Chemistry of Protein Assays

Chemistry of of Pr Protein As Assays

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Most colorimetric protein assay methods can be divided into two groups based on the type of chemistry involved: those involving protein-copper chelation with secondary detection of the reduced copper and those based on protein-dye binding with direct detection of the color change associated with the bound dye. Copper-based Thermo Scientific Protein Assays: Pierce BCA Protein Assays Pierce Modified Lowry Assay Dye-based Thermo Scientific Protein Assays: Pierce Coomassie (Bradford) Assay Pierce Coomassie Plus (Bradford) Assays Pierce 660 nm Assays These products are well-characterized, robust assays that provide consistent, reliable results. Nevertheless, each assay reagent has its limitations; having a basic understanding of the chemistries involved with each type of assay is essential for selecting an appropriate method for a given sample and for correctly evaluating results. Copper-based Assay Chemistries Dye-based Assay Chemistries

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Copper-based Assay Chemistries Peptides and the Biuret Reaction Copper-based protein assays, including the BCA and Lowry methods, depend on the well-known "biuret reaction", whereby peptides containing three or more amino acid residues form a colored chelate complex with cupric ions (Cu2+) in an alkaline environment containing sodium potassium tartrate. This became known as the biuret reaction because it is chemically similar a complex that forms with the organic compound biuret biuret (NH2-CO-NH-CO-NH2) and the cupric ion. Biuret, a product of excess urea and heat, reacts with copper to form a light blue tetradentate complex.

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Diagram of the biuret reaction. By reducing the copper ion from cupric to cuprous form, the reaction produces a faint blue-violet color.

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Chemistry of Protein Assays


Structures of urea, biuret and peptide. Because polypeptides have a structure similar to biuret, they are able to complex with copper by the biuret reaction. Single amino acids and dipeptides do not give the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce the light blue to violet complex that absorbs light at 540nm. One cupric ion forms a colored coordination complex with four to six nearby peptides bonds. The intensity of the color produced is proportional to the number of peptide bonds participating in the reaction. Thus, the biuret reaction is the basis for a simple and rapid colorimetric reagent of the same name for quantitatively determining total protein concentration. The working range for the biuret assay is 5-160mg/mL, which is adequate for some types of industrial applications but not nearly sensitive enough for most protein research needs.

Bicinchoninic Acid (BCA) Protein Assays The BCA Protein Assay was introduced by Smith, et al. in 1985. Since then it has become the most popular method for colorimetric detection and quantitation of total protein. One particular benefit is that, unlike other methods available at that time (e.g., Bradford and Lowry assays), the BCA Protein Assay is compatible with samples that contain up to 5% surfactants (detergents). In addition, the BCA Assay responds more uniformly to different proteins than the Bradford method. The BCA Protein Assay combines the protein-induced biuret reaction (see above) with the highly sensitive and selective colorimetric detection of the resulting cuprous cation (Cu1+) by bicinchoninic acid (BCA). Thus, two steps are involved. First is the biuret reaction, whose faint blue color results from the reduction of cupric ion to cuprous ion. Second is the chelation of BCA with the cuprous ion, resulting in an intense purple color. The purple colored reaction product is formed by the chelation of two molecules of BCA w ith one cuprous ion. The BCA/copper complex is water-soluble and exhibits a strong l inear absorbance at 562nm with increasing protein concentrations. The purple color can be measured at any wavelength between 550nm and 570nm with minimal (less than 10%) loss of signal. The BCA reagent is approximately 100 times more sensitive (lower limit of detection) than the biuret reagent.

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The reaction of BCA with cuprous ion. Two molecules of BCA bind to each molecule of copper that had been reduced by a peptide-mediated biuret reaction.

The reaction that leads to BCA color formation as a result of the reduction of Cu2+ is especially influenced by the presence of three particular amino acid residues in proteins: cysteine/cystine, tyrosine and tryptophan. Apparently these amino acids enhance copper reduction independently and in the biuret reaction, thereby causing formation of a colored BCA-C u1+ chelate. However, studies performed

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with di- and tripeptides indicate that these produce more color than can be accounted for by the four individual BCA-reactive amino acids. In other words, the peptide backbone (and thus the total amount of protein) is the major contributor to the reduction of copper in the biuret reaction and color development in the BCA assay. Slight protein-to-protein variation in the BCA protein assay results from differences among proteins in composition with respect to these three amino acids. The binding of BCA to cuprous ion effectively removes the weakly chelated peptides of the biuret reaction. Those peptide groups are then free to bind another molecule of cupric ion. Therefore, if bicinchoninic acid and copper are present in large excess (as they always are in BCA protein assay reagents), the protein assay does not reach an end-point. In addition, the rate of BCA color formation is dependent on the incubation temperature. C onsequently, the key to obtaining accurate results with the BCA assay method is to assay standards and unknown samples simultaneously so that they both receive identical incubation time and temperature. Assuming that the assay is performed in this way, the assay characteristic enables one to speed development or wait longer for desired colored development as needed.

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Substances that reduce copper will also produce color in the BCA assay, thus interfering with the accuracy of the protein quantitation. Reagents that chelate the copper also interfere by reducing the amount of BCA color produced with protein. Certain single amino acids (cysteine or cystine, tyrosine and tryptophan) will also produce color and interfere in BCA assays. Tech Tips and specialized versions of BCA protein assay products address one or another of these sampleincompatibility issues.

Lowry Protein Assays The Lowry protein assay is named after Oliver H. Lowry, who developed and introduced it (Lowry, et al., 1951). It offered a significant improvement over previous protein assays and his paper became one of the most cited references in life science literature for many years. The Modified Lowry Protein Assay uses a stable reagent that replaces two unstable reagents described by Lowry. Essentially, the assay is an enhanced biuret assay involving copper chelation chemistry. Although the mechanism of color formation for the Lowry assay is similar to that of the BCA protein assay, there are several significant differences between the two. The exact mechanism of color formation in the Lowry assay remains poorly understood. The assay is performed in two distinct steps. First, protein is reacted with alkaline cupric sulfate in the presence of tartrate for 10 minutes at room temperature. During this incubation, a tetradentate copper complex forms from four peptide bonds and one atom of copper (this is the "biuret reaction"). Second, a phosphomolybdic-phosphotungstic acid solution is added. This compound (called Folin-phenol reagent) becomes reduced, producing an intense blue color. It is believed that the color enhancement occurs when the tetradentate copper complex transfers electrons to the phosphomolybdic-phosphotungstic acid complex. The blue color continues to intensify during a 30 minute room temperature incubation. It has been suggested that during the 30 minute incubation, a rearrangement of the initial unstable blue complex leads to the stable final blue colored complex which has higher absorbance (Lowry, et al. 1951; Legler, et al. 1985). The final blue color is optimally measured at 750nm, but it can be measured at any wavelength between 650nm and 750nm with little loss of color intensity. It is best to measure the color at 750nm since few other substances absorb light at that wavelength. For small peptides, the amount of color increases with the size of the peptide. The presence of any of five amino acid residues (tyrosine, tryptophan, cysteine, histidine and asparagine) in the peptide or protein backbone further enhances the amount of color produced because they contribute additional reducing equivalents to further reduce the phosphomolybdic/phosphotungstic acid complex. With the exception of tyrosine and tryptophan, free amino acids wi ll not produce a colored product with the Lowry reagent, however, most dipeptides can be detected. In the absence of any of the five amino acids listed above in the peptide backbone, proteins containing proline residues have a lower color response with the Lowry reagent due to the amino acid interfering with complex formation.

Unlike in the BCA assay, the secondary binding step in the Lowry method does not involve detachment of the peptide-copper chelate. Therefore, the Lowry method is effectively an end-point assay. Although it is always best to include internal standards in any protein assay, it is possible to obtain reasonable protein estimations with this assay method by comparing to a previously-plotted standard

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curve. The protocol requires that the Folin phenol reagent be added to each tube precisely at the end of the ten minute incubation. At the alkaline pH of the Lowry reagent, the Folin phenol reagent is almost immediately inactivated. Therefore, it is best to add the Folin phenol reagent at the precise time while simultaneously mixing each tube. Because this is somewhat cumbersome, some practice is required to obtain consistent results. This also limits the total number of samples that can be assayed in a single run. If a ten second interval between tubes is used, the maximum number of tubes that can be assayed within ten minutes is sixty (10 seconds/tube x 60 tubes = 600 seconds or 10 minutes). The Lowry assay reagent forms precipitates in the presence of detergents or potassium ions. When potassium ions are the cause, the problem can sometimes be overcome by centrifuging the tube and measuring the color in the supernatant. Most surfactants cause precipitation of the reagent even at very low concentrations. One exception is sodium dodecyl sulfate (SDS), which is compatible with the reagent at concentrations up to 1% in the sample. Chelating agents interfere by binding copper and preventing formation of the copper peptide bond complex. Reducing agents and free thiols also interfere by reducing the phosphotungstate-phosphomolybdate complex, immediately forming an intensely blue colored product upon their addition. The Modified Lowry Protein Assay Reagent must be refrigerated for long-term storage, but it must be warmed to room temperature before use. Using cold Modified Lowry Protein Assay Reagent will result in low absorbance values.

Dye-based Assay Chemistries Coomassie Dye (Bradford) Protein Assays Use of Coomassie G-250 dye in a colorimetric reagent for the detection and quantitation of total protein was first described by Dr. Marion Bradford in 1976 (Bradford, 1976). Thermo Scientific Pierce Coomassie and C oomassie Plus Protein Assay Products are variants of the reagent first reported by Bradford.

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Chemical structure of Coomassie dye. Formulated in a low-pH phosphoric acid buffer, this colloidal form of Coomassie dye is the basis for Bradford protein assay reagents. In the acidic environment of the reagent, protein binds to the Coomassie dye. This results in a spectral shift from the reddish/brown form of the dye (absorbance maximum at 465nm) to the blue form of the dye (absorbance maximum at 610nm). The difference between the two forms of the dye is greatest at 595nm, so that is the optimal wavelength to measure the blue color from the Coomassie dye-protein complex. If desired, the blue color can be measured at any wavelength between 575nm and 615nm. At the two extremes (575nm and 615nm) there is a loss of about 10% in the measured amount of color (absorbance) compared to that obtained at 595nm.

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Absorbance spectra for protein standards in the Thermo Scientific Pierce Coomassie Plus (Bradford) Protein Assay. Protein standards are 0, 125, 250, 500, 750, 1000, 1500, 2000µg/mL of bovine serum albumin, respectively. The 2000µg/mL line is drawn thicker than the others to orient the sequence. Notice that an i nverse relationship between protein concentration and absorbance occurs below 525nm (maximum at 465nm). Development of color in Bradford protein assays is 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. Free amino acids, peptides and low molecular weight proteins do not produce color with Coomassie dye reagents. In general, the m ass of a peptide or protein must be at least 3000 daltons to be detectable with this reagent. In some applications this can be an advantage. For example, the Coomassie Protein Assay has been used to measure "high molecular weight proteins" during fermentation in the beer brewing industry.

Coomassie dye binding assays are the fastest and easiest to perform of all protein assays. The assay is performed at room temperature and no special equipment is required. Standard and unknown samples are added to tubes containing preformulated Coomassie assay reagent and the resultant blue color is measured at 595nm following a short room temperature incubation. The Coomassie dye-containing protein assays are compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents encountered in protein samples. The main disadvantage of Coomassie based protein assays is their incompatibility with surfactants at concentrations routinely used to solubilize membrane proteins. In general, the presence of a surfactant in the sample, even at low concentrations, causes precipitation of the reagent. In addition, the Coomassie dye reagent is highly acidic, so proteins with poor acid-solubility cannot be assayed with this reagent. Finally, Coomassie reagents result in about twice as much proteinto-protein variation as copper chelation-based assay reagents. The ready-to-use liquid Coomassie dye reagents should be mixed gently by inversion just before use. The dye in these liquid reagents forms loose aggregates within 60 minutes in undisturbed solutions. Gentle mixing of the reagent by inversion of the bottle will uniformly disperse the dye and ensure that aliquots are homogeneous. After binding to protein, the dye also forms protein-dye aggregates. Fortunately, these protein-dye aggregates can be dispersed easily by mixing the reaction tube. This is common to all liquid Coomassie dye reagents. Because these aggregates form relatively quickly, it is also best to routinely mix (vortex for 2-3 seconds) each tube or plate just before measuring the color.

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Pierce 660 nm Protein Assay Introduced in 2008, the Thermo Scientific Pierce 660 nm Protein Assay is a dye-based reagent that offers the same convenience as Coomassie-based assays while overcoming several of their disadvantages. In particular, the Pierce 660 nm Assay is compatible with most detergents and produces a more linear response curve. The detailed assay chemistry is proprietary, but the essential mechanism can be summarized as follows. The reagent contains a proprietary dye-metal complex in an acidic buffer. The dye-metal complex binds to protein in the acidic condition, causing a shift in the dye's absorption maximum, which is measured at 660 nm.

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The reagent is reddish-brown and changes to green upon protein binding. The color produced in the assay is stable and increases in proportion to a broad range of increasing protein concentrations. The color change is produced by the deprotonation of the dye at low pH facilitated by protein-binding interactions through positively charged amino acid groups and the negatively charged deprotonnated dye-metal complex. The assay binds to proteins in a manner similar to Coomassie dye. Thus, it has similar protein-to-protein variability to Coomassie (Bradford) assay methods. However, unlike Coomassie-based assays, the Pierce 660 nm Protein A ssay is fully compatible with nonionic detergents typically used in protein samples. In fact, when used with the Ionic Detergent Compatibility Reagent (IDCR), the Pierce 660 nm Assay is also compatible with sample containing Laemmli SDS sample buffer with bromophenol blue and other buffers containing common ionic detergents.

References 1. Bradford, M.M. (1976). Anal. Biochem. 72 , 248-254. 2. Smith, P.K., et al. (1985). Anal B iochem. 150 , 76-85. 3. Krohn, R.I. (2002). The C olorimetric Detection and Quantitation of Total

Protein, Current Protocols in Cell Biology , A.3H.1-A.3H.28, John Wiley & Sons, Inc. 4. Krohn, R.I. (2001). The C olorimetric Determination of Total Protein, Current Protocols in Food Analytical Chemistry , B1.1.1-B1.1.27, John Wiley & Sons, Inc. 5. Lowry, O.H., et al. (1951). J Biol Chem. 193 , 265-275. 6. Legler, G., et al. (1985). Anal. Biochem. 150 , 278-287.

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Chemistry of Protein Assays

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