Proteins, analysis color reactions

A variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

Monitoring of solubility and dissolution Gravimetric analysis, a protein assay method (involving color reaction with bicinchoninic acid) and UV absorbance measurements at 214 nm Chemical analysis using TNBS reagent Fluorescence spectrophotometry... 

Ammonia, some amines, and some proteins and peptides will also yield a colored product but will not generate CO2. Thus, the colorimetric analysis is not specific for amino acids unless CO2 release is measured or the amino acids are purified and freed from interfering materials (the usual procedure). The color reaction with ninhydrin is used extensively in manual and automated procedures. 

R27. Rosenthal, H. L., and Kawakami, T., Effect of high salt concentrations on color production of the biuret reaction for protein analysis. Am. J. Clin. Pathol. 26, 1169-1173 (1956). 

Immunoblot Analysis of Cross-Reactivity of Monoclonal Anti-rat Hepatoma Thymidylate Synthase Antibodies After PAGE/SDS (12.5% polyacrylamide, 100 pg of crude extract protein or 1 pg of purified endogenous/recombinant thymidylate synthase protein per lane), proteins were transferred to PVDF membrane. Thymidylate synthase was detected by treatment with a mixture of monoclonal antibodies (produced by clones 27-1, 27-7and 111-40 and mixed at 1 1 1 ratio) and goat anti-mouse IgG (H h- L) - HRP conjugate (BioRad) as a secondary antibody. Color reactions were developed using goat anti-mouse Amplified Opti-4CN Detection Kit (Bio-Rad). 

Ninhydrin-Color Reaction. This reaction is commonly used for qualitative analysis of a-amino acids, peptides, and proteins. 

Both universal staining procedures and specific detection techniques can be performed after (electro) transfer or (electro)blotting of proteins (also called Western blotting) from the gel matrix (which sometimes hinders protein analysis) to a nitrocellulose or polyvinylidenedifluoride membrane, to which they are bound and immobilized. On the membrane, protein molecules are faster and better accessible for the interactions with the applied specific antibodies. The antigen-antibody complexes are visualized by a second antibody (against the first antibody) with an attached enzyme label, which catalyzes the color reaction in the place of the protein zone. 

Figure 15.4. Principle of HDX-MS analysis The protein complex is diluted in deuterium oxide to start the exchange reaction. After the incubation time at room temperature, the exchange reaction is quenched by lowering both pH and temperature. To study the topology of deuterium incorporation, the protein complex is digested with pepsin under quenched conditions. The peptides generated can be analyzed using ESI or MALDI ionization. During the mass spectrometric analysis the number of deuterium atoms incorporated is determined to calculate the exchange rate along the sequence of the protein (See color insert).

Because the amount of time required for a given amino acid to elute from a standard column is reproducible, the identities of the amino acids in a peptide can be determined. The amount of each amino acid in the sample is determined by measuring the intensity of the purple color resulting from its reaction with ninhydrin. Figure 26.3 shows the results of amino acid analysis of a standard equimolar mixture of 17 a-amino acids. Typically, amino acid analysis requires about 100 picomoles (2-3 /xg) of sample for a protein containing about 200 residues. 

The enzyme attached to antibody 2 is critical for quantitative analysis. Figure 19-14 shows two ways in which the enzyme can be used. The enzyme can transform a colorless reactant into a colored product. Because one enzyme molecule catalyzes the same reaction many times, many molecules of colored product are created for each analyte molecule. The enzyme thereby amplifies the signal in the chemical analysis. The higher the concentration of analyte in the original unknown, the more enzyme is bound and the greater the extent of the enzyme-catalyzed reaction. Alternatively, the enzyme can convert a nonfluorescent reactant into a fluorescent product. Colorimetric and fluorometric enzyme-linked immunosorbent assays are sensitive to less than a nanogram of analyte. Pregnancy tests are based on the immunoassay of a placental protein in urine. 

Such a procedure adapted from Gross and Morell (1966) and Blumenfeld and Gallop (1962) is as follows. Visser et al. (1971) treated 2.5 to 10 mg of a modified elastase with 1 N NHjOH-HCl, adjusted to pH 9 by addition of sodium hydroxide, for 2 hr at 25°C. The excess hydroxylamine was removed either by dialysis or by precipitation of the protein at pH 3.0. The protein was then dissolved, brought to pH 8.0 by addition of NaOH and treated with an equal volume of a 1 % solution of l-fluoro-2,4-dinitrobenzene in ethanol. The pH of the solution was maintained at 8 by the continuous addition of NaOH. The reaction is complete when no additional alkali must be added for 5 min. The mixture is then extracted three times with ether and the aqueous phase subjected to the conditions of the Lossen rearrangement (i.e. heating to 100°C under alkaline condition (0.1 N NaOH) for 10 min). Acid hydrolysis followed by amino acid analysis permits the identification of either diaminopropionic or diaminobutyric acid which would result from either aspartate or glutamate modification, respectively. Diaminopropionic and diaminobutyric acids may be estimated on the short column of the amino acid analyzer. Diaminopropionic acid emerges with histidine. (Color values do not seem to be available.)... 

---