The Biuret and Lowry Assays

The abundance and ease of purification made bovine serum albumin (BSA) an early standard in protein chemistry, and BSA is widely used as protein standard in biuret, Lowry, and Bradford assays as well as a molecular weight... 

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. 

Many methods are available for measuring TCA-soluble peptides. Possibly the easiest is to measure the absorbance of the solution at 280 nm, as the absorbance at this wavelength is a function of the aromatic amino acid content of the solution. This approach requires a UV spectrophotometer, and the sensitivity of the assay is likely to be lower than that of some of the colorimetric assays. There are also several colorimetric peptide assays that can be applied to this type of peptidase assay, such as the Biuret, Lowry, and Bradford dye-binding methods (for comparison see Piyachomkwan and Penner, 1995). All of these methods measure a relative value rather than an absolute amount of peptide in solution. The results should thus be reported in terms of equivalents, such as BSA equivalents when using a calibration curve prepared using a BSA standard solution. 

In the Biuret reaction, a purple colour develops when the protein is treated with alkaline copper sulphate. This reaction is dependent on peptide bonds and not on the side chains of individual amino-acids present. In the Folin-Ciocalteu reaction, the protein is treated with tungstate and molybdate under alkaline conditions and the formation of a complex such phenylalanine and tyrosine gives rise to a blue colour. Lowry developed one of the most widely used protein assays in which a combination of the above reactions is involved07, l8). 

Folin-Ciocalteu (Lowry) Assay. The quantitative Folin-Ciocalteu assay (also often called the Lowry assay ) can be applied to dried material as well as to solutions. In addition, the method is sensitive samples containing as little as 5 /Ug of protein can be analyzed readily. The color formed by the Folin-Ciocalteu reagent is thought to be caused by the reaction of protein with the alkaline copper in the reagent (as in the biuret test) and the reduction... 

Protein was determined by the method of Lowry, using a calibration curve which had been obtained with a TK solution standardized by the Biuret method. TPP was determined by radioactivity measurements. The transketolase assay was carried out in the presence and absence of added thiamine diphosphate/Mg++. A stimulation of 13 % of enzymatic activity was found when the coenzyme was added, indicating a loss of bound coenzyme upon gel filtration. Assuming a linear relationship between enzymatic activity and thiamine diphosphate molecules bound to apotransketolase the value found for thiamine diphosphate was corrected by these 13 %. 

Folin-Ciocalteau or Lowry method While the biuret method is sensitive in the range 0.5 to 2.5 mg protein per assay, the Lowry method is 1 to 2 orders of magnitude more sensitive (5 to 150 pg). The main disadvantage of the Lowry method is the number of interfering substances these include ammonium sulfate, thiol reagents, sucrose, EDTA, Tris, and Triton X-100. 

The final colour in the Lowry method is a result of two reactions. The first is a small contribution from the biuret reaction of protein with copper ions in alkali solution. The second results from peptide-bound copper ions facilitating the reduction of the phos-phomolybdic-tungstic acid (the Folin reagent) which gives rise to a number of reduced species with a characteristic blue colour. The amino acid residues which are involved in the reaction are tryptophan and tyrosine as well as cysteine, cystine and histidine. The amount of colour produced varies slightly with different proteins. In this respect it is a less-reliable assay than the biuret method, but it is more reliable than the absorbance method since A280 may include contribution from other species, and also the absorption of a given residue is dependent on its environment within the protein. 

Zhou, P. and Regenstein, J. M. (2006). Determination of total protein content in gelatin solutions with the Lowry or Biuret assay. /. Food Sci. 71(8), 474- 79. 

Many physicochemical assays are established to quantify the protein mass. It is determined by exploiting the extinction coefficient in optical density measurements or by colorimetric assays such as the Bradford, Lowry, bicinchoninic (BCA), and biuret assay [13, 14]. Albeit easy to perform, these colorimetric assays suffer from inaccuracies that are due to the use of inappropriate standards like bovine serum albumin. If relevant standards are not available, quantitative amino acid analysis [6], the (micro-)Kjeldahl nitrogen method [14, 15] or gravimetry as very accurate but time-consuming alternatives can be applied. 

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