Biuret Procedure Research Articles

Albumin-based or albumin-linked calibrators cause a positive bias in serum proteins assayed by the biuret method

Assay of total serum protein by the biuret method calibrated with albumin standards according to the reference method provides results with a positive bias approximately 3%-5% exceeding the total error of 3.4% allowable for total protein in serum analysis made by analysers using two-part reagents and short-term procedures. We used two types of two-part biuret reagents utilised in a short-term measurement in analysers with albumin or serum calibrators, in which protein was attested by the Kjeldahl method. Tests with potentially interfering substances proved that serum blanking used in a short-term biuret procedure is not capable of sufficiently eliminating effects of serum interferents. A short-term blanking is evidently capable of suppressing only an absorbance caused by serum-present coloured and turbid interferents, but its capacity to transform them (oxidise, hydrolyse, saponify, etc.) to some other not-interfering substances is very low compared with a long-term blanking. Lipids and bilirubin are responsible for significant positive bias of total protein in normal serum samples (approximately 3%) and even a greater positive offset in lipaemic and icteric sera (approximately 5%). We verified that interference tests based on a normal serum spiked with endogenous lipids and bilirubin give quite false and misleading results in the biuret reaction. A pure albumin, not depending on its bovine/human origin, gives absorbance responding only to its copper complexes with protein with a biuret regent, while its absorbance with a serum also includes the absorbance of interferents present in serum. The simplest way to improve current short-term biuret procedures is the use of a human serum calibrator with total protein attested by the Kjeldahl method. A serum calibrator, behaving analogously to serum samples, compensates for a positive bias in most normal sera. Reagents with a greater concentration of active biuret components (copper and alkali, reference method included) seem to be unnecessarily aggressive to proteins and are responsible for a lower accuracy when used in short-term measurements. Standard Reference Material 927c based on pure bovine albumin is still recommended and used as the primary standard for assays of total protein by colourimetric methods. The albumin calibrator is responsible for a positive bias of approximately 3%-5% in serum total protein assayed by the biuret reaction both in the reference and in current methods. Its substitution by a serum calibrator attested by the Kjeldahl method could solve this drawback. Clin Chem Lab Med 2009;47:91-101.

Novel UV assay for protein determination and the characterization of copper–protein complexes by mass spectrometry

A very simple, highly selective and sensitive assay of proteins based on the biuret absorption in the ultraviolet region has been developed. The well-known biuret assay is based on the reaction of proteins with copper ions under strong alkaline conditions to form a copper–protein complex. Yet, copper ions may seriously interfere with the determination if the measurement is made in the UV range. In the present approach, proteins mobilize copper ions from insoluble salts at different pH values, and the copper–protein complexes are investigated by UV spectrophotometry and mass spectrometry. Upon using copper phosphate, free copper ions do not interfere with the determination from 540 to 240 nm. Copper absorbance slowly increases from 240 to 190 nm where a blank with the reagents is recommended. A maximum absorbance for the copper–protein complex was found at 226 nm and high pH value. The stoichiometries of the copper–protein complexes measured directly with a mass spectrometer are pH dependent: half of the peptides without any histidine residue chelate just a single Cu 2+ ion at pH 7.4 but each such peptide mobilizes from 1 to 6 Cu 2+ ions at pH 10.3. To determine proteins, 1–1.5 ml of 1.8% KOH solution with 0–20 μg ml −1 protein is treated with 25 mg of copper phosphate powder. The mixture is powerfully stirred, centrifuged, and the absorbance of the supernatant is measured at 226 nm in 1 cm quartz cuvettes against a blank of the reagents. The color system obeys Beer's law in the range 0.1–20 μg ml −1 protein at this wavelength. The molar absorptivity value proved to be a characteristic of each protein being analyzed. Therefore, individual proteins should be used to plot calibration curves. This assay proved to be over 100 times more sensitive than the classical biuret procedure. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of the method has been verified by the analysis of some biological materials.

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.

External Quality Assessment of Total Urinary Protein Estimation in the United Kingdom

Three surveys of total urinary protein quantitation have been carried out in 350 UK laboratories. The seven specimens comprised buffered saline or normal human urine with added human serum albumin or human serum, or urine from individuals with nephrotic syndrome. Principal method groups were: turbidimetry (57%), dye binding (25%) and biuret (15%). For all surveys, overall between-laboratory agreement was poor (CV 22.8% to 57.1%), with ranges of results from 24-fold (0.83-20 g/L) to 366-fold (0.05-18.3 g/L); there was no improvement with time. The most popular method (sulphosalicylic acid turbidimetry) consistently performed the worst, and performance of the direct biuret procedure was also unacceptable; both methods should be discontinued. There were no significant differences in performance between the other major method groups, and none can be specifically recommended. Within the individual calibrant groups, least variation was observed with human serum. A common calibrant for all participants yielded significantly better between-laboratory agreement for all methods except sulphosalicylic acid turbidimetry.

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.

A Collaborative Study to Develop a Standardized Food Protein Solubility Procedure

ABSTRACTA colaborative study was conducted to develop a rapid, simple and reliable procedure for determining the solubility of food protein products, e.g., spray‐dried whey protein concentrate, sodium caseinate, egg white protein and soy protein isolate. The procedure was developed by modifying the nitrogen solubility index (NSI) procedure. Protein content and soluble protein were determined by micro‐Kjeldahl or biuret procedures with standard deviations of 0.83‐4.12 for all proteins except caseinate which had a value of 13.95. Although the biuret and micro‐Kjeldahl procedures generally provided comparable accuracy and precision for protein content and solubility of certain proteins, the biuret procedure exhibited considerable error and variability for other proteins.

Spinal fluid protein revisited: a reappraisal of the biuret procedure.

In this study our previously described selected method, a biuret procedure with deproteinization, for the determination of spinal fluid protein is thoroughly discussed against the background of the results found with a number of Lowry modifications. The use of various separation techniques, i.e. deproteinization, ultrafiltration and chromatography (HPLC), for protein analysis led to the question as to whether low molecular weight proteins in cerebrospinal fluid play an important role or not with respect to the choice of a selected method for the determination of total protein.

A rapid biuret assay for protein of whole fatty tissues

A rapid biuret procedure is described which avoids the turbidity that occurs with protein analysis of intact fatty tissues. Recovery is complete and absorbancy linear with both concentration of the soluble crystalline serum albumin standard and the volume of homogenate of a variety of tissues. This method has been used successfully for the determination of protein concentrations of homogenates of whole rat heart, liver, kidney, brain, lung, and the following muscles: gastrocnemius, interior and exterior obliques, red and white vastus lateralis, and soleus.

The influence of growing season on the determination of protein in hard red spring wheat by the biuret reaction

AbstractProtein was determined by a biuret procedure in three series of Hard Red Spring wheat samples, two of which represented different growing seasons, the third being drawn from randomised railcar lots. When calibrations were based on samples drawn from single individual seasons, differences in calibration slopes caused significant errors in the prediction of protein in samples drawn from different seasons. The analysis of the randomised lots of wheat using calibrations based on single individual seasons gave errors which were intermediate between those obtained for samples from individual seasons. However, these were closer to the results obtained using the calibration prepared from samples of the season, the wheat from which the randomised lots were mainly composed. The precision of the biuret test for all three series was superior to that of the Kjeldahl test.

Determination of High Molecular Weight Proteins in Beer Using Coomassie Blue

The Coomassie blue method of Bradford is an excellent means of measuring proteins in beer. Kjeldahl nitrogen, the Lowry method, and the biuret procedure have limited applicability to the measuremen...

Determination of High Molecular Weight Proteins in Beer Using Coomassie Blue

The Coomassie blue method of Bradford is an excellent means of measuring proteins in beer. Kjeldahl nitrogen, the Lowry method, and the biuret procedure have limited applicability to the measurement of proteins in beer. Current procedures using the Coomassie blue method relate protein concentration to color developed with standards such as bovine serum albumin and γ-globulin. These standards cannot be directly related to beer proteins. Significant correlation was achieved between Coomassie blue dye-binding and the high molecular weight nitrogen fraction of beers isolated by gel filtration. The correlation was linear over a wide variety of beers and chillproofing methods. The Coomassie blue method for high molecular weight nitrogen might be a better criterion for measuring proteins in beer than the Kjeldahl method presently used.

Sample viscosity can be a source of analytical error when discrete sampler-dilutors are used.

Total protein concentration in the serum of a patient with hyperviscosity syndrome differed as measured by the biuret procedure in the DuPont aca (80 g/L) and the SMA 12/60 (105 g/L), owing to viscosity-dependent errors with the aca sampling system; the magnitude depended on sample temperature and volume of sample aspirated. This kind of error was not observed with the SMA 12/60 and was far less severe when a Micromedic sampler-dilutor was tested. It could be eliminated in the case of the aca by adding sample to test packs with a syringe rather than with the aca automated sampler-dilutor. We thus recommend use of the syringe method when unusually viscous samples (serum or other body fluids) are analyzed in the aca.

A Stopped-Flow, Reaction-Rate Method for Total Serum Protein

Abstract A reaction-rate method for total serum protein is described. The method uses a stopped-flow analyzer to obtain information on the rate of formation of the biuret reaction product. Measurement times can be as low as 100 ms after initiation of the reaction in the stopped-flow system. However, reaction times of 10 s are shown to give higher precision with relative standard deviations in the range of 0.1 to 1.4%. The results for total serum protein determinations are also accurate as shown by comparison with values obtained by equilibrium-based biuret procedures.

Measurement of total protein in urine: Comparison of two dye-binding procedures with a gel filtration/modified biuret method

Two dye-binding methods for the determination of urinary total protein concentration have been compared to a biuret-based procedure requiring a preliminary gel filtration step. The biuret-based procedure is claimed to be both specific and sensitive for the measurement of protein in urine. With those urines having relatively low protein concentrations, the dye-binding methods provide values about one-half of those given by the modified biuret procedure. This difference disappears when urines containing high levels of protein are measured. The lower results obtained at normal protein levels are most likely caused by the inability of dye-binding methods to measure proteins of high carbohydrate content (such as uromucoid) accurately. In spite of this limitation, both dye-binding methods appear suitable for routine use. In contrast to the biuret-based procedure, we found the dye-binding methods to be free from interferences by compounds known to appear in urine in high concentrations.

Relationship of protein concentration and water content of equine serum and plasma samples.

A highly significant correlation between the water content and protein concentration of equine serum and plasma samples was demonstrated over a wide range of concentrations. A close correlation was also observed between protein concentration as estimated by refractometry and as determined by the biuret procedure for equine serum and plasma samples.

Polarographic determination of total serum protein

The total serum protein is accurately determined with high sensitivity by a catalytic polarographic method with trans-dichloro-bis(N,N′-dimethylethylenediamine) Rh(III) chloride as reagent. It is shown that microlitre serum samples can be analyzed, giving correlation with the biuret colorimetric technique which is the recommended standard total protein method of analysis. Analysis of 17 human serum samples with varying albumin/globulin ratios gave a correlation coefficient (r) of 0.9910 relative to the biuret procedure.

Polarographic determination of total serum protein

The total serum protein is accurately determined with high sensitivity by a catalytic polarographic method with trans-dichloro-bis(N,N′-dimethylethylenediamine) Rh(III) chloride as reagent. It is shown that microlitre serum samples can be analyzed, giving correlation with the biuret colorimetric technique which is the recommended standard total protein method of analysis. Analysis of 17 human serum samples with varying albumin/globulin ratios gave a correlation coefficient ( r) of 0.9910 relative to the biuret procedure.

Improved Biuret Procedure for Routine Determination of Urinary Total Proteins in Clinical Proteinuria

This communication describes and evaluates an improved routine methodology for quantitating clinical proteinuria. Based on investigations of Piscator and of Savory et al., a modified Tsuchiya's reagent (ethanolic HCI-phosphotungstic acid) is used to precipitate proteins at 56 degrees C, followed by biuret spectrophotometry at 540 nm. The accuracy of the proposed procedure was assessed by comparisons with results obtained by using an ultrafiltration membrane that retains solutes with an average molecular weight in excess of 10 000 for separating of urinary proteins before they are measured with the biuret reaction. Precision of the method (coefficient of variation) is typically 2-3%.

26] Appendix: An accurate gravimetric determination of the concentration of pure proteins and the specific activity of enzymes

Publisher Summary In studies of subunit structure, equivalent weight and mechanism of action of enzymes, an accurate measure of the weight-concentration of the highly purified protein in solution is required. Concentration is usually measured spectrophotometrically, utilizing chromophores associated with the protein itself or chromophores produced in the Folin–Lowry or biuret procedures. In any spectrophotometric assay of protein concentration, it is necessary to know the relationship between the weight-concentration of the protein and the absorbance measured. The chapter describes an absolute gravimetric method for determining the weight-concentration of a protein solution. This method is useful in standardizing the measurement of concentration of a pure protein by any spectrophotometric method. One of the most important physical characteristics of an enzyme is its specific activity. Because of the convenience of measuring the concentration of protein by means of its A 280 , it is suggested that, where possible, the specific activity of an enzyme be expressed in terms of the empirical A 280 rather than in terms of milligram per milliliter or milligram of protein.

Evaluation and Elimination of the Interference by Starch in the Biuret Determination of Wheat Protein

In the biuret determination of wheat protein, starch, color substances and lipids extractable with an alkaline solution such as biuret reagent were found to interfere with the biuret method. Comparative evaluation of their effects revealed that starch, a major component of wheat, has the most significant effect on the biuret method. In the presence of starch, the correlation between Kjeldahl protein and the optical density of biuret was poor.It was found that several organic solvents effectively retarded solubilization of wheat starch in the biuret reaction mixture. By treatment of the wheat samples with these solvents prior to the biuret procedure, the effect of starch was completely eliminated and the optical density of biuret became closely correlated with Kjeldahl protein.