The need to determine protein concentration in solution is a routine requirement during protein purification. The only truly accurate method for determining protein concentration involves acidic hydrolysis of a portion of the sample and subsequent amino-acid analysis of the hydrolysate. However, this is very time-consuming, particularly if multiple samples are to be analysed. Fortunately, there are quicker methods that give a reasonably accurate assessment of protein concentrations.

Most of these (Table 1) are colorimetric methods, where a portion of the protein solution is reacted with a reagent that produces a coloured product. This coloured product is then measured spectrophotometrically and the observed absorbance related to the amount of protein present by appropriate calibration. However, these methods are not absolute, since the development of colour is often at least partly dependent on the amino-acid composition of the protein(s). The presence of prosthetic groups (e.g. carbohydrate) also influences colorimetric assays. A standard calibration curve using bovine serum albumin (BSA), is commonly used due to its low cost, high purity and ready availability. However, since the amino-acid composition of BSA will differ from the composition of the sample being tested, any concentration values deduced from the calibration graph can only be approximate.

Table1. Summary of protein concentration assays

Ultraviolet (UV) Absorption

The aromatic amino-acid residues tyrosine and tryptophan in a protein exhibit an absorption maximum at a wavelength of 280 nm. Since the proportions of these aromatic amino acids in proteins vary, the extinction coefficients are characteristic parameters of individual proteins that can be estimated based on their sequence.

The direct determination of protein concentration is relatively sensitive, as it is possible to measure protein concentrations as low as 10 μg cm⁻³ . Unlike colorimetric assays, this method is non-destructive, i.e. having made the measurement, the sample in the cuvette can be recovered and used further. This is particularly useful when one is working with small amounts of protein and cannot afford to waste any. However, the method is subject to interference by the presence of other compounds that absorb at 280 nm. Nucleic acids fall into this category, having an absorbance as much as 10 times that of proteins at this wavelength. Hence the presence of only a small per centage of nucleic acid (absorption maximum at ~260 nm) can greatly influence the observed absorbance of aromatic amino-acid side chains. However, if the absorbances at both wavelengths 260  and 280 nm are measured, it is possible to apply a correction factor.

A further great advantage of the UV absorption method is its usefulness for continuous non-destructive monitoring of protein contents, for example in chromatographic column effluents.

All contemporary column chromatography systems have in-line UV spectrometer units (typically using multiple fixed wavelengths, for example 230, 260 and 280 nm) that monitor protein elution from columns.

Lowry (Folin–Ciocalteau) Assay

Historically, the Lowry assay has been the most commonly used method for deter mining protein concentration, but has nowadays been replaced by the more sensitive methods described below. The Lowry method is reasonably sensitive, detecting down to 10 μg cm −3 of protein, and the sensitivity is moderately constant from one pro tein to another. When the Folin–Ciocalteau reagent (a mixture of sodium tungstate, molybdate and phosphate), together with a copper sulfate solution, is mixed with a protein solution, a blue-purple colour is produced that can be quantified by its absorbance at 660 nm. As with most colorimetric assays, care must be taken such that other compounds that interfere with the assay are not present. For the Lowry method, this includes TRIS and zwitterionic buffers such as PIPES and HEPES, as well as EDTA. The method is based on both the Biuret and the Folin–Ciocalteau reaction. In the Biuret reaction, the peptide bonds of proteins react with Cu 2+ under alkaline conditions to produce Cu + . The Folin–Ciocalteau reaction is poorly understood, but essentially involves the reduction of phosphomolybdotungstate to hetero-polymolybdenum blue by the copper-catalysed oxidation of aromatic amino acids. The resultant strong blue colour is therefore partly dependent on the tyrosine and tryptophan content of the protein sample.

The Bicinchoninic Acid Method

This method is similar to the Lowry method in that it also depends on the conversion of Cu 2+ to Cu + under alkaline conditions. Complexation of Cu + by bicinchoninic acid (BCA) results in an intense purple colour with an absorbance maximum at 562 nm. The method is more sensitive than the Lowry method, being able to detect down to 0.5 μg protein cm⁻³ . Perhaps more importantly it is generally more tolerant of the presence of compounds that interfere with the Lowry assay; hence it enjoys higher popularity than the original Lowry method.

The Bradford Method

This method relies on the binding of the dye Coomassie Brilliant Blue to protein. At low pH, the free dye has absorption maxima at 470 and 650 nm; however, when bound to protein, the absorption maximum is observed at 595 nm. The practical advantages of the method are that the reagent is simple to prepare and that the colour develops rapidly and is stable. Although it is sensitive down to 20 μg protein cm −3 , it is only a relative method, as the amount of dye binding appears to vary with the con tent of the basic amino acids arginine and lysine in the protein. This makes the choice of a standard rather difficult. In addition, many proteins will not dissolve properly in the acidic reaction medium.

Kjeldahl Analysis

 This is a general chemical method for determining the nitrogen content of any compound. It is not normally used for the analysis of purified proteins or for monitoring column fractions, but is frequently used for analysing complex solid samples and microbiological samples for protein content. The sample is digested by boiling with concentrated sulfuric acid in the presence of sodium sulfate (to raise the boil ing point) and a copper and/or selenium catalyst. The digestion converts all the organic nitrogen to ammonia, which is trapped as ammonium sulfate. Completion of the digestion stage is generally recognised by the formation of a clear solution. The ammonia is released by the addition of excess sodium hydroxide and removed by steam distillation in a Markham still. It is collected in boric acid and titrated with standard hydrochloric acid using methyl-red–methylene-blue as indicator. It is possible to carry out the analysis automatically in an autokjeldahl apparatus. Alternatively, a selective ammonium ion electrode may be used to directly deter mine the content of ammonium ion in the digest. Although Kjeldahl analysis is a precise and reproducible method for the determination of nitrogen, the determination of the protein content of the original sample is complicated by the variation in the nitrogen content of individual proteins and by the presence of nitrogen in contaminants such as DNA. In practice, the nitrogen content of proteins is generally assumed to be 16% by weight. This method has the major limitation that it can be fooled by non-protein sources of nitrogen; this was revealed during the 2008 Chinese milk scandal. Use of the Kjeldahl method by regulatory agencies allowed unscrupulous manufacturers to add melamine to infant milk formula, artificially increasing the apparent protein content.

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