[Microbiological transformation of biuret].
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In Vitro Techniques↗
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[Total blood protein determination by means of the Biuret method in the presence of synthetic colloids].
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Blood Proteins↗
Effects of low and high relative molecular protein mass on four methods for total protein determination in urine.
Four commonly used methods for the determination of total protein in urine were compared. These were two biuret methods using different precipitants, a Ponceau S method and a Coomassie Brilliant Blue method. The protein content of the urines was also evaluated by sodium dodecylsulphate polyacrylamide gel electrophoresis. The biuret method with ethanolic phosphotungstic acid as precipitant correlated best with the Coomassie Brilliant Blue method (r = 0.944; p less than 0.001) but less well with the Ponceau S (r = 0.895; p less than 0.001) or biuret-trichloroacetic acid (r = 0.874; p less than 0.001) methods. For urines with normal electrophoretic protein patterns, the imprecise biuret-trichloroacetic acid method (cv = 18.5%) gave the greatest number of false high results (23 in 36 urines) as assessed by electrophoresis. False low results were common in low relative molecular mass (Mr) proteinuria, especially with the biuret-tricholoroacetic acid and Ponceau S methods. High Mr proteinuria rarely caused false low results. Discrepancies between methods appear to have resulted from incomplete precipitation of low Mr protein by trichloroacetic acid.
Azo Compounds↗
Evaluation of assay procedures for prediction of passive transfer status in lambs.
OBJECTIVE: To compare 4 assay procedures for prediction of passive transfer status in lambs. ANIMALS: Thirty-one 1-day-old Sardinian lambs. PROCEDURE: Serum IgG concentration was determined by use of single radial immunodiffusion. The following were determined: serum total protein concentration as measured by refractometry (ie, refractometry serum total protein concentration), serum total protein concentration as determined by the biuret method (ie, biuret method serum total protein concentration), serum gamma-globulin concentration as determined by serum protein electrophoresis, and serum gamma-glutamyltransferase (GGT) activity as measured by spectrophotometry. Accuracy of these assays for estimation of serum IgG concentration in 1-day-old lambs was established by use of linear regression analysis. RESULTS: Refractometry serum total protein concentration, biuret method serum total protein concentration, and serum gamma-globulin concentration were closely and linearly correlated with serum IgG concentration. The natural logarithm (ln) of serum GGT activity was closely and linearly correlated with serum IgG concentration (ln). Refractometry serum total protein concentration, biuret method serum total protein concentration, and gamma-globulin concentration accounted for approximately 85%, 91%, and 95% of the variation in serum IgG concentration, respectively. Serum GGT activity (ln) accounted for approximately 92% of the variation in serum IgG concentration (ln). CONCLUSIONS AND CLINICAL RELEVANCE: For prediction of passive transfer status in 1-day-old lambs, serum GGT activity or biuret method serum total protein concentration determination will allow for passive transfer monitoring program development. Immediate refractometry serum total protein concentration determination is beneficial in making timely management and treatment decisions. Serum gamma-globulin concentration determination can be used as a confirmatory test.
Animals↗
Standards for total serum protein assays--a collaborative study.
We have studied the standardization of total serum protein assay with the biuret reaction. Standard solutions were prepared from lyophilized preparations of human serum albumin and bovine serum albumin, with corrections made for volatile material and ash contents. These solutions and a solution of crystalline albumin standard were analyzed with a new stable biuret reagent, to establish absorptivity values (values for the absorbance of a 1 g/liter final reaction mixture). The mean values obtained were 0.302, 0.292, and 0.290 for human serum albumin, bovine serum albumin, and the crystalline albumin, respectively. We believe that the established absorptivity value will improve the accuracy of serum protein determinations. We studied the linearity of the relation between color produced and protein concentration, with use of the solutions described above and a serum pool. The color adheres to Beer's law up to the highest concentration tested: 3 g/liter for HSA and BSA, and 2.8 g/liter for serum in the final reaction mixture. The new biuret reagent has been stable for one year at room temperature. We recommend the use of bovine serum albumin as a primary standard for serum protein assays. It is inexpensive, easily available, and exhibits the best linearity in the biuret reaction.
Animals↗
The usefulness and limitations of hand-held refractometers in veterinary laboratory medicine: an historical and technical review.
Medical hand-held refractometers have been used in veterinary practice since their development in the 1960s. They have become ubiquitous for the measurement of protein and urine solute concentrations because of their rapidity of analysis, ease of use, and relatively low cost. Refraction of light offers advantages for the determination of solute concentrations because the measurement requires no chemical alteration of the specimen. Numerous authors have reported that the results of protein estimation by refractometry for domestic mammals correlate well with those obtained by the biuret method, although others have reported both higher and lower refractometric results compared with biuret results. Major discrepancies between biuret and refractometric results have been reported for avian samples. Some of the variation in reported results may be due to differences in design by refractometer manufacturers. Another possible source may be variation in the biuret reagent mixture and assay conditions. Refractometers also can be used to calculate serum water concentration. A table that converts index of refraction to serum water concentration can be used to convert electrolyte concentration from mmol/L of serum to mmol/L of serum water, a more accurate indicator of effective electrolyte concentration. Refractometers are especially useful for determining urine specific gravity on veterinary samples because they require relatively small sample volumes. Specific gravity continues to be the most common unit for reporting total solids concentration. Some solutes, such as acetone, may cause false increases in specific gravity by refractometry, as they increase refraction but are less dense than water.
Journal Article↗
Protein measurement of particulate and solubilized ovine liver membranes.
Protein concentration measurements are critical in biochemical work with cellular membranes, including the determination of cell surface receptor concentration in human malignant tissues obtained at surgery or after biopsy. In this study we compared the results of protein concentration measurements in ovine liver cellular membranes using either particulate preparations or membranes solubilized with four different detergents. In all cases protein was determined by two different indirect methods (Lowry's Folin phenol method and Bradford's Coomassie Brilliant Blue dye binding method) and compared to the direct biuret method. Our results indicate that the direct biuret method gives the highest protein concentrations followed by the method of Lowry. Maximal concentrations (approaching those obtained by the direct biuret method) were obtained after membrane solubilization with Triton X-100 (3-5%). It is suggested that either the direct biuret method (whenever protein concentrations permit it) or the method of Lowry after solubilization of membranes with Triton X-100 (3-5%) should be used preferentially for the determination of membrane protein samples.
Animals↗
Effect of aggregates on albumin standardization.
In order to investigate the consequences of presence of aggregates in human albumin standards, pools of monomer, dimer and polymer albumin were prepared and quantitated by three total-protein methods (biuret, Folin-Lowry and spectrophotometry at 279 nm) and by four different albumin methods (dye-binding by bromcresol green, electroimmunoassay, radial immunodiffusion and automated immunoprecipitation). Biuret was chosen as the reference method and the monomer was used as the standard in all methods. Both the total-protein and albumin methods gave values for aggregated albumin different from the biuret values. The maximal bias occurred in radial immunodiffusion where quantitation of the dimer and polymer pools gave only 67% and 48% of the biuret values, respectively. In five commercial albumin preparations investigated, the content of di- and polymer varied from 3 to 34%. Uncritical use of albumin preparations in standardization may thus introduce bias in the measurements.
Albumins↗
[Determination of proteinuria in children: changes in results according to the methods used (author's transl)].
Five methods for determination of proteinuria are compared : gravimetry, colorimetry (bromocresol green, Biuret test, Lowry's test), opacimetry (sulfosalicylic acid). Determinations were performed on normal urine, overloaded with albumin or gammaglobulins or on abnormal urine, after varying several parameters (pH, osmolarity, glucose, fructose, red blood cells, hemoglobin. leukocytes, germs). Some methods give non linear results depending on the concentrations (gravimetry, sulfosalicylic acid, bromocresol green). Some systematically give over-results (Biuret test, Lowry's test). Some almost exclusively determine albumin levels (bromocresol green, sulfosalicylic acid). In some, results are altered by glucose or fructose (Biuret, Lowry) or by red blood cells (Biuret, Lowry, sulfosalicylic acid). None of the tested methods can be used as a reference method. The method using sulfosalicylic acid is suitable for routine determination; however, results should be interpreted according to the parameters mentioned above.
Albuminuria↗
A comparison of methods for determining total body protein.
The aim of the study was to find the optimal method (with respect to convenience and accuracy) for determining total protein in whole-body homogenates of rats. Three different protein extraction methods and five different protein concentration methods were assessed. The results were compared against a reference value measured by complete amino acid analysis after acid hydrolysis. The data demonstrated that extraction with 5% (w/v) sodium dodecylsulfate (SDS) in 0.5 N NaOH was far superior to that with water alone or to 6 N guanidine-HCl. A comparison of the Biuret, Bradford, and bicinchoninic acid methods on the SDS-NaOH-extracted samples showed that the Biuret method was optimal, giving a value that was 90% of the reference value with a small variation (2.4% of the mean). The Kjeldahl method gave the correct protein concentration only when a nitrogen factor of 5.51 +/- 0.03 (N = 5) was applied. The results suggest that extraction with SDS-NaOH followed by the Biuret procedure is a good method for measuring protein concentrations in whole body rat homogenates.
Amino Acids↗
Ring cleavage and degradative pathway of cyanuric acid in bacteria.
The degradative pathway of cyanuric acid [1,3,5-triazine-2,4,6(1H,3H,5H)-trione] was examined in Pseudomonas sp. strain D. The bacterium grew with cyanuric acid, biuret, urea or NH4+ as sole source of nitrogen, and each substrate was entirely metabolized concomitantly with growth. Enzymes from strain D were separated by chromatography on DEAE-cellulose and three reactions were examined. Cyanuric acid (1 mol) was converted stoichiometrically into 1.0 mol of CO2 and 1.1 mol of biuret, which was conclusively identified. Biuret (1 mol) was converted stoichiometrically into 1.1 mol of NH4+, about 1 mol of CO2 and 1.0 mol of urea, which was conclusively identified. Urea (1 mol) was converted into 1.9 mol of NH4+ and 1.0 mol of CO2. The reactions proceeded under aerobic or anoxic conditions and were presumed to be hydrolytic. Data indicate that the same pathway occurred in another pseudomonad and a strain of Klebsiella pneumoniae.
Ammonia↗
[Quantitative determination of total urinary protein utilizing the principle of Coomassie Brilliant Blue G250 binding to protein (author's transl)].
Coomassie Brilliant Blue G250 was used for the determination of total urinary protein and compared with the biuret-procedure. In the Coomassie Brilliant Blue G250-assay 0.10 ml urine are added to 5.0 ml Coomassie Brilliant Blue G250 reagent and the sample is read against the reagent blank. The Coomassie Brilliant Blue G250 method seems to be superior to the biuret-procedure because of better reproducibility, higher sensitivity and simpler handling. The upper limit or urinary protein excretion in 49 healthy subjects was 120 mg/24 h. To evaluate the clinical significance of the Coomassie Brilliant Blue G250 method the urinary protein concentration of 134 patients with metabolic, systemic and organ diseases was compared with the biuret-procedure. The regression line was y = 0.827 X + 8.713 mg/l with a correlation coefficient of r = 0.966. The type of proteinuria (mixed, glomerular, tubular) had no influence on the protein value measured with the Coomassie Brilliant Blue G250 method. However in selective proteinuria, like Bence-Jones protein excretion, there is no correlation between the two methods, because the concentration of Bence-Jones protein is underestimated. Lower protein values were also frequently obtained for patients with diabetes mellitus. Some specimens from patients with chronic renal failure treated by haemodialysis showed elevated values. In spite of these limitations the Coomassie Brilliant Blue G250 method might become the method of choice for the determination of total urinary protein.
Bence Jones Protein↗
Photometric determination of total protein in lipemic sera.
We compared various procedures and biuret reagents suggested for determination of protein in lipemic sera. A modified procedure, consisting of the precipitation of serum protein with acetone and dissolution of the precipitate in a biuret reagent, has been developed. The sample (0.02 ml) is mixed with 0.1 ml of distilled water and 2 ml of acetone. The mixture is shaken, centrifuged, and the precipitate dissolved in 1.5 ml of ethylenediaminete-traacetate-chelated biuret reagent. This method is suitable for both clear and lipemic sera. Values obtained with the procedure proposed are in good agreement with values by the Kjeldahl method.
Biuret↗
Effects of sodium dodecylsulphate, dye concentration and paraprotein on coomassie blue dye-binding assays for protein in urine.
Various Coomassie Blue reagents, containing either increased dye concentration or added sodium dodecylsulphate, were compared with a biuret method for the assay of total protein in urine. When immunoglobulin free light chain protein or immunoglobulin paraprotein were present, results from the Coomassie Blue methods were up to 50% lower than with the biuret method; increased dye concentration did not improve comparability substantially, but the addition of sodium dodecylsulphate reduced the bias to about 20%. When neither free light chain protein nor immunoglobulin paraprotein was present, results from the Coomassie Blue methods were only about 30% lower. The addition of sodium dodecylsulphate reduced this bias to 10%. Correlations between the biuret and the Coomassie Blue method were best when the Coomassie Blue reagent contained 40 mg/L sodium dodecylsulphate (r better than 0.98 in all groups; p less than 0.001).
Binding, Competitive↗
Protein, cholesterol, acid phosphatase and aspartate aminotransaminase in the seminal plasma of turkeys (Meleagris gallopavo) producing normal white or abnormal yellow semen.
Turkeys which produce yellow semen have abnormal ductuli efferentes' epithelial morphology, with blebbing of cytoplasmic material into the ductal lumen. This could possibly increase the activity or concentration of seminal plasma components. In the present study, seminal plasma from 270 Large White breeder turkeys was evaluated for protein and cholesterol concentrations and the activities of acid phosphatase and asparate aminotransaminase. In a separate experiment, protein concentrations of turkey seminal plasma were estimated by biuret or Bradford methods. Bradford estimates were 46.6% less than those obtained with the biuret assay, using bovine serum albumin as the standard. Estimates of seminal plasma protein concentration in the main study were obtained using the Bradford method, and should be adjusted accordingly when compared with other studies using the biuret technique. Abnormal yellow seminal plasma, compared to normal white seminal plasma, had elevated levels of total protein and cholesterol and increased activities of acid phosphatase and aspartate aminotransaminase. Overall means were: 14.3 mg/ml, 38.9 mg/dl, 232.6 IU/ml, 81.0 IU/ml, respectively. Correlation coefficients for cholesterol concentration, acid phosphatase and aminotransaminase activity with protein concentration were +0.65, 0.70 and 0.50 (P less than 0.0001), respectively. Specific activities of both enzymes showed a significant reduction as seminal plasma protein increased, indicating a disproportionate increase in proteins other than these enzymes in yellow seminal plasma.
Acid Phosphatase↗
Gene sequence and properties of an s-triazine ring-cleavage enzyme from Pseudomonas sp. strain NRRLB-12227.
Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of the s-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO(2) and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of Escherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent K(m) of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4, 6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.
Amidohydrolases↗
Comparison of a commercial method for total protein with a candidate reference method.
The biuret method for total protein has been compared on the Boehringer Mannheim Diagnostics (BMD) Hitachi 717 Analyzer with a candidate reference method in efforts to standardize the BMD method. The methods were compared using 115 paired serum specimens collected during morning rounds at the Roger Williams Hospital. Results from the test method were compared to the reference method using a statistical procedure for quantifying bias between analytical methods. Linear regression statistics were also calculated. The Hitachi 717 biuret method shows no bias when compared to the reference method (z = -0.90), and there is acceptable correlation between the two methods (y = 0.86, m = 0.86, r = 0.896). The Hitachi method is linear to 15.0 g per dL and demonstrates excellent precision (CV less than or equal to 2.14 percent, N = 140). The Hitachi 717 biuret method has been found to be excellent in all respects and its use is recommended as a convenient and accurate means of measuring total protein.
Blood Chemical Analysis↗
Evaluation of three methods of protein analysis for serum and heart homogenates.
The Biuret, Ultraviolet and Lowry techniques for the determination of total protein content were compared in 31 samples of patient serum and in 30 rat heart tissue homogenates. Statistically significant differences were found to exist between values obtained with each method. Human serum and heart homogenate protein were both lowest when assayed by the Lowry procedure, while serum and homogenate values were highest with the Ultraviolet and Biuret method, respectively. Possible sources of these discrepancies are discussed. Although the Biuret method may not be as sensitive as the other two methods, it proved to be the most reproducible technique for determining total protein concentration in the particular types of samples used in this study.
Animals↗