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PubMed1981

Criticism of the use of Coomassie Brilliant Blue G-250 for the quantitative determination of proteins.

The quantitative determination of proteins in biological fluids, using Coomassie Brilliant Blue G-250, was evaluated. Compared with the biuret method, the Coomassie Blue G-250 method needs a much shorter time for analysis and has a greater sensitivity. The sensitivity of the dye for albumin is significantly greater than for globulins. The standard curves for the biuret method are more linear than those for the Coomassie Brilliant Blue G-250 method. The Coomassie Brilliant Blue G-250 method produces a precipitate, which sticks to the walls of the cuvet and results in an intolerable carry-over. Therefore, the use of the Coomassie Brilliant Blue G-250 method for the quantitative determination of proteins in urine and serum is not recommended.

Chemical Phenomena↗
PubMed2001

Comparison of four methods for determination of total protein concentrations in pleural and peritoneal fluid from dogs.

OBJECTIVE: To compare 4 techniques for determination of total protein concentrations in peritoneal and pleural effusions from dogs. SAMPLE POPULATION: 23 peritoneal and 12 pleural fluid samples from 35 dogs with various abnormalities. PROCEDURE: Samples were collected into tubes containing EDTA, centrifuged, and stored at -20 C until total protein concentrations were assessed. Protein concentration in each sample was determined by use of urine test strips, refractometry, and Bradford and biuret techniques. Accuracy of each method was determined, using dilutions of human control sera. RESULTS: There was good correlation among results of all quantitative procedures. Results of the biuret technique were more accurate than results of the Bradford assay. Refractometry underestimated protein concentration in samples with < 20 g of protein/L. Results of urine test strips correctly classified effusion samples into 2 groups on the basis of total protein concentrations less than or greater than 20 g/L. CONCLUSIONS AND CLINICAL RELEVANCE: Results of any of these 4 techniques can be used to rapidly and efficiently differentiate peritoneal and pleural fluid from dogs into transudates and exudates on the basis of total protein concentration less than or greater than 20 g/L, respectively.

Animals↗
PubMed1991

Chemiluminescence assays of Cu2Zn2 superoxide dismutase mimicking Cu-complexes.

The aqueous decay of K3CrO8 was used to compare the reactivity of Cu2Zn2 superoxide dismutase and two active centre analogues where the first shell atoms around the copper are four unsaturated nitrogens. Unlike the acetate or biuret type Cu(II) chelates these di-Schiff-base complexes had an identical reactivity compared to that of the intact enzyme. Nanomolar concentrations of copper coordinated in these complexes were sufficient to inhibit the K3CrO8 induced chemiluminescence by 50%. Furthermore, a lucigenin amplified chemiluminescence assay based on isolated polymorph nuclear leucocytes in the absence and presence of whole, unseparated blood was developed and successfully employed. CuPu(Im)2 and CuPu(Py)2 equivalent to 0.5 and 0.8 SOD units, only, were required to inhibit the photon emission by 50% in the absence of bovine serum albumin. Even in the presence of 600 microM albumin mimicking the competitive copper chelation in biological fluids Cu-Pu(Py)2 and CuPu(Im)2 remained active, whereas the carboxylate- and biuret type chelates Cu(Sal)2 and Cu(Ser)2 reacted like CuSO4. The same reactivity of these low M, SOD mimics was seen in human blood.

Acridines↗
PubMed1980

Haemolymph protein concentration of Scylla serrata : assessment of quantitative methods and intra-individual variability.

The suitability of micro-Kjeldahl, biuret and Folin-Ciocalteu methods of protein determination in analysing the haemolymph protein concentration of Scylla serrata was assessed. It was found that biuret method is simple, reliable and the results are consistent. Intra-individual factor as a function of time of day is also responsible for individual variability in the haemolymph protein concentration of the crab.

Animals↗
PubMed1999

Colorimetric protein assay techniques.

There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

Colorimetry↗
PubMed1999

Intramolecular synergism of the inhibiting action of polydisulfide antioxidants in the system ferritin--H2O2--tetramethylbenzidine.

Polydisulfides of urea (PDSU), thiourea (PDSTU), biuret (PDSB), and gallic acid (PDSG) and their monomer analogues (urea, biuret, and gallic acid) inhibited (in a competitive manner) tetramethylbenzidine (TMB) peroxidation catalyzed by ferritin in 0.1 M acetate buffer, pH 4.2, containing 10% dimethylformamide. Their efficiency characterized in terms of inhibition constants, Ki, increased in the following order PDSU < PDSB approximately PDSTU << PDSG. This order is determined by the reactivity of monomers with respect to HO* radicals which are the main oxidizing agents in the system ferritin--H2O2. Polydisulfide antioxidants exhibit the intramolecular synergism of the inhibiting action (non-additivity of antiradical activity relative to their monomers) that was quantitatively characterized by alpha = (Ki)pol/(Ki)mon x n, where n is the number of monomers in the polymeric inhibitors. The alpha values increased from 1.5 up to 5.18 in the following order: PDSG < PDSU < PDSB. Significantly higher inhibiting efficiency of polydisulfide antioxidants as compared to monomer forms and synergism of the inhibitory action offer promising opportunities of their use as quenchers of free radical processes in biochemical systems.

Animals↗
PubMedDate not supplied

[Determination of the low protein concentration in biological fluids. Methodological aspects (the use of mathematical analysis)].

We investigated the effect of albumin/globulin ratio on the analytic error of protein determination. Several methods were used: Bradford, Lowry, pyrogallol-red, biuret, and sulfosalicylic acid. Reagents used in Lowry method exhibit higher affinity to globulins, whereas reagents used in other methods have higher affinity to albumins. Although the biuret method demonstrated equal efficiency in determining concentrations of various proteins low sensitivity of this method limits its clinical applicability. For other methods there was non-linear dependence of the analytic error on protein composition in the sample. Combination of two methods for protein determination (using one calibrator) completely removes the analytic error.

Albumins↗
PubMed1989

[The photometric determination of total bile protein].

Studies of bilirubin absorption spectra by protein measurements with the use of the biuret test and Lowry's method have shown that bilirubin influences much the protein absorption spectrum, provoking higher results in examinations of the bile. To eliminate bilirubin effects, the authors recommend bilirubin extraction with ethyl-acetone mixture in a 1:1 ratio after protein sedimentation with trichloroacetic acid. The biuret test with bilirubin-free protein yields results compatible with those obtained by nonphotometric techniques not involving bilirubin effects.

Bile↗
PubMed1989

Estimation of reference intervals for total protein in cerebrospinal fluid.

Protein in cerebrospinal fluid (CSF) was measured by a modified biuret procedure and in two automated instruments, the Du Pont aca and the Kodak Ektachem. The latter's dry-slide reagent was also evaluated for precision, linearity, and effect of potential interferents. In vitro, ampicillin and vancomycin increase the apparent value for CSF protein as measured with the Ektachem slides. We excluded patients with disorders of the central nervous system, and we estimated the central 95% percentile reference intervals for CSF protein for each of the three methods. We found no age or gender dependence of values. By the biuret procedure, the reference interval is 140 to 620 mg/L.

Adult↗
PubMed1988

[Antibiotic production by variants of Penicillium chrysogenum isolated after protoplast formation and exposure to mutagens at the protoplast stage].

A procedure for protoplast formation in the penicillin-producing organism Penicillium chrysogenum was developed. The yield of the protoplasts was high, the protoplasts were stable and capable of regeneration. Two types of the protoplast regeneration were revealed. The spores and protoplasts were treated with UV light and N-nitroso-N'-methyl biuret and their effect on production of the antibiotic by the isolated variants was studied. It was shown that the protoplasts of P. chrysogenum were more liable to the mutagenic effect of UV light and nitroso methyl biuret than the fungus conidia. It is possible to use this specific feature in intensification of selection aimed at isolation of highly productive strains of P. chrysogenum.

Culture Media↗
PubMed1986

[Induced mutagenesis in Escherichia coli--a producer of penicillin acylase].

The action of certain chemical mutagens such as N-nitroso methyl urea, N-nitroso ethyl urea, N-nitroso dimethyl urea and N-nitroso methyl biuret, as well as physical ones such as UV light on Escherichia coli producing penicillin acylase was studied. High mutagenic activity of N-nitroso methyl biuret with respect to this culture was shown.

Amidohydrolases↗
PubMed1986

Urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a Hitachi 726 automated analyzer.

In this new method for determining urinary protein, the reaction is complete within 10 min at 37 degrees C. This method is applicable to automated as well as manual measurements. Protein concentration and absorbance at 600 nm are linearly related throughout a wide range of concentrations, 10 to 16 000 mg/L. However, the chromogenicity of the gamma-globulins in this method is 70% of that of albumin, as estimated from results by a biuret method. Within-run CVs were less than 3.3%; the day-to-day CV was 2.9%. Errors due to interfering components in urine are less than 2%. The normal range for urinary protein as measured by this method was from 28 to 141 mg/day. Results by this method (y) and by a trichloroacetic acid-biuret method (x) correlated well (n = 80, r = 0.995; y = 0.99x - 2.9).

Adult↗
PubMed1986

[Protein determination in urine--a critical review].

Seven methods for protein determination in urine were systematically checked, viz. turbidimetric assay with benzethonium chloride, turbidimetry with trichloroacetic acid of various concentrations, micellar complex formation with Coomassie Brilliant Blue G-250, formation and gel filtration of protein complexes with copper, reaction of protein tannin compounds with iron, and biuret reaction after different precipitation procedures. The evaluation was mainly based on the following criteria: identical reaction with various proteins, stable and linear absorbance with concentration, complete recovery, and greatest possible freedom from interferences. On this basis, the biuret reaction must now be regarded as the method of choice. Albumin and gamma-globulins are reliably estimated after precipitation by perchloric acid or by an ethanol-diethylether mixture, whereas all proteins, including uromucoid, may be measured after precipitation by Tsuchiya's reagent. Interference from urinary pigments can be avoided by reading versus a sample blank.

Benzethonium↗
PubMed1976

Improved method for determining serum protein concentrations in the far ultraviolet.

We examined the feasibility of measuring absorbance in the far ultraviolet for serum protein determination with a medium-priced, double-beam spectrophotometer. The method has been simplified by using an automatic dilutor to dilute the serum, and a flow-through cell and recorder in conjunction with the spectrophotometer. Because it is only necessary to dilute the serum and determine its absorbance, the procedure is quite rapid. As compared to the biuret reaction, interferences from hemolysis have been about halved. Interferences owing to light scattering by turbid sera have also been appreciably decreased by use of an anionic detegent (sodium dodecyl sulfate) as a serum diluent. Comparisons of the present method to the biuret and Kjeldahl methods indicate that it is relatively accurate, and is practical for routine use.

Blood Proteins↗
PubMed2006

Felinine stability in the presence of selected urine compounds.

The stability of felinine, an amino acid present in feline urine, was investigated. Synthetic felinine was unstable in the urine of a selection of mammals. Felinine was found to stable in feline urine in which urea had been degraded. Synthetic felinine was found to react specifically with urea and did not react with urea analogues such as biuret or thiourea or other nucleophilic compounds such as ammonia which is more nucleophilic or acetamide and water which are less nucleophilic than urea. The reaction of urea and felinine was independent of pH over the range of 3-10. Urea did not react with N-acetyl-felinine suggesting a felinine N-terminal interaction with urea. Mass spectral analysis of the reaction products showed the presence of carbamylated felinine and fragmentation ions derived from carbamyl-felinine. The physiological relevance of felinine carbamylation is yet to be determined.

Animals↗
PubMed1981

Spectrophotometric study of total protein-albumin methods applied to cerebrospinal fluid.

A spectrophotometric study was carried out for three proteins assays when modification of their serum procedures using bromcresol green, bromcresol purple and biuret reagents were applied to the determinations of total proteins and albumin in cerebrospinal fluids. A novel concentration device wherein the sample itself was used as the primary diluent for the three reagents concentrated to contain the proper amounts of chemicals in smaller volumes than suggested in their serum procedures allowed reasonable absorbance signals to be obtained. Low molecular weight molecules were separated from the albumin and globulins of the fluids by centrifugal ultrafiltration using a 25K cutoff and spectra were obtained for both high and low molecular weight fractions. Some materials were obtained in the separated ultrafiltrates which gave reactions with all three reagents, reactions which either overlapped the spectra of the albumin reactions or superimposed the spectra obtained with the total protein reaction. A screening procedure for cerebrospinal fluid total proteins or centrifugally ultrafiltered albumin appears reasonable as an inference from studies made, although further elucidation of the low molecular weight fractions in needed as a confirmation device.

Albumins↗
PubMed1980

Three automated quantitative assays for serum proteins.

Three quantitative assays for detection of proteins are reported. One is an adaptation to the Auto-Analyzer of the widely used method of Lowry et al. In another procedure, the product of the reaction of proteins with a modified biuret reacts with the phosphomolybdic--phosphotungstic reagent of Folin and Ciocalteu. The third method, performed on hydrolyzed sample, is based on the measurement of proline (Pro) and the conversion of the Pro content into protein. The universal presence of proline in serum proteins suggested this assay. The values for total serum proteins as assayed by the three procedures are similar. The second and the third assay for proteins, described in this paper, can also be performed manually. Studies on interfering substances and their elimination as well as on the sensitivity of the assays are reported.

Animals↗
PubMed1997

Serum protein determination by high-performance gel-permeation chromatography.

A general high-performance gel-permeation chromatography (HPGPC) method was developed to determine protein in human serum with improved sensitivity and speed. The optimum UV wavelength for protein detection was found to be 210 nm, by comparing the protein values obtained by varying the UV wavelength of the HPLC detection system with the protein values obtained from spectrophotometric protein assays, i.e., the bicinchoninic acid (BCA) method and the biuret method. The analysis time was less than 1 min. Since this HPGPC serum protein assay method is simple and rapid, it is expected to be particularly well adapted for use in clinical laboratories.

Adolescent↗