A shortened procedure of the diphenylamine reaction for the measurement of deoxyribonucleic acid by using light activation

The diphenylamine reaction used for the measurement of DNA was found sensitive to light activation but only when acetaldehyde was omitted from the reagent. The increase in sensitivity of the overall reaction was dependent on several factors, namely, the intensity and the emission spectrum of the light used for the activation of the incubation medium. This property made possible reduction of the time needed for the development of the final blue coloration to 4 hr, with illumination values ranging from 10,000 to 15,000 lx. Such a shortened procedure was applied both to highly purified DNA preparations and to DNA crude extracts.     

The use of the L-serine sulfhydrylase assay for the estimation of cystathionine beta-synthase

The reaction of diphenylamine (DPA) with deoxyribose has been widely used in the determination of DNA and purine deoxynucleotides. Since the publication of the original method by Dische (1), several reports (2–5) have described modifications which have reduced or eliminated interferences and have increased sensitivity. The procedure of Blakley (5) has been employed for the measurement of ribonucleotide reductase activity in which ribonucleoside triphosphates are reduced to 2′-deoxyribonucleoside triphosphates in the presence of a dithiol and vitamin B₁₂ coenzyme (6–8). The interference to color development caused by dithiols is eliminated by an alkylation step in which the sample is boiled with phosphate-buffered chloroacetamide.     

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.     

Volatile amines in mouse brain: a radioassay with picogram sensitivity

An isolation procedure and a radioassay for volatile amines in gram quantities of brain tissue are described. The radioassay utilizes ³H-2,4-dinitrofluorobenzene to form radioactive diphenylamine derivatives which are separated by thin-layer chromatography. The limit of sensitivity of the radioassay is in the picogram range. Seven volatile amines, including two not previously reported, are quantitated in adult mouse brain.     

A simplified diphenylamine colorimetric method for growth quantification

Cell growth needs to be monitored in biological studies and bioprocess optimization. In special circumstances, such as microbial fermentations in media containing insoluble particles, accurate cell growth quantification is a challenge with current methods. Only the Burton method is applicable in such circumstances. The original Burton method was previously simplified by adopting a two-step sample pretreatment in perchloric acid procedure to eliminate the need for DNA extraction. Here, we further simplified the Burton method by replacing the previous two-step perchloric acid pretreatment with a new and one-step diphenylamine reagent pretreatment. The reliability and accuracy of this simplified method were assessed by measuring the biomass of four model microorganisms: Escherichia coli, Streptomyces clavuligerus, Saccharomyces cerevisiae, and Trichoderma reesei grown in normal media or those containing solid particles. The results demonstrate that this new simplified method performs comparably to the conventional methods, such as OD₆₀₀ or the previously modified Burton method, and is much more sensitive than the dry weight method. Overall, the new method is simple, reliable, easy to perform, and generally applicable in most circumstances, and it reduces the operation time from more than 12 h (for the previously simplified Burton method) to about 2 h.     

Optimization of Folin-Ciocalteu reagent concentration in an automated Lowry protein assay

The Lowry method (G. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951, J. Biol. Chem.193, 265–275) for protein concentration measurement has been automated to permit assay of samples with concentrations from 1 to 400 μg/ml. Calibration with solutions of bovine serum albumin resulted in a nonlinear (quadratic) curve. The quantity of color developed in the assay was found to be strongly dependent on the concentration of the Folin-Ciocalteu phenol reagent. Color yield peaked sharply at a reagent concentration 40% lower than that used in the Lowry procedure. Optimization of the reagent concentration is necessary to obtain maximum sensitivity from the Lowry assay.     

Fluorometric Determination of Deoxyribonucleic Acid in Bacteria with Ethidium Bromide

A simple, sensitive, and rapid method is presented for the determination of deoxyribonucleic acid (DNA) in both gram-positive and gram-negative bacteria. It is based upon the fluorometric determination of DNA with ethidium bromide after alkaline digestion of the bacteria to hydrolyze the interfering ribonucleic acid. The assay takes less than 2 hr. Its sensitivity is at least 0.2 μg of DNA in a final solution of 4 ml and it uses commonly available filter or double monochromator fluorometers. Judicious choice of light source and filters allows an additional 10-fold increase in sensitivity with a filter fluorometer. Turbidity caused by bacteria or insoluble polysaccharides does not interfere with the fluorescence measurements. There was no significant difference between the results obtained with this method and those obtained with the indole and diphenylamine methods when these assays were applied to Escherichia coli and sucrose- or glucose-grown Streptococcus mutans. The method was also tested by determining the specific growth rate of E. coli. This new procedure should be especially useful for the determination of bacterial DNA in dilute suspensions and for the estimation of bacterial growth or DNA replication where more conventional methods are not applicable or sensitive enough.     

Properties of Anabaena variabilis cells grown in the presence of diphenylamine

Anabaena variabilis cells have been cultivated in the presence of diphenylamine (12 mg/l) which inhibits the biosynthesis of β-carotene, echinenone and zeasanthin. The content of chlorophyll a is also reduced by diphenylamine. The biosynthesis of myxoxanthophyll is, however, stimulated by this reagent.

The membrane fragments prepared from Anabaena cells grown in the presence of diphenylamine have the activities of both Photosystem 1 (NADP⁺ reduction with DCIP-ascorbate as electron donor) and Photosystem 2 (DCIP reduction with 1,5-diphenylcarbazide as electron donor).

The fluroescence spectra of these cells at 77°K show peaks at 696 and 731 nm and a shoulder around 687 nm. The fluorescence intensity at 687 and 696 nm is higher in these cells than in normal-Anabaena cells.     

DNA measurement by mithramycin fluorescence in chromatin solubilized by heparin

The mithramycin fluorescence procedure described by B. T. Hill and S. Whatley (1975, FEBS Lett., 56, 20–23) for DNA measurement tends to underestimate DNA concentrations in biological samples as compared to the results obtained by the diphenylamine reaction. This discrepancy disappears when DNA is first solubilized, by buffer containing heparin, from either cell homogenates or nuclear preparations. The optimal conditions for maximal fluorescence are 8 mm Mg²⁺, 10 μg/ml mithramycin, and heparin to DNA ratios ≥0.15 ($\text{w}\text{w}$). Background fluorescence is reduced 90% by dextran-coated charcoal adsorption of unbound mithramycin. The limit of sensitivity of the assay is 0.3 μg/ml and fluorescence is linear up to 30 μg DNA/ml.     

Purification and partial characterization of citrate synthase from Phaseolus vulgaris mitochondria

Citrate synthase (E.C. 4.1.3.7) has been isolated from bean mitochondria by an improved procedure. The purified enzyme had a specific activity of 50. In most respects (e.g. sedimentation constant, K_ms, pH sensitivity and ionic strength inhibition) the enzyme is similar to that prepared from mammalian sources. The feature distinguishing the plant enzyme from the others was its inhibition by several sulfhydryl reagents. The substrates conferred either complete protection (acetyl coenzyme A) or partial protection (oxalacetic acid) against the inhibition. Dithiothreitol (DTT) was capable of partially reversing the inhibition. The efficacy of DTT varied with the sulfhydryl reagent and was inversely related to the period of incubation of the enzyme with the reagent.     

The estimation of deoxyribonucleic acid in the presence of sialic acid: application to analysis of human gastric washings

1. Sialic acid has been found to interfere with three colorimetric reactions used for the estimation of DNA: a modified diphenylamine reaction at 100° (Dische, 1930), the nitrophenylhydrazine method (Webb & Levy, 1955) and the diphenylamine reaction at 30° (Burton, 1956). 2. Evidence is presented that sialic acid is present in hydrolysates obtained from gastric wash-out material. 3. A mathematical method for correcting for interference from sialic acid in the diphenylamine reaction at 30° is described. 4. The diphenylamine reaction has been modified to make it suitable for the estimation of DNA in the presence of sialic acid. The modifications are to increase the concentration of diphenylamine to 2% and to perform the reaction at 6–13° for 48hr. These modifications increase the sensitivity 25% above Burton's (1956) modification of the diphenylamine reaction. 5. The precipitation, extraction and recovery of DNA from gastric wash-out material have been investigated.     

An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines.

The use of 2,4,6-trinitrobenzenesulfonic acid (TNBS) as a reagent for determining the concentrations of amines has been widely accepted (1–3) since its introduction in 1960 by Satakeet al. (4). The original procedure has since been modified by Mokrasch (5) to permit the determination of amines, amino acids, and proteins in mixtures. In both procedures the trinitrophenylation reaction is followed by a quenching step, after which the amino content is related to the increase in absorbance at 340 nm (4) or 420 nm (5). We have studied the trinitrophenylation reaction and have found that amino content can be related directly to the absorbance of the trinitrophenylation reaction mixture after a relatively short incubation period (15–30 min). Therefore, it is unnecessary to quench this reaction. We describe herein an extremely convenient procedure for the determination of amines, amino acids, and proteins where the quenching step employed by previous investigators has been eliminated. The proposed method has a greater sensitivity than previously described techniques employing TNBS.     

Infection intensity-dependent accuracy of reagent strip for the diagnosis of Schistosoma haematobium and estimation of treatment prevalence thresholds

BackgroundReagent strip to detect microhematuria as a proxy for Schistosoma haematobium infections has been considered an alternative to urine filtration for individual diagnosis and community-based estimates of treatment needs for preventive chemotherapy. However, the diagnostic accuracy of reagent strip needs further investigation, particularly at low infection intensity levels.MethodsWe used existing data from a study conducted in Tanzania that employed urine filtration and reagent strip testing for S. haematobium in two villages, including a baseline and six follow-up surveys after praziquantel treatment representing a wide range of infection prevalence. We developed a Bayesian model linking individual S. haematobium egg count data based on urine filtration to reagent strip binary test results available on multiple days and estimated the relation between infection intensity and sensitivity of reagent strip. Furthermore, we simulated data from 3,000 hypothetical populations with varying mean infection intensity to infer on the relation between prevalence observed by urine filtration and the interpretation of reagent strip readings.Principal findingsReagent strip showed excellent sensitivity even for single measurement reaching 100% at around 15 eggs of S. haematobium per 10 ml of urine when traces on reagent strip were considered positive. The corresponding specificity was 97%. When traces were considered negative, the diagnostic accuracy of the reagent strip was equivalent to urine filtration data obtained on a single day. A 10% and 50% urine filtration prevalence based on a single day sampling corresponds to 11.2% and 48.6% prevalence by reagent strip, respectively, when traces were considered negative, and 17.6% and 57.7%, respectively, when traces were considered positive.Conclusions/SignificanceTrace results should be included in reagent strip readings when high sensitivity is required, but excluded when high specificity is needed. The observed prevalence of reagent strip results, when traces are considered negative, is a good proxy for prevalence estimates of S. haematobium infection by urine filtration on a single day.     

A convenient manual trinitrobenzenesulfonic acid method for monitoring amino acids and peptides in chromatographic column effluents.

The trinitrobenzenesulfonic acid (TNBS) method of R. Fields (1971, Biochem. J., 124, 581–590) has been modified for the manual detection of amino acids and peptides in chromatographic column effluent by changing the reaction conditions to 1 mm TNBS in 0.4 m potassium borate buffer, pH 9.2, at room temperature for 30 to 50 min. The reaction with amines and the spontaneous hydrolysis of TNBS are stopped by neutralization to pH 6.25 with sodium monobasic phosphate (0.33 m). Sodium sulfite (3 mm) is added to increase the absorptivity of the product. The TNBS reagent blank is less than 0.100 A₄₂₀ after 50 min of reaction. Since the ΔA₄₂₀ of the reagent blank is ～0.002/min before quenching the reaction, and zero afterward, the time required for reaction and for absorbance measurements need not be controlled precisely. Alkaline hydrolysis of peptides is carried out prior to detection to increase the sensitivity of the method. This procedure is convenient for the manual determination of 5 to 100 nmol of amino acids in the 50–100 samples required to define a chromatographic elution profile.     

Fluorometric quantification of DNA in cells and tissue

The validation of a simple and rapid DNA solubilization procedure is described. Quantitative extraction of intact, polymerized DNA was achieved by cell lysis or tissue homogenization in an ammonium hydroxide-Triton X-100 solution. The solubilization procedure inactivates endogenous DNAase and increases the fluorescence-enhancement activity of the extracted DNA, thereby eliminating the need for enzyme treatment or exposure to high salt solutions. The extracts can be utilized directly in a sensitive fluorescence-enhancement assay with bisbenzimidazole (Hoechst 33258) reagent. Estimates of DNA cell content were unaffected by the number of cells lysed or the volume of lysate employed in the assay. In all cases, the solubilized DNA estimates were linear and parallel to the bovine DNA standard. The optimum range for estimation of DNA in this assay is 5-150 ng. In addition, estimates of DNA obtained with this method and the standard diphenylamine assay were in excellent agreement. This simple, one-step DNA extraction procedure can be utilized in conjunction with Hoechst reagent to obtain quantitative estimates of DNA levels in cell or tissue extracts.     

A simple assay for measurement of urinary p-aminobenzoic acid in the oral pancreatic function test.

A simple method for the measurement of p-aminobenzoic acid (PABA) in urine following oral administration of N-benzoyl-l-tyrosyl-p-aminobenzoic acid (Bz-ty-PABA), a synthetic peptide used for the assessment of exocrine pancreatic function, was investigated using p-dimethylamino cinnamaldehyde (DACA) as a chromogenic reagent. DACA reacted with PABA in acidic solution and the red dye which developed had an absorption spectrum with maximum at 550 nm. Urine containing PABA and its conjugated metabolites was hydrolyzed with HCl, DACA solution was added to a portion of the hydrolysate, and the color intensity was determined. When Bz-ty-PABA was administered orally to healthy humans and patients with pancreatic insufficiency, urinary PABA content determined by the DACA method was in good agreement with that by the diazo-coupling method. This DACA method is preferable to the diazo-coupling method with respect to the simplicity and rapidity of the procedure and to the stability of the reagent solution. It is especially useful for the above application in the clinical laboratory.     

Colorimetric determination of acetaldehyde in the presence of formaldehyde

A procedure is described that enables use of the p-phenylphenol color reaction to determine acetaldehyde in the presence of formaldehyde. The sample is first treated with an acidic 2,4-pentanedione reagent, which selectively removes formaldehyde. The method is applicable to blochemical reactions using tissue preparations.     

Comparison of two approaches for quantitative O-linked glycan analysis used in characterization of recombinant proteins

The principal aim of this study was to demonstrate the optimization and fine-tuning of quantitative and nonselective analysis of O-linked glycans released from therapeutic glycoproteins. Two approaches for quantitative release of O-linked glycans were examined: ammonia-based β-elimination and hydrazinolysis deglycosylation strategies. A significant discrepancy in deglycosylation activity was observed between the ammonia-based and hydrazinolysis procedures. Specifically, the release of O-glycans from glycoproteins was approximately 20 to 30 times more efficient with hydrazine compared with ammonia-based β-elimination reagent. In addition, the ammonia-based reagent demonstrated bias in the release of particular glycan species. A robust quantitative hydrazinolysis procedure was developed for characterization of O-glycans. The method performance parameters were evaluated. It was shown that this procedure is superior for quantitative nonselective release of O-glycans. Identity confirmation and structure elucidation of O-glycans from hydrophilic interaction chromatography (HILIC) fractions was also demonstrated using linear ion trap Fourier transform mass spectrometry (LTQ FT MS) with mass accuracy below 1 ppm.     

A new salting-out technique for colorimetric free fatty acid assays.

A colorimetric assay for measuring long chain (C > 10) free fatty acids in plasma is described. The basic assay procedure of R. J. Schimmel and E. Knobil ((1970) Amer. J. Physiol.218, 1540–1547) was modified in three ways: 1) by not buffering the cobalt-triethanolamine reagent, 2) by replacing potassium sulfate with lithium sulfate, and 3) by making a single reagent of chloroform, heptane, and methanol. In the absence of the buffer system, the soap complex was chemically more stable. A loss of sensitivity incurred by omitting the buffer system was corrected by using a salt solution of 20% (w/w) Na₂SO₄/10% (w/w) Li₂SO₄ instead of saturated Na₂SO₄/K₂SO₄. The number of pipettings was reduced by making a single reagent of chloroform, heptane, and methanol. Because of improved assay stability, large numbers of samples (90) can be processed simultaneously. The method is adequately linear over the range of 100–1250 μequiv/liter. The standard deviation is independent of the mean over the range of 250–1000 μequiv/liter and is about 40 μequiv/liter (n = 15).     

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.