The Biuret method

This assay is not affected by the variation of the amino acid content of proteins as it is based on the complex of peptide bonds with in alkaline solution and is robust, albeit the least sensitive method, with a useful range of 1 to 5 mg. 

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The same is true for specimens of serum from the newborn. Those who attempt to apply the biuret method to newborn sera, end up with values in the normal infant, resembling those for... 

Some drugs act as Indicators (e.g. phenolphthaleln, vegetable laxatives) and affect tests carried out at a particular pH. The presence of sulfobromophthaleln dye (BSP) In serum will Interfere with serum protein determined by the biuret method. 

The method is more sensitive than the biuret method and has an analytical range from 10 ju,g to 1.0 mg of protein. Using the method outlined below this is equivalent to sample concentrations of between 20 mg l-1 and 2.0 g l-1. The relationship between absorbance and protein concentration deviates from a straight line and a calibration curve is necessary. The method is also subject to interference from simple ions, such as potassium and magnesium, as well as by various organic compounds, such as Tris buffer and EDTA (ethylenediamine-tetraacetic acid). Phenolic compounds present in the sample will also react and this may be of particular significance in the analysis of plant extracts. 

Protein Determination. Protein was determined by the biuret method (10) and expressed as % of dry weight. The standard was bovine serum albumin (BDH). 

There are numerous substances that are administered intravenously and have a direct effect on biochemical analysis. Obviously, glucose or electrolyte concentrations will be spuriously elevated if the specimen is taken from the same vein into which these substances are being administered. The presence of sulfobromophthalein dye (BSP) in serum or plasma will interfere with protein determined by the biuret method. The... 

Students will isolate intact mitochondria from beef heart and fractionate them to prepare submitochondrial particles. Each fraction will be characterized by protein estimation by the biuret method and measurement of malate dehydrogenase and monoamine oxidase activity. 

This unit describes four of the most commonly used total protein assay methods. Three of the four are copper-based assays to quantitate total protein the Lowry method (see Basic Protocol 1 and Alternate Protocols 1 and 2), the bicinchoninic acid assay (BCA see Basic Protocol 2 and Alternate Protocols 3 and 4), and the biuret method (see Basic Protocol 3 and Alternate Protocol 5). The fourth is the Coomassie dye binding or Bradford assay (see Basic Protocol 4 and Alternate Protocols 6 and 7), which is included as a simple and sensitive assay, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acidic pH. A protein assay method should be chosen based on the sensitivity and accuracy of method as well as the condition of the sample to be analyzed. 

Mycelium was washed twice with 0.9% (w / v) NaCl followed by three extraction steps. In each extraction step the mycelium was boiled for 10 min with 1M NaOH and the supernatant was collected after centrifugation (16). The amount of protein in the pooled supernatant was measured based on the biuret method (17). 

The Biuret method depends on the reaction of compounds containing several peptide bonds with Cu2+ under alkaline conditions to form a violet-coloured species. 

Berg et al. (B5a) correlated the results of gastric biopsies with chemical and electrophoretic analysis of gastric juice. They determined total proteins by the biuret method and total hexoses colorimetrically by the orcinol technique, and performed horizontal paper electrophoresis followed by amido black and PAS staining. Their work corroborated previous findings (G19) that carbohydrate materials staining with PAS were localized in the center of the electrophoretic partition of gastric juice with some extension to adjacent areas. They also observed that... 

Several s pec tropho tome trie methods are available for the determination of protein in solution. The biuret method is based on the reaction of Cu with peptides in alkaline solution to yield a purple complex that has an absorption... 

The protein concentrations were determined either by the biuret method, using serum albumin as standard (Gornall et al., 1949), or with the Folin reaction (Lowry et al., 1951). 

The determination of total serum protein is an important clinical measurement used in diagnosing liver malfunctions. Although the Kjeldahl method is capable of high precision and accuracy, it is too slow and cumbersome to be used routinely in determining total serum protein. The Kjeldahl procedure, however, has historically been the reference method against which other methods are compared. Methods commonly used include the Biuret method and the Lowry... 

F5. Fine, J., The biuret method of estimating albumin and globulin in serum and urine. Biochem. J. 89, 799-803 (1935). 

Albumin and globulins can be analyzed by the same procedure as for total proteins after fractionation by salting out with either sodium sulfate or sodium sulfite. More detailed information may be required about the protein fractions (a, j8, 7 globulins), in which case starch gel electrophoresis can be used to separate the proteins. Micro-Kjeldahl analysis of proteins is used when highly accurate data are required the biuret method is accurate to about 4%. 

Lowry method. The Lowry method is the most generally accepted owing to its sensitivity, simplicity, and reproducibility it is 20 times more detectable than the UV absorption method and 100 times more sensitive than the biuret method. Since the color development in the Lowry method depends on the content of aromatic amino acid residues, the variation of response with different proteins is surely greater than in the biuret method but generally less than in UV adsorption measurement. 

The biuret method The bimet method is a colorimetric technique specific for proteins and peptides. Copper salts in alkaline solution form a pruple complex with substances containing two or more peptide bonds. The absorbance produced is proportional to the number of peptide bonds that are reacting and therefore to the number of protein molecules present in the reaction system. Thus, the biuret reaction with proteins is suitable for the determination of total protein by spectrophotometry (at 540-560 nm). The method is used extensively in clinical laboratories, particularly in automated analyzers in which protein concentration can be measured down to 0.1-0.15gl. The use of bovine or human serum albumin to standardize the biuret method is well established. ITigh-purity albumin contains only amino acids its nitrogen content is a constant fraction... 

Other methods Although other methods have been used for total serum protein quantification, none is more convenient or practical than the biuret method. These methods are reviewed here predominantly as background information for the assay of total protein in urine or CSF. They are dependent on four general properties of proteins. 

The biuret method is based on the fact that proteins (and, as a rule, all substances containing two or more peptidic bonds) react with copper to form a colored complex whose absorption (2max = 454 nm), in the presence of excess copper, is proportional to the amount of protein present. The reagent is obtained by dissolving 1-5 g of copper(II) sulfate and 6g of sodium potassium tartrate tetrahydrate in 3% sodium hydroxide. Bovine serum albumin is used as standard. The most serious drawback of this method is its poor sensitivity. 

Methods of analysis. The total protein concentration of crude solutions, including cellular proteins, was determin by the biuret method (72). With dilute xanthan solutions, this method was not sensitive enough, and a spectrofluorometric assay was carried out. This method consisted of an acid hydrolysis of the samples followed by derivatization with o-phthaldialdehyde and HPLC analysis on a Cl8 reverse column of the amino acids released, using spectrofiuorescence as the detection nKxie. 

Aqueous solution (0.6 wt%) of a protein (ovalbumin, gelatin) and 0.2 g of PMS (Cp s = 10 wt%) or 0.02 g of nanosilica A-300 was mixed for 1 h and then centrifuged. The equilibrium concentration of protein in the liquid was determined by the Biuret method. A distribution function /(-AG) of Gibbs free energy (AG) of the protein adsorption was calculated using the kernel of the integral equation in the form of the Langmuir equation. 

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