Dye-binding method

Observations that the presence of protein affects the colour change of some indicators used in acid-base titrations led to the development of methods for the quantitation of proteins based on these altered absorption characteristics of such dyes. As the presence of protein alters the colour produced by these indicators when measuring pH, so in the quantitation of proteins using dye-binding methods the control of pH is vital. 

Since the caseins differ in lysine content (14, 24, 11 and 9 residues for asl-, xs2-, / - and tc-caseins, respectively) they have different dye-binding capacities. This feature may be of some commercial significance in connection with dye-binding methods for protein analysis if the ratio of the caseins in the milks of individual animals varies (as it probably does). It should also be considered when calculating the protein concentration of zones on electrophoretograms stained with these dyes. 

Addition of specific compounds to milk has been used to allow spec-trophotometric measurement of lactose as the osazone (Wahba 1965) and fat by fluorescence (Bakhiren and Butov 1968 Konev and Kozlova 1970). The dye-binding method for measuring protein in milk is based on the ability of sulfonic acid dyes to complex with the basic amino acid residues of milk proteins at low pH (Fraenkel-Conrat and Cooper 1944). Dye binding correlates well with Kjeldahl (Sherbon 1970) and infrared (Mogot et al. 1982 Grappin et al. 1980) results, but variations are caused by the different compositions of the different milk proteins (Ashworth 1966 Vanderzant and Tennison 1961). 

Sherbon, J. W. 1970. Dye binding method for protein content of dairy products. J. Assoc. Offic. Anal. Chem. 53, 862-864. 

For greatest accuracy of the estimates of the total protein concentration in unknown samples, it is essential to include a standard curve in each run. This is particularly true for the protein assay methods that produce nonlinear standard curves (e.g., Lowry method, Coomassie dye-binding method). The decision about the number of standards used to define the standard curve and the number of replicates to be done on each standard depends upon the degree of nonlinearity in the standard curve and the degree of accuracy required of the results. In general, fewer points are needed to construct a standard curve if the color response curve is linear. For assays done in test tubes, duplicates are sufficient however, triplicates are recommended for assays performed in microtiter plates due to the increased error associated with microtiter plates and microtiter plate readers. 

The development of color in the Coomassie dye-binding methods has been associated with the presence of certain basic amino acids (primarily arginine, lysine, and histidine) in the protein. Van der Waals forces and hydrophobic interactions also participate in the binding of the dye by protein. The number of Coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein. In the... 

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. 

The dye-binding method with Acid Orange 12 was very similar in results to succinic anhydride, showing regression equations between ( near to ) SA and DAN. The DB method also underestimated FDNB lysine in the severely heat-damaged samples. 

Table 11. Dye binding methods used for assaying proteins...

EN45 lhara, H., Nakamura, H., Aoki, Y., Aoki, T. and Yoshida, M. (1991). Effects of serum-isolated vs synthetic bilirubin-albumin complexes on dye-binding methods for estimating serum albumin. Clin. Chem. 37, 1269-1272. 

When free in aqueous solution, these dyes exhibit limited fluorescence, but upon binding DNA, their fluorescence increases markedly. Ethidium binds to RNA and DNA while DAPI and Hoechst 33258 selectively interact with DNA. Hoechst 33258 at a final concentration of 1 pg/mL in 0.05 M phosphate, pH 7.4, with 2 M NaCl has been used to quantitate as little as 10-ng double-stranded DNA.18 Because these dye-binding methods involve intercalation between the bases of double-stranded DNA they are not useful for the quantitation of single-stranded DNA. 

Dye-Binding Methods. Dye-binding methods are based on the ability of proteins to bind dyes, such as Coumassie brilliant blue (CBB). The unequal affinities and binding capacities of individual proteins for dyes are a limitation and are complicated further by the inability to define a consistent material for use as a calibrator. The dye-binding method of greatest contemporary interest uses CBB G-250 for assay of total protein in CSF or urine. CBB binds to protonated amine groups of amino acid residues in the polypeptide chain, resulting in decreased absorbance at 465 nm and increased absorbance at 595 nm. 

The low levels of protein in CSF limit the methods that are used to measure total protein in it. Turbidimetric methods and versions of the CBB dye-binding method are commonly used for this purpose. The most serious disadvantage of turbidimetric methods is the requirement for 0,2 to 0.5 mL of sample. Coomassie Brilliant Blue (CBB) methods are sensitive enough for use with samples of as little as 25 XL> but they... 

Washed cell pastes were diluted with fresh buffer using a ratio of 3 ml of buffer for each 1 g of cell paste. A French Cell Press operated at 16,000 lb/in was used to disrupt cells. The crude enzyme preparation was then centrifuged at 10,444 X g for 30 min at 0°C, and the supernatant was retained as a cell-free extract (CFE). This extract was carried through a series of activation and purification steps and the methlonlnase activity was assayed after each step using a modification of the method of Tanaka t al. (52). Protein was assayed by the dye-binding method of Bradford (62), and enzyme solutions were dialyzed in tubing prepared by procedures described by Brewers at al. (63). 

Bromcresol green (BCG) is also firmly boimd to albumin. The difference spectrum of buffered BCG with and without added albumin shows a narrow peak at 615 nm (B3). Under suitable conditions BCG offers the most sensitive means of determining albumin by a dye-binding method. In addition, measurements at the red end of the visible spectrum suffer minimum interference by bilirubin and the blood pigments (B3, E.19). 

Another assay, the Bradford assay, also known as the Coomassie dye binding method, was first described in 1976 [19]. In an acidic environment, proteins will bind to Coomassie dye and cause a shift from the reddish brown color (465 nm) to the blue dye protein complex read at 595 nm. The development of the color is attributed to the presence of the basic amino acids arginine, lysine, and histidine. Van der Waals forces and hydrophobic interactions account for the dye binding and the number of Coomassie blue dye molecules bound is roughly proportional to the number of positive charges on the molecule. A protein molecular weight of about 3 kDA is required for successful color development. The Bradford assay dose response is nonlinear and this method demonstrates the greatest difference in reactivity with BSA compared to BGG. 

Protein-dye binding methods Enzymes are stained with the dye Coomassie Brilliant Blue G-250 to produce color yields that exhibit absorption maxima at 595 nm 2-100 ng ml" ... 

Dye-binding methods use the ability of dyes to bind to charged amino acid residues of the proteins. The proteins may be separated as previously described and then treated with the dye. Dyes used include amido black and orange G. On binding to the protein they form an insoluble complex that may be removed by centrifugation. The amount of residual color in the supernatant is measured by spectrophotometry and calibrated by reference to the Kjeldahl method. 

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