Potential interference of hydrogen peroxide in the 2,2'-bicinchoninic acid protein assay

At a concentration of 20-800 nmol/0.1 ml hydrogen peroxide instantly reacts with a 2,2'-bicinchoninic acid copper color reagent. It also reacts with a reformulated reagent at pH 7 but the color develops less rapidly. While the effect may interfere with protein estimations at alkaline pH, the effect of pH 7 may be used to determine the possible extent of hydrogen peroxide interference after treatment with catalase.     

Mechanism of dye response and interference in the Bradford protein assay

Bradford Coomassie brilliant blue G-250 protein-binding dye exists in three forms: cationic, neutral, and anionic. Although the anion is not freely present at the dye reagent pH, it is this form that complexes with protein. Dye binding requires a macromolecular form with certain reactive functional groups. Interactions are chiefly with arginine rather than primary amino groups; the other basic (His, Lys) and aromatic residues (Try, Tyr, and Phe) give slight responses. The binding behavior is attributed to Van der Waals forces and hydrophobic interactions. Assay interference by bases, detergents, and other compounds are explained in terms of their effects upon the equilibria between the three dye forms.     

Carbohydrate interference in assays based on the periodate-coupled thiobarbituric acid reagent

Interference with a procedure for measuring free sialic acids was encountered during a study on a neuraminidase in a rabies vaccine. This led to an investigation of the ability of carbohydrates to interfere in thiobarbituric acid-based assays. In addition to glucose and glycerol, 2-ketohexoses and 6-deoxyhexoses caused little interference, while glycosides caused strong interference. The generation of substances capable of forming pigments after reaction with thiobarbituric acid was due to the fact that the amount of periodic acid added to samples was less than the amount needed for complete degradation of the carbohydrate present. Glyceraldehyde, glycolaldehyde, and hydroxymalonaldehyde appeared to be the substances reponsible for the interfering colors.     

Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80

Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances.     

Plant and algal interference in bacterial beta-D-galactosidase and beta-D-glucuronidase assays.

Several commonly occurring freshwater and marine plants and algae were screened for beta-D-galactosidase and beta-D-glucuronidase activities by using a 60-min enzyme assay based on the hydrolysis by these enzymes of 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl- beta-glucuronide, respectively. All freshwater plant extracts tested showed beta-D-galactosidase activity several at relatively high levels, and a number also showed beta-D-glucuronidase activity. A number of the macroalgae showed no activity of either enzyme, but those showing beta-D-galactosidase activity also showed beta-D-glucuronidase activity. The majority of microalgae showed some beta-D-galactosidase activity, but few showed beta-D-glucuronidase activity. Further studies, using the commercial Colilert test and the marine water formulation of Colilert, revealed that 2 of 11 of the microalgal species and several of the plant extracts tested caused positive reactions. It was concluded that several plant extracts and algae could significantly interfere with the detection of coliform bacteria and Escherichia coli with the use of rapid assays, on the basis of their production of beta-D-galactosidase and beta-D-glucuronidase, respectively. The significance of the plant and algal interferences in tests such as Colilert is dependent on the levels of enzymes released under natural conditions, the dilution which they may undergo, and the numbers of algal cells present. This also applies to interferences in rapid enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the bicinchoninic acid protein assay to eliminate lipid interference in determining lipoprotein protein content.

The bicinchoninic acid (BCA) assay method for the determination of protein has been investigated for its utility in measuring the protein content of plasma lipoproteins. Although other methods, principally those based on the method of Lowry et al. (1951, J. Biol. Chem. 193, 265-275) have been extensively used for this purpose, the tolerance of the BCA method to many commonly encountered detergents and buffers offers a definite advantage over the Lowry-based methods. In this study, lipoprotein protein values obtained by the BCA method were compared to a standard modification of the Lowry et al. procedure since this assay forms the basis of much of the relevant literature. The standard BCA assay was found to overestimate the protein content of very low density lipoprotein by approximately 70% and low density lipoprotein by approximately 30%; high density lipoprotein values compared favorably. Overestimations by the BCA assay paralleled the relative phospholipid content of the lipoprotein fractions. This apparent lipid effect was eliminated by the addition of 2% sodium dodecyl sulfate to samples prior to the analysis. In the presence of this detergent, BCA assay measurements for these three lipoprotein fractions were 97, 90, and 98%, respectively, of the reference assay values.     

Alkaline phosphatase interference in immuno-enzymatic assays

BackgroundAlkaline phosphatase (ALP) enzymes are widely used as signal amplifiers in immunoenzymatic methods. Conditions that cause ALP elevations, such as bone or liver diseases, can cause interference in immunoenzymatic methods. We aimed to examine ALP''s effect on immunoenzymatic assay by adding isolated pure ALP to the prepared serum pool.MethodsWe prepared a serum pool and divided it into 4 groups. By adding isolated pure ALP at different concentrations to each group, we obtained sample groups containing ALP enzyme at concentrations of 85 U/L, 340 U/L, 870 U/L, and 1570 U/L. 20-repetition of bhCG, ferritin, FT4, TSH, troponin I, and Vit B12 tests were performed in each group. The coefficient of variation, bias, and total error was calculated. All groups were compared by using the Friedman test for paired samples.ResultsAfter ALP addition, the calculated total error values of FT4, bhCG and troponin I tests were above the acceptable error limits. There were statistically significant differences in bhCG, FT4, troponin I, and Vit B12 tests compared to the baseline ALP level (P<0.0125).ConclusionsIsolated ALP elevations can be a source of interference for immunoenzymatic methods.     

pH and buffering in the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay

The HCO3/CO3(2-) buffer used in the bicinchoninic acid (BCA) protein assay has only weak buffering capacity at the recommended pH (11.25). Consequently the assay is rather sensitive to interference from effectively acid or alkaline samples, particularly in the micro method. Adjustment of pH in these alkaline solutions of high [Na+] is complicated by Na+ errors on the pH electrode. Hence it is recommended to prepare the buffers from known amounts of NaHCO3 and Na2CO3, and to reduce the pH to around 10.7; this offers much better buffering capacity with only a limited reduction in color development.     

Ascorbic acid reduction of microtubule protein disulfides and its relevance to protein S-nitrosylation assays

The biotin switch assay was developed to aid in the identification of S-nitrosylated proteins in different cell types. However, our work with microtubule proteins including tubulin and its associated proteins tau and microtubule-associated protein-2 shows that ascorbic acid is not a selective reductant of protein S-nitrosothiols as described in the biotin switch assay. Herein we show that ascorbic acid reduces protein disulfides in tubulin, tau, and microtubule-associated protein-2 that are formed by peroxynitrite anion. Reduction of microtubule-associated protein disulfides by ascorbic acid following peroxynitrite treatment restores microtubule polymerization kinetics to control levels. We also show that ascorbic acid reduces the disulfide dithiobis(2-nitrobenzoic acid), a reagent commonly used to detect protein thiols. Not only do we describe a new reactivity of ascorbic acid with microtubule proteins but we expose an important limitation when using the biotin switch assay to detect protein S-nitrosylation.     

Sensitivity and variability of the Bradford protein assay in the presence of detergents

The effects of Triton X-100, sodium dodecyl sulfate (SDS), and urea on the response of Coomassie blue G to 16 different proteins and peptides of Mr 1140 to 146,000 were studied to assess the significance of protein conformation and of ionic and nonionic interactions for the dye response to individual proteins. Triton X-100 at a final concentration of 0.008% (v/v) increased the sensitivity of the Bradford assay toward all proteins of Mr 5700 or higher by an average 33%. Increases ranged from +11% with myelin basic protein to +128% with aprotinin. The relative range of absorbance of proteins and deviations from bovine serum albumin decreased by approximately 25%. Triton X-100 appears to facilitate nonionic interactions of the dye with proteins of limited capacity for ionic binding. Conformation of proteins also seemed to be of some significance because the chaotropic agent urea (0.16 M final concentration) increased sensitivity of the assay by 14%. Sensitivity of the assay was lowered by SDS (0.004% final concentration, w/v) by an average 75% from that of the control assay. The results indicate that the incorporation of low concentrations of a nonionic detergent may be useful in improving sensitivity and variability of the Bradford assay.     

Interference of the detergent Tween 80 in protein assays.

The nonionic detergent Tween 80, which has been widely used to stimulate protein secretion in bacterial and fungal systems, caused interferences in three protein determination methods. The OD595 developed in the Coomassie blue dye-binding assay with a variety of purified proteins in the presence of Tween 80 was 1.6 to 3.4 times greater than that observed without detergent. These differences could not be attributed totally to the rapid color development in the assay with Tween 80 alone. Crude concentrated extracellular bacterial proteins shaken overnight with Tween 80 yielded an altered fractionation pattern on size exclusion chromatography and 10-fold increased color with an absorption spectrum in the dye-binding assay different from that of bacterial proteins shaken without detergent. In the bicinchoninic acid method, the detergent caused a 2- to 3-fold increase in OD562 due largely to contaminating peroxides which could be removed by treatment with catalase. In the Folin phenol method, the detergent caused a slight precipitate, but residual interference was not detectable in filtered assay mixtures.     

General practitioner obstetrics in Bradford.

The standard of obstetrics care by general practitioners in Bradford was assessed by reviewing the case records of all women who in 1988 were booked for delivery under their general practitioner but subsequently required transfer to consultant care. A total of 5885 women were delivered in Bradford during 1988. Of 1289 booked under their general practitioner, 637 required transfer to consultant care. In 259 cases transfer occurred during labour; only 37 of these women were visited by their general practitioner. Many of the problems that precipitated transfer were predictable and some were considered preventable: 263 of the women transferred were considered unsuitable for booking by general practitioners. The perinatal mortality among women booked under their general practitioner was 10.1/1000 and the stillbirth rate 7.8/1000. These figures are high and suggest a need for tighter controls over the qualifications and experience of doctors participating in a fully integrated system of obstetric care.     

A test for endogenous interference in superoxide dismutase assays

Endogenous interfering substances can be detected by applying the techniques of parallel-line analysis of variance to the assay of superoxide dismutase (SOD) activities in crude tissue extracts. The technique also allows expression of specific activities in terms of units of activity as defined by commercially available standards as opposed to definition by the specific assay conditions utilized. This analysis may be broadly applied to many of the indirect SOD assays currently in use. In the current investigation, SOD was assayed by its ability to inhibit the rate of reduction of acetylated ferricytochrome c by superoxide anion ($\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$) generated via the xanthine-xanthine oxidase reaction. The parallel-line analysis was effective in detecting the presence of interference by exogenous ascorbate in the reaction system, and by unidentified endogenous reactants within extracts of whole blood and ocular choroidal tissues of the rainbow trout, Salmo gairdneri.     

Sialic acid interference with the thiobarbituric acid method for measuirng 3-deoxyoctulosonic acid.

Sialic acid produced an interfering color in an assay designed for measuring 3-deoxyoctulosonic acid. The color yield from sialic acid was not as high as that from 3-deoxyoctulosonic acid but was high enough to cause concern about problems arising when sialic acid-containing substances are present in preparations of bacterial lipopolysaccharides. The instabilityof 3-deoxyoctulosonic acid in acidic media led to reduced color yields. In general, the results suggested that data generated by the application of the thiobarbituric acid method to the measurement of 3-deoxyoctulosonic acid should be approached with caution.     

Glucose suppression of deoxyribose interference in the thiobarbituric acid determination of sialic acid.

Glucose has been demonstrated to suppress the reaction of deoxyribose in the thiobarbituric acid determination of sialic acid. Suppression occurs at the periodate oxidation step in which glucose apparently competes with deoxyribose. This suppression is augmented by sodium chloride and trichloroacetic acid. With these three substances, deoxyribose reactivity can be completely eliminated at concentrations up to 75 μm with only a 25% decrease in sialic acid reactivity.Mixtures of deoxyribose and sialic acid and hydrolyzed extracts of rat organs gave reaction products with an absorption spectrum closely resembling that given by a sialic acid standard. Provided that the spectral characteristics of the colored product from an unknown sample are verified, sialic acid can be determined directly from absorbance at 550 nm without interference by deoxyribose.     

Water-induced hydrophobicity of soy protein materials containing 2,2-diphenyl-2-hydroxyethanoic acid

Biodegradable soy protein isolate (SPI), containing 2,2-diphenyl-2-hydroxyethanoic acid, films (SB) were successfully prepared with bis-(2-hydroxyethyl)sulfide as a plasticizer by compression molding at 155 degrees C and 15 MPa. By immersing the SB in distilled water for 26 h, we prepared the films (coded as SB-WM) having good water resistance. The films were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, dynamic mechanical thermal analysis, and tensile testing to evaluate their structure and properties. Moreover, the surface of the SB-WM films was analyzed by X-ray photoelectron spectroscopy and contact angle measurement. SB-WM films exhibited significantly higher contact angle than SB. The results revealed that a lotus-like nanoscale structure was created in SB-WM films, with increased hydrophobicity, through the process of the solvent-induced microphase separation during the immersion in water. More stable compound diphenylhydroxymethane could form from 2,2-diphenyl-2-hydroxyethanoic acid of SB in water, leading to the hydrophobicity of the SB-WM materials. A "green" and easy method for fabricating hydrophobic materials from soy protein has been provided in this work.