish proteinaceous peaks from other peaks containing uv-absorbing compounds.     

Reversible oxidation of glyceraldehyde 3-phosphate dehydrogenase thiols in human lung carcinoma cells by hydrogen peroxide

Human lung carcinoma cells (A549) were oxidatively stressed with mildly-toxic or non-toxic amounts of hydrogen peroxide (H2O2, 0.1 mM to 120 mM) for 5 min. Hydrogen peroxide exposure resulted in a dose dependent inhibition of binding (pH 7) of the thiol reagent iodoacetic acid (IAA) to a 38 kDa cell protein. Incubation of cells in saline for 60 min following H2O2 removal restored the ability of IAA to bind to the protein. Treatment with 20 mM dithiothreitol or 2 M urea also restored IAA binding, but 10% Triton X102 or 1 mM Brij 58 had no effect. Increasing to pH 11 during the IAA binding also increased thiol availability. Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) has been identified as the protein undergoing thiol/disulfide redox status and enzymic activity changes.     

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.     

A comparison of five of the methods commonly used to measure protein concentrations in fish sera

The concentration of protein in the sera of rainbow trout, Salmo gairdneri , brown trout S. trutta and Atlantic salmon S. salar has been measured by six standard techniques viz refractometry, copper sulphate specific gravity, automated and manual biuret, optical density and Lowry et al. phenol reagent and the results compared. Good correlation was obtained in most cases and interconversion formulae are given between each method in the three salmonid species. The concentrations obtained with the refractometer and optical density methods were approximately one and a half times those obtained with the others.     

Protein measurement using bicinchoninic acid: elimination of interfering substances

Protein quantitation based on bicinchoninic acid (BCA) is simple, sensitive, and tolerant to many detergents and substances known to interfere with the Lowry method. However, certain compounds often used during protein purification do interfere with the BCA protein assay. The response of the BCA chromophore to various interfering substances has provided insight into the mechanism of protein quantitation by BCA. Certain substances (e.g., glucose, mercaptoethanol, and dithiothreitol) elicit a strong absorbance at 562 nm when combined with the BCA working reagent. The absorbance appears to be identical to the normal response elicited by protein. Other agents (e.g., ammonium sulfate and certain ampholytes) diminish the protein-induced color development and shift the wave-length of the color response. Both types of interference can be eliminated by selectively precipitating protein with deoxycholate and trichloroacetic acid (A. Bensadoun and D. Weinstein (1976) Anal. Biochem. 70,241-250) prior to reaction with bicinchoninic acid. The modifications described here permit quick, efficient removal of many interfering substances that are commonly utilized during protein purification.