Determining the exact quantity of proteins in a solution is very often necessary in the biochemical practice. There are many ways to measure protein concentration. In chromogenic methods, the absorbance of a coloured product formed by the protein and an organic molecule is measured. Protein concentration can also be determined from the protein’s own (intrinsic) UV absorbance. Note, however, that these methods may give different results for different proteins of the same concentration. Also, different methods can yield somewhat different results for the same protein. There is no absolute photometric protein concentration assay. All methods have advantages and disadvantages and we must choose among them by taking the following aspects into consideration: specificity, sensitivity, the measurable range of concentration, the accuracy, the nature of the protein to be examined, the presence of materials interfering with the measurement, and the time required for the measurement.
Molecules with two or more peptide bonds react with Cu2+ ions in alkaline solution and form a purple complex. Nitrogen atoms of the peptide bonds form a coordination bond with the metal ion. The quantity of the complexes formed is proportional to the number of peptide bonds.
In practice, the determination of protein concentration is done using a calibration curve created using samples of known concentration. The protein treated with biuret reagent is measured at 540 nm after the purple product is formed.
The advantages of the method include that only few materials (e.g. Tris and amino acid buffers) interfere with it, it can be done in a short time and does not depend on the amino acid composition of the protein. Its disadvantages are its low sensitivity and that it requires at least 1 mg of protein.
In this sensitive technique, a coloured product is formed similarly to the biuret reaction, but a second reagent (Folin-Ciocalteu reagent) is used in addition to strengthen the colour. The strong blue colour is created by two reactions: (1) formation of the coordination bond between peptide bond nitrogens and a copper ion and (2) reduction of the Folin-Ciocalteu reagent by tyrosine (phosphomolybdic and phosphotungstic acid of the reagent react with phenol). The measurement is carried out at 750 nm.
As in the biuret reaction, a calibration curve is created (for example using BSA, bovine serum albumin), and the concentration of the unknown protein is determined from the curve.
The advantages of the method include that it is quite sensitive and is able to detect even 1 µg of protein. Its disadvantages are that it takes rather long to carry out, is disturbed by various materials (including ammonium sulphate, glycine and mercaptans) and that the incubation time is critical. As different proteins contain different amounts of tyrosine, the amount of the coloured product will also be different. As a consequence, this method is more suited to compare the concentration of solutions of the same protein than to absolute measurement.
Despite being relatively new, probably this is the most widely used protein assay. The method is based on the ability of the Coomassie Brilliant Blue dye to bind to proteins in acidic solution (via electrostatic and van der Waals bonds), resulting in a shift of the absorption maximum of the dye from 465 to 595 nm.
The advantages of the method include that it is highly sensitive, is able to measure 1-20 µg of protein and is very fast. Only relatively few materials interfere with it (it works even in presence of urea or guanidine hydrochloride) but, importantly, detergents do. Even traces of detergent (e.g. cleaning products) can invalidate the results. Its disadvantages are that it depends strongly on amino acid composition and that it stains the cuvettes used.
This method is based on the fact that two of the aromatic amino acids, tryptophan and tyrosine, show a peak in absorbance around 280 nm. It has the advantage of being quick and easy. Since it needs no chemical reaction to be performed, it is widely used for detection of proteins or peptides during their separation by chromatography. As proteins contain different ratios of aromatic amino acids, per se it is more suited to the comparison of solutions of the same protein and less to absolute measurement. The latter requires the knowledge of the molar extinction coefficients of proteins. For many proteins, these were determined and can be found in the literature. Moreover, if we know the number of tyrosine and tryptophan amino acids in the protein of interest, since their absorption values are additive, it is possible to calculate the molar extinction coefficient.
This method has a moderate sensitivity with a material requirement of around 50 µg. It is disturbed by anything that has an absorbance at 280nm—most commonly, DNA. (Nucleic acids have an absorption maximum at 260 nm and their absorption at 280 nm is still considerable.) By using the correction introduced by Warburg and Christian, we can account for the error caused by nucleic acids. Absorption is measured at 260 and 280 nm and protein concentration can be calculated with the following equation:
cprot(mg/mL) = 1.55 * A280nm ─ 0,76*A260nm
Proteins and peptides also show high absorbance between 220 and 240 nm, originating from peptide bonds and carboxyl groups. This wavelength range can be used for quantitative assessment only if the solution is pure, because a large number of other substances also have high absorbance in this range.