Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins

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1 Anal Bioanal Chem (2008) 391: DOI /s x ORIGINAL PAPER Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins Christos D. Georgiou & Konstantinos Grintzalis & George Zervoudakis & Ioannis Papapostolou Received: 19 December 2007 / Revised: 13 February 2008 / Accepted: 18 February 2008 / Published online: 8 March 2008 # Springer-Verlag 2008 Abstract We investigated the mechanism of Coomassie brilliant blue G-250 (CBB) binding to proteins in order to develop a protein assay with the maximum possible sensitivity. We found that the neutral ionic species of CBB binds to proteins by a combination of hydrophobic interactions and heteropolar bonding with basic amino acids. On the basis of these findings, we developed a very sensitive hydrophobic assay for proteins (at the nanogram level) using the hydrophobic reagents ammonium sulfate and trichloroacetic acid under ph conditions that increase neutral species concentration in the assay reagent in order to enhance the binding of more CBB dye molecules per protein molecule than in previous CBB-based assays. Keywords Coomassie brilliant blue G-250. Proteins. Quantification Introduction Several methods for protein determination have been developed but those most commonly used today are based on the reaction of Coomassie brilliant blue G-250 (CBB) [1] and alkaline Cu(II) [2] with proteins. The most recent modifications of these methods are the Sedmak and Grossberg [3] assay and the bicinchoninic acid (BCA) [4] assay, having a C. D. Georgiou (*) : K. Grintzalis : I. Papapostolou Department of Biology, Section of Genetics, Cell Biology and Development, University of Patras, Patras, Greece c.georgiou@upatras.gr G. Zervoudakis Department of Greenhouse Crops and Floriculture, Technological Institute of Mesologgi, Nea Ktiria, Mesologgi, Greece sensitivity limit of 1 and 20 μg, respectively. The method of Sedmak and Grossberg, in particular, replaces the 4.7% ethanol + 8.5% (w/v) phosphoric acid solvent of CBB used in the Bradford [1] assay by either 0.6 N HCl or 3% (w/v) perchloric acid (PCA), with both assay reagent versions achieving nearly equal sensitivity [3]. In the present study we investigated the mechanism of CBB binding to proteins in order to develop a new CBB-based protein assay. We present a simple and very sensitive assay in three versions that detects proteins at the nanogram level. Materials and methods Materials CBB was from SERVA (Heidelberg, Germany). Bovine serum albumin (BSA), lysozyme (chicken egg white, grade I), cytochrome c (horse heart), hemoglobin (horse blood), pepsin (porcine stomach mucosa) and Bradford reagent (product no. B6916) were from Sigma-Aldrich (St. Louis, MO, USA). PCA, trichloroacetic acid (TCA), trifluoroacetic acid (TFA) and ammonium sulfate (AS) were from Merck (Darmstadt, Germany). Methods Spectral study of CBB binding to proteins For studying CBB ionic species binding to proteins in the presence and absence of AS and TCA, we prepared the anionic species by dissolving 9 mg CBB per milliliter of 5 mm phosphate buffer, ph 7.0, followed by centrifugation at 12,000 g for 5 min. From the resulting supernatant, a 75 fold dilution (approximate absorbance of 0.33 at

2 392 Anal Bioanal Chem (2008) 391: nm) was used for the spectral studies described in Results and discussion. Bradford and Sedmak and Grossberg assays For the Bradford and the Sedmak and Grossberg assays, the reagents used in the present study were prepared as described for the original methods [1, 3], using for the latter the 0.6 N HCl-based CBB reagent. For comparing these assays with the standard version of our assay (the hydrophobic CBB TCA/AS assay, see later), the assay reaction mixture was scaled down to 1 ml final volume using 0.05 ml BSA protein sample volume. Hydrophobic CBB TCA/AS assay Assay reagents Assay stock reagents are prepared by dissolving 60 mg CBB in 100 ml 1 N HCl (designated as CBB HCl stock) for preparing the hydrophobic CBB TCA reagent for the standard and microplate assays, and in 100 ml 2 N HCl (designated as CBB 2HCl stock) for preparing the hydrophobic CBB AS reagent for the microassay. The CBB HCl and CBB 2HCl stock solutions are stirred for 40 min, filtered through Whatman no. 1 filter paper and stored at 4 C (stable for months when protected from light). Standard and microplate assays Before use, the hydrophobic CBB TCA reagent (20 ml) was prepared by adding 20 ml CBB HCl stock solution to 1% absolute ethanol and 2% TCA (with 0.4 g TCA or 0.4 ml 100% TCA), followed by adjustment of the ph of the resulting mixture to 0.4 by addition of approximately 1.67 g solid Na 3 PO 4 12H 2 Owith continuous stirring. The reagent was cleared from the blue particulate formed by centrifugation at 5,000 g for 5 min at room temperature. A low concentration of ethanol (1 2%) maintains the hydrophobic CBB TCA reagent stable at room temperature (protected from light) for at least 3 weeks without affecting assay sensitivity. The standard assay (performed in a final volume of 1 ml) consists of mixing 0.05 ml protein solution with 0.95 ml hydrophobic CBB TCA reagent. After 5 10-min incubation at room temperature, the absorbance of the mixture is measured at 610 nm against an appropriate reagent blank. In this study, the absorbance was measured in a 1.4-ml glass microcuvette (internal dimensions 45 mm 4 mm 10 mm, from Starna Optiglass, Hainault, UK), using a Hitachi UV vis U-1800 spectrophotometer (Hitachi High-Technologies Europe, Germany). A normal 3-ml cuvette can be also used after proportional scaling up of the assay mixture volumes. For making a linear standard curve, BSA (or lysozyme, cytochrome c, hemoglobin, pepsin) standard solutions of 2 60 μg/ml (or μg/0.05 ml) are used. The microplate assay is used when very low volume protein samples are available. The assay consists of mixing 5 μl protein sample with 0.25 ml hydrophobic CBB TCA reagent in a 96-well plate. For maximum assay accuracy, 5 μl protein sample buffer is used as a reagent blank, and a multipipettor (e.g., a 12-channel ml multipipettor) is used for dispensing 0.25 ml of the hydrophobic CBB TCA reagent in the microplate wells. After 5 10-min incubation at room temperature, the absorbance of the mixtures in the microplate wells is read using a microplate reader (MRX microplate reader, Dynex Technologies,VA, USA) with its filter set at 595 nm. For making the linear standard curve, BSA (or lysozyme, cytochrome c, hemoglobin, pepsin) standard solutions of ng/5 μl sample (or μg/ml) were used. Although the standard and microplate assays are phdependent, they are unaffected by the high ionic strength and ph of the protein sample because it is used in these assays in a very small volume. Microassay Before use, the hydrophobic CBB AS reagent (20 ml) is prepared by mixing 10 ml CBB 2HCl stock solution with 10 ml 2 N HCl, and adding it to 2% absolute ethanol and 1.36 M AS (with 3.6 g AS) with continuous stirring. If any blue particulate is formed, it is removed by centrifugation at 5,000 g for 5 min at room temperature. Ethanol (at 2%) maintains the hydrophobic CBB AS reagent stable at room temperature (protected from light) for at least 3 weeks without affecting assay sensitivity. The microassay is used when low concentration protein samples are available in high volumes. The assay is performed in a final volume of 1 ml as follows. A maximum of 0.5 ml protein solution is mixed with 0.5 ml hydrophobic CBB AS reagent. After 5 10-min incubation at room temperature, the absorbance of the mixture is measured at 610 nm against an appropriate reagent blank. For making the linear standard curve, BSA (or lysozyme, cytochrome c, hemoglobin, pepsin) standard solutions of μg/ml (or μg/0.33 ml) were used. It should be noted that because the microassay is ph-dependent (optimum ph ) and the hydrophobic CBB AS reagent is diluted 2 times by the sample, the latter must not have high ionic strength and ph that may influence the optimum assay ph. Protein samples in 0 50 mm phosphate buffer, ph 7.0, are appropriate for this assay. Statistical analysis All data are reported as the mean ± the standard error from at least triplicate experiments. In all protein concentration measurements, the standard error was less than 10% of the mean value for all proteins (BSA, lysozyme, cytochrome c, hemoglobin and pepsin) and all assay versions (standard assay, microplate assay, microassay) used. The significance was determined using Stu-

3 Anal Bioanal Chem (2008) 391: dent s unpaired t test, with a value of P<0.05 considered to be significant. Results and discussion In this study we investigated the mechanism of CBB binding to proteins in order to develop a new CBB-based protein assay with the highest possible sensitivity. Protein interaction with the CBB ionization species and the effect of AS and TCA We explored in more detail how CBB dye binds to proteins since the previously proposed mechanism of the Bradford assay [5] has raised some unanswered crucial questions which obscure the molecular details of this mechanism. CBB has been shown to exist in three ionic species: the doubly protonated cationic (red), the neutral (green) and the unprotonated anionic (blue) species [1, 5]. Under very acid conditions, the dye is most stable as the doubly protonated (cationic) species. Although it has been shown that the anionic species is not present at the low ph existing in the Bradford assay reagent, it has been presumed that it is only this species that complexes with proteins [1, 5], without providing any explanation for this apparent paradox. It has also been suggested that primarily the basic amino acids (Arg and to a lesser degree Lys and His) and secondarily the aromatic amino acids (Trp, Phe, Tyr) participate in the binding of the anionic species to proteins. Protein CBB dye binding has been also postulated to be due to the development of hydrophobic interactions and van der Waals forces between proteins and the anionic species of the CBB dye [5] without any evidence being presented for this hypothesis. For studying which CBB ionic species binds to proteins, we prepared the CBB anionic species as described in Materials and methods, and by sequential titration of this species with HCl we prepared the cationic and neutral species (Fig. 1). As shown in this study and as has also been reported elsewhere [5], the cationic, the neutral and the anionic species exist at ph<0.39, ph~1.3 and ph>1.3, respectively (Fig. 1, panel a). The anionic species (absorption peak at 590 nm) absorbs approximately threefold more than the cationic species and approximately 1.9-fold more than the neutral species (Fig. 1, panel a). Addition to the anionic species of a high amount of BSA (e.g., up to 0.6 mg) shifts its 590 nm peak to 615 nm, but the absorbance increase is not proportional to the concentration of BSA and the absorbance is not substantially higher than the absorbance of the free anionic species, suggesting low strength of binding to proteins (Fig. 1, panel b, spectra a c). The soformed anionic species protein complex because of its distinct absorption peak confirms the previous observation that the anionic species binds to proteins [5]. In addition, we found that the neutral species binds to proteins as well, and shifts its 650 nm peak to 615 nm (Fig. 1, panelb).the absorption wavelength of this neutral species protein complex is ph-dependent because as the ph of formation of the Fig. 1 a Absorption spectra of the various ionization species of Coomassie brilliant blue G-250 (CBB). The anionic species (CBB in 5 mm phosphate buffer, ph 7.0, dashed spectrum) is gradually converted to the neutral (peak at 650 nm) and the cationic (peak at 470 nm) species by titration with HCl, 0.05 N (ph 1.3), 0.1 N (ph 1.0), 0.2 N (ph 0.69), 0.4 N (ph 0.39), 0.6 N (ph 0.23) and 1 N (ph 0), corresponding to absorption spectra 1, 2, 3, 4, 5 and 6 respectively. b Absorption spectra of the CBB ionic species in the presence of 0.2 mg bovine serum albumin (BSA; in a final reaction volume of 1 ml). Spectra a, b and c result from the binding of the anionic species with 0.2, 0.4 and 0.6 mg BSA, respectively

4 394 Anal Bioanal Chem (2008) 391: neutral species (by gradual titration with HCl) decreases, the peak of the neutral species protein complex shifts from 615 to 630 nm (Fig. 1, panel b, spectra 5, 6). That is, the absorption peak of the neutral species protein complex lies between the absorption peaks of the neutral and the anionic species. The absorbance at 615 nm of the neutral species protein complex is much higher (2 3-fold) than the absorbance of the neutral species that it results from, depends on its concentration (Fig. 1, spectra 1 4), and its maximum value reaches a plateau (Fig. 1, panel b, spectra 1, 2). Interestingly, the maximum absorbance value at 615 nm of the neutral species protein complex that results from the binding of proteins to CBB completely converted to the neutral species is equal to the absorbance value at 615 nm of the neutral species protein complex that results from the binding of an equal amount of protein (0.2 mg) to the same amount of CBB completely converted to the anionic species (Fig. 1, panel b, spectra 1, 2, a). This leads to the conclusion that protein molecules pull out the maximum number of dye molecules they can bind from the CBB dye, either as anionic or as neutral species, they convert them to the CBB dye protein complex with a peak at 615 nm, and they cause the protein-bound dye molecules to absorb a certain maximum amount at 615 nm regardless of the ionic species (anionic or neutral) they result from (Fig. 1, panel b, spectra 1, 2, a). Thus, the absorbance of the CBB dye protein complex depends on the number of bound dye molecules and not on any absorbance-enhancing effect of the protein on the bound dye. These findings are not in agreement with previous claims (1) that proteins bind exclusively to the anionic species and (2) that the resulting CBB dye protein complex has an absorption peak at the same wavelength as the anionic species [5]. It is this spectral wavelength difference that makes possible the quantification of the protein dye complex. Furthermore, the cationic species does not bind to proteins as is shown by the unchanged absorbance at 470 nm of the cationic species in the presence and absence of BSA (Fig. 1, spectra 5, 6). Hydrophobic interactions enhance binding of CBB neutral species to proteins Since hydrophobic interactions have been suggested but not proven to play an important role in the binding of proteins to CBB, we tested the binding of BSA to the neutral and the anionic species in the presence of AS and TCA, two reagents with known ability to dehydrate proteins and induce hydrophobic and van der Waals interactions [5, 6]. We obtained difference spectra from spectra in the presence of a constant amount of BSA complexed with a certain amount of neutral and anionic species at various concentrations of AS and TCA, from which the corresponding spectra without BSA were subtracted (Figs. 2 and 3). In order to obtain visible changes in the spectra of the anionic and neutral species with proteins, we used 0.2 mg BSA (in a final volume of 1 ml) after establishing the experimental conditions that do not cause protein precipitation. We ended up using 0.4 N HCl to prepare the neutral species, with which various concentrations of AS (0 1 M) and TCA (0 1%) without BSA or with 0.2 mg BSA were mixed. The peak absorbance value in the absorbance difference spectra (without and with BSA) of the neutral species of (spectrum 1, resulting also from subtraction of spectrum 4 in Fig. 1, panel a from spectrum 4 in Fig. 1, panel b) is increased at 615 nm to a maximum of 0.19 (approximately 2.5-fold) at 0.7 M AS (Fig. 2, panel a, spectrum 4), and at 620 nm to a maximum of (approximately 2 fold) at 0.8% TCA (Fig. 2, panel b, spectrum 4). Therefore, binding of proteins to the neutral Fig. 2 Absorbance difference spectra of the neutral ionization species (formed in 0.4 N HCl) in the absence of BSA and in the presence of 0.2 mg BSA (in a reaction volume of 1 ml) at various concentrations a of ammonium sulfate (AS; 0, 0.25, 0.5, 0.7 M, spectra 1, 2, 3, 4, respectively) and b of trichloroacetic acid (TCA; 0,0.3,0.5,0.8%, spectra 1, 2, 3, 4, respectively). The inserts show the difference between the maximum absorbances of the peaks of the spectra and the absorbance of spectrum 1 (blank), designated by the corresponding spectrum number, as a function of the concentration of AS and TCA

5 Anal Bioanal Chem (2008) 391: Fig. 3 Absorbance difference spectra showing the absorbance of the anionic species protein (BSA) complex in low/high ionic strength (5/ 700 mm) phosphate buffer (a) and in the absence and presence of AS (b) and TCA (c). a The anionic species protein complex is shown at low ionic strength (absorbing at 650 nm, in the presence of 0.2, 0.4, 0.6 mg BSA, spectra a, b, c, respectively) and at high ionic strength (absorbing at 630 nm, in the presence of 0.2 mg BSA, spectrum 1). b The anionic species BSA (0.2 mg) complex is shown at high ionic strength in 0, 0.5 and 0.7 M AS (spectra 1, 2, 3, respectively). c The anionic species BSA (0.2 mg) complex is shown at high ionic strength in 0, 0.3 and 0.8% TCA (spectra 1, 2, 3, respectively) species in the absence of AS/TCA shifts its absorbance near that of the anionic species, forming a CBB species with an absorbance maximum at 615 nm (Fig. 2, spectra 1), which changes slightly (to 620 nm) only in the presence of TCA. Thus, the spectral properties of the protein-bound neutral species show that it behaves as a pseudo-anionic species. The maximum net absorbance values of (at 615 nm) and (at 620 nm) in the presence of 0.7 M AS and 0.8% TCA, respectively, are both even higher than the maximum absorbance difference (0.13) in for solutions without BSA and with 0.2 mg BSA of the spectrum of the neutral species formed in the highest concentration at 0.05 N HCl (spectrum 1inFig.1, panel b minus spectrum 1 in Fig. 1, panel a). This result shows that both AS and TCA (the first more effectively) cause even more molecules of the neutral species to bind to the protein than in their absence. This can be explained by the increase of hydrophobic interactions that AS and TCA cause between neutral species and protein (BSA) molecules, resulting in the binding of more neutral species molecules per protein molecule than in the absence of AS/ TCA. TCA is thought to precipitate proteins by dehydration and denaturation, which cause an increase in hydrophobic and van der Waals interactions, while AS is known to increase these interactions by its water molecule ordering (kosmotropic) function [6, 7]. It should be noted that the TCA effect was not repeated with equimolar amounts of acetic acid ( without and with FeCl 3 ) or TFA, which proves that neither chloride ions in the presence or absence of acetic acid nor fluoride bound to acetic acid plays any role in the binding of neutral species to proteins (data not shown). As far as the spectral behavior of the anionic species protein complex in the presence of AS/TCA is concerned, the anionic species binds to proteins, and, interestingly enough, it forms an anionic species protein complex with maximum absorbance at 650 nm irrespective of protein concentration (Fig. 3, panel a). Thus, this complex has the same absorbance maximum as the neutral species. This can be explained by the neutralization of one of the existing two negatively charged sulfonic groups in the CBB anionic species (most likely the one distant from the positively charged quaternary nitrogen of CBB) by the positively charged guanidino group of Arg (or by the amino group of Lys and the imidazole amino group of His) present in proteins and BSA. The binding of BSA to the anionic species is weaker than that to the neutral species because the absorbance maximum of the anionic species BSA complex is twofold less than that of the neutral species BSA complex for the same protein concentration (0.2 mg BSA) (Fig. 3, panel a, spectrum a, Fig. 2, spectra 1). The wavelength of the maximum absorbance of the anionic species protein complex is ionic-strength-dependent, shifting from 650 nm at low ionic strength (in 5 mm phosphate buffer, ph 7.0) to 630 nm at high ionic strength (in 0.7 M phosphate buffer, ph 7.0) (Fig. 3, panel a, spectra a, 1) without any change in its maximum absorbance value. This high ionic strength was imposed by the need to keep the ph of the aqueous solution of the anionic species constant at 7.0 when studying the effect of AS and TCA on its complexation with BSA. An additional ionic-strength-dependent shift was observed for AS at 0.7 M from 630 to 615 nm (Fig. 3, panel b, spectra 1 3), and to a lesser degree for TCA at 0.8% (or 0.05 M) from 630 to

6 396 Anal Bioanal Chem (2008) 391: nm (Fig. 3, panel c, spectra 1 3), both without any change in the maximum absorbance values. The latter observation suggests that the binding of proteins to the anionic species is not enhanced by hydrophobic interactions and proceeds via electrostatic interactions with Arg, Lys and His amino groups. It should be noted that ionic strength seems to be crucial as well for the absorbance maximum of the neutral species protein complex at 615 and 625 nm in the presence of 0.7 M AS and 2% TCA, respectively, and this can possibly be due to an ionic-strength-dependent weakening of the heteropolar bond of the proton-neutralized sulfonic group proximal to the quaternary nitrogen and of the electrostatic bond between the other sulfonic group and Arg, making the neutral species protein complex in the presence of AS/TCA behave spectrally more as a pseudo-anionic species. The neutral species is the active protein-binding species in the CBB-based assays It has been claimed that the only CBB ionic species that binds to proteins when using the Bradford and the Sedmak and Grossberg reagents is the anionic species [1, 5]. When CBB is dissolved either in 0.6 N HCl (as in the reagent in the Sedmak and Grossberg [3] assay) or in 8.5% phosphoric acid (in the presence of 4.7% ethanol, as for the reagent in the Bradford [1] assay), it has been claimed that it is converted only to the cationic species [5]. If, then, the only species existing in the previously mentioned reagents is the cationic species, which has been already shown not to bind to proteins, how can it be justified that it is the nonexisting, in these reagents, anionic species that binds to proteins? Since we have already shown that neutral and anionic species both bind to proteins, we propose that the Bradford and the Sedmak and Grossberg assay reagents must contain either one of these ionic species. Specifically, they should contain only the neutral species (expected to be formed at ph 0.64 and 0.23 of the Bradford and the Sedmak and Grossberg reagents, respectively) and not the anionic species (because it exists above ph 1.3), and in quantities in excess of the protein quantities detected by these assays. The presence of the neutral species in the Sedmak and Grossberg and the Bradford assay reagents was verified by their difference spectra as a peak absorbance at 650 nm (Fig. 4, spectra 1, 2). Moreover, the neutral species concentration was related to reagent assay ph: the higher the assay reagent ph, the higher the concentration of the neutral species, which is expected since a ph increase causes the transition of the cationic to the neutral species (Fig. 1, panel a, spectra 1 5). Mechanism of protein CBB dye binding The neutral species is the most hydrophobic among the three CBB ionic species, and it is with this species that the Fig. 4 Identification of the CBB neutral species (650 nm) in the Sedmak and Grossberg and the Bradford assay reagents by their absorbance difference spectra 1 and 2, respectively, and their relation to the corresponding assay reagent ph. Absorbance difference spectrum 1 for the Sedmak and Grossberg reagent was obtained from the spectrum of this reagent minus the spectrum of the same reagent the 0.6 N HCI of which was previously adjusted to 2 N, and absorbance difference spectrum 2 for the Bradford reagent was obtained from the spectrum of this reagent minus the spectrum of the same reagent to which 2 N HCI was added hydrophobic interactions with proteins attain their maximum degree. Hydrophobicity is a combination of the kosmotropic (water molecule ordering) nature of molecules such as AS and low ph via its dehydration effect on proteins [8]. The TCA hydrophobicity effect and van der Waals forces are also exerted via dehydration of proteins [5, 6]. The presence of salting-out ions such as (NH 4 ) 2 SO 4 (as well as Na 2 SO 4 and MgSO 4, the so-called kosmotropic compounds) enhances the hydrophobic interactions as a function of their concentration [7]. Hydrophobic interactions depend also on the concentration of the neutral species (and of proteins). The more neutral species molecules that are available, the more of these molecules that will bind per protein molecule. Regarding the binding mechanism of proteins with CBB, the data from this study show that it depends on the particular ionic species and on the type of associations they can develop with proteins (Fig. 5). CBB has two negatively charged sulfonic groups and a positively charged quaternary nitrogen-carbon group in its anionic species and one, proton-neutralized, sulfonic group in its neutral species. Arg (and the other basic amino acids Lys and His) can react with the neutral or the anionic species because of its positively charged guanidino group in the ph range (0 1.3) of their formation. Specifically, Arg could react preferably

7 Anal Bioanal Chem (2008) 391: Fig. 5 Mechanism of CBB dye binding to proteins. a Neutral species binds to proteins both by hydrophobic interactions (via Phe, Trp, etc.) and by electrostatic attraction between the dissociated sulfonic group distant from the quaternary nitrogen primarily and the positively charged guanidino group of Arg (and secondarily with Lys, His). Upon protein binding, the absorption peak of the neutral species shifts to nm possibly owing to a weakening of the heteropolar bond of the proton-neutralized sulfonic group proximal to the quaternary nitrogen (dashed arrow), making it behave spectrally as a pseudo-anionic species. The development of hydrophobic interactions was shown by the increase of the absorbance of the protein dye complex formed in the presence of the kosmotropic AS and TCA (see Fig. 2). b The anionic species binds to proteins primarily by electrostatic attraction between the dissociated sulfonic group distant from the quaternary nitrogen and Arg (Lys, His), forming a complex absorbing at 650 nm and thus having spectral properties similar to those of the neutral species. Reaction of the quanidino group of Arg with the dissociated sulfonic group proximal to the positively charged quaternary nitrogen seems not to be favored owing to electrostatic repulsions. If both sulfonic groups of the anionic species were able to react with Arg, Lys or His, the resulting protein dye complex would be expected to behave spectrally as the cationic species (470 nm). The absence of hydrophobic interactions in the protein dye complex was shown by the fact that its absorbance was not increased in the presence of AS and TCA (see Fig. 3). c The cationic species does not form a complex with proteins because its absorbance at 470 nm does not change. This is expected because both sulfonic groups of this species are unavailable (proton-neutralized) for reaction with Arg, Lys or His group. The interaction of Arg with both proton-neutralized sulfonic groups of the basic species is very limited, possibly causing their destabilization (weak dissociation). Such bond destabilization may be also caused by high ionic strength (e.g., caused by high concentrations of AS and TCA). Proteins interact primarily with the neutral species in the reagents used in the Bradford and the Sedmak and Grossberg assays (1) by hydrophobic interactions with the hydrophobic amino acids Trp and Phe (and the polar aromatic amino acid Tyr) and (2) by electrostatic interactions between basic amino acids and the sulfonic groups of CBB. They can also interact with the anionic species only via heteropolar bonding between Arg and anionic CBB. Although CBB exists primarily as cationic species in the reagents used in the Bradford and the Sedmak and Grossberg assays, this ionic species cannot react with the basic amino acids of proteins nor can it react with them via hydrophobic interactions. with the sulfonic group distant from the positively charged quaternary nitrogen of CBB, leading to the formation of a heteropolar bond, and to a lesser degree (owing to possible development of positive charge repulsions between Arg and the quaternary nitrogen of CBB) with the other sulfonic group of the anionic species. Arg could react with the negatively charged sulfonic group distant from the positively charged quaternary nitrogen of the neutral species and also destabilize its other, proton-neutralized, sulfonic CBB hydrophobic assay Having clarified the mechanism of CBB binding to proteins, we used this mechanism in order to develop a new CBB protein assay with the highest possible sensitivity. We created a new CBB reagent which, in contrast to the Bradford and the Sedmak and Grossberg reagents, combines high concentration of neutral species with the hydrophobicity of AS and TCA (since it was shown that both promote the strong binding of proteins to neutral

8 398 Anal Bioanal Chem (2008) 391: species). The assay was developed in standard assay, microassay and microplate assay versions. For developing the standard version of the new assay we used an assay reagent to sample protein volume ratio of 20:1, a ratio usually used for CBB-based assays. As the solvent for CBB we chose HCl to test the effect of AS/TCA concentration and ph on binding of CBB neutral species to proteins for the following reasons: (1) HCl is a strong acid and its ph can be easily adjusted over the ph range 0 1 of neutral species formation and (2) HCl is a very effective solvent for CBB. We used 1 N HCl (instead of 0.6 N as used in the Sedmak and Grossberg assay reagent) since at this concentration we were able to increase the neutral species concentration by 1.6-fold in comparison with that of the Sedmak and Grossberg reagent (Fig. 4, spectrum 1, Fig. 6, spectra 1). This concentration increase was due to the increased solubility of CBB in 1 N HCl and not to its ph (0) since at 1 N HCl the neutral species concentration is very low (Fig. 1, panel a, spectrum 6). We did not choose phosphoric acid as the solvent for CBB because (1) its ph Fig. 6 Relation of neutral species concentration (absorbance at 650 nm, derived from absorbance difference spectra) in the CBB HCl reagent without and with various concentrations of AS or TCA (CBB HCl AS or CBB HCl TCA reagents, respectively) and ph in each of the resulting reagents with BSA binding (assessed by BSA standard curve slopes in terms of absorbance units at 610 and 620 nm/μg BSA for AS and TCA, respectively). Absorbance difference spectra of the CBB HCl AS (a) and CBB HCl TCA (b) reagents at various AS (0, 0.2, 0.4, 0.6, 0.7 M, spectra 1, 2, 3, 4, 5, respectively) and TCA (0, 0.5, 1, 1.5, 2%, spectra 1, 2, 3, 4, 5, respectively) were obtained from the spectra of these reagents minus the spectrum of CBB HCl reagent, the 1 N HCl of which was adjusted to 2N HCl (CBB 2HCl reagent). Base line spectrum 6 represents the difference spectrum arising from substraction of the spectrum of CBB 2HCl from itself, and spectrum 1 is actually the difference spectrum of the spectrum of reagent CBB HCl (i.e., without AS/TCA) minus the spectrum of CBB 2HCl. Insert a shows the correlation between the neutral species net concentration change (expressed as the absorbance difference at 650 nm of spectra 2 5 over spectrum 1) and the ph change as a function of AS/TCA concentration. Insert b shows the correlation between the BSA standard curve slope fold change [over the BSA slope in the absence of AS and TCA, corresponding to absorbance units difference at 610 and 620 nm (ΔA 610 and 620 nm )/μg BSA, respectively] and the neutral species concentration fold change (expressed as the ratio of the absorbance at 650 nm of spectra 2 5 over spectrum 1) as a function of AS/TCA concentration

9 Anal Bioanal Chem (2008) 391: over the range of neutral species formation (ph 0 1) is not easily regulated as it is a weak acid and (2) its limited solubility for CBB needs to be enhanced by 4.7% ethanol (as in the Bradford assay) but has the disadvantage of decreasing hydrophobic interactions [9, 10]. We then used a CBB stock dissolved in 1 N HCl reagent (designated as CBB HCl reagent) to formulate reagents CBB HCl AS and CBB HCl TCA with various concentrations of AS and TCA, respectively, in order to test the effect of AS and TCA on the neutral species concentration and ph in relation to the degree of CBB binding to BSA (assessed by corresponding standard curve slopes). It was shown that neutral species concentration increases as a function of increasing AS concentration (Fig. 6 panel a, spectra 1 5), and this increase is proportional to the ph increase (Fig. 6 panel a, insert a). This ph increase is actually caused by the increasing concentration of AS. AS at 0.68 M causes a maximum BSA standard curve slope fold of 28.6 (over the BSA slope in the absence of AS), which is not identical to the concentration fold increase (2.3-fold) of neutral species (Fig. 6 panel a, insert b) as would be expected if its concentration was the only factor causing this fold increase. Instead, the observed 12 times higher BSA standard curve slope at 0.68 M AS can be attributed mainly to an increase in the hydrophobicity caused by AS and to a lesser degree to the increase of neutral species concentration caused by the increase of ph. This is true at AS concentration up to 0.68 M (giving a reagent ph of 0.23) above which the neutral species concentration drops abruptly despite the reagent ph increase (Fig. 6 panel a, insert a) possibly owing to precipitation of neutral species by AS. On the other hand, the neutral species concentration decreases as a function of TCA concentration (Fig. 6 panel b, spectra 1 5) although the corresponding reagent ph remains relatively constant (Fig. 6 panel b, insert a). This can be attributed to the fact that TCA in excess of 0.5% causes gradual precipitation of neutral species (experimentally observed), although the unchanged ph is expected to keep the neutral species concentration constant. The observed maximum increase of the BSA standard curve slope at 2% TCA by 20.5-fold (over the BSA slope in the absence of TCA) despite the neutral species concentration decrease by 0.6-fold (Fig. 6 panel b, insert b) can be attributed to the increase in hydrophobicity caused by TCA. This result shows that hydrophobicity is more important than the concentration of the neutral species for increasing the sensitivity of any CBB-based assay, and that TCA (at 2%) is a more effective hydrophobic agent than AS (at 0.68 M). This is because the 1.4-fold greater BSA slope with the CBB HCl AS reagent in comparison with the CBB HCl TCA reagent corresponds to a 3.8-fold higher neutral species concentration between these reagents (Fig 6, spectra 5). Thus, the hydrophobicity degree (BSA slope) per neutral species concentration for TCA is 2.7-fold (i.e., 3.8/1.4) higher than for AS. The importance of hydrophobicity for developing a CBB-based protein assay was verified by the outcome of the inclusion of 5% ethanol in the CBB HCl AS and CBB HCl TCA reagents in an attempt to increase the maximum solubility of neutral species at 0.68 M AS and 2% TCA, respectively. It was found that although the concentration of neutral species in the CBB HCl AS and CBB HCl TCA reagents increased by 21 and 50%, respectively, the corresponding BSA standard curve slopes decreased by 30 and 17%, respectively (data not shown), which can be attributed to the known weakening of hydrophobic interactions by ethanol [9, 10]. Ethanol (4.7%) is also used in the reagent in the standard Bradford [1] assay, and this may be one of the reasons for the low sensitivity of this assay. In developing a sensitive CBB assay for proteins it is necessary to maximize the slope of its BSA standard curve while keeping the concentration of neutral species low (and thus that of the reagent blank at the absorption wavelength peak of the protein neutral species complex) in order to be able to detect very small absorbance changes over the reagent blank background. The higher the ratio of the slope of the BSA standard curve to the neutral species concentration for any CBB reagent the better. For example, this ratio is approximately 0.05 for the Bradford and the Sedmak and Grossberg reagents, while for the CBB HCl, CBB HCl AS and CBB HCl TCA reagents this ratio is 0.01, 0.13 and 0.35, respectively. Since the CBB HCl TCA reagent has the highest ratio and the lowest neutral species concentration, we investigated the possibility to increase further the slope of its BSA standard curve as a consequence of its neutral species concentration increase caused by increasing its ph. For this, we tested NaOH, NH 4 OH and Na 3 PO 4, and we chose the latter because (1) it caused insignificant dilution of the reagent solution and (2) its adjusted ph is stable since Na 3 PO 4 is a weak-acidderived salt. The maximum BSA standard curve slope is obtained at CBB HCl TCA reagent ph 0.4, and represents a 47% increase over the slope obtained without adjusting the reagent ph, and concurs with a 29% increase of neutral species concentration (Fig. 7 panel a, insert, spectra a, b). Moreover, the presence of Na 3 PO 4 (approximately 0.19 M) in CBB HCl TCA reagent causes a shift of the absorption wavelength peak of the protein neutral species complex from 620 nm to 610 nm, possibly owing to ionic strength increase by Na 3 PO 4. The absorbance of this complex at 610 nm remains constant at least for 20 min (Fig. 7 panel b, spectrum 1, insert, slope 1). In addition, the BSA standard curve slope (0.063 absorbance units at 610 nm/μg BSA) obtained by this CBB HCl TCA (ph 0.4) reagent, designated as hydrophobic CBB TCA reagent, is equal to the

10 400 Anal Bioanal Chem (2008) 391: CBB HCl AS reagent derived slope [Fig. 6 panel a, insert b, data 5, which cannot be further increased by increasing the reagent ph because of neutral species precipitation (data not shown)]. It should be noted that the hydrophobic CBB TCA reagent has the advantage of having a threefold lower reagent background (in terms of neutral species concentration) than the CBB HCl AS reagent (since their neutral species concentrations are and absorbance units at 610 nm, respectively, Fig. 7 panel a, insert b, Fig. 6 panel a, insert a, data 5). Low assay reagent background results in high assay sensitivity because it allows monitoring with high accuracy of very small absorbance differences (due to small protein concentrations) above this background. This accuracy depends on the photometric instrumentation in use, and for a usual spectrophotometer it is usually ±0.005 at 1.0 absorbance units. So, for a reagent blank background at 650 nm between 0.1 and 0.2 absorbance units the accuracy is ± to ±0.001 absorbance units, which means that, e.g., a minimum absorbance of absorbance units above the reagent blank can be measured with an accuracy ±5 10%. This accuracy is even higher at the 610 nm absorption peak of the protein neutral species (using the hydrophobic CBB TCA reagent) since this peak is 40 nm away from the absorption peak at 650 nm of the neutral species. The hydrophobic CBB TCA reagent, although suitable for the standard assay and microplate assay versions, is not suitable for a microassay version needed to measure large volumes of low-concentration protein samples (e.g., by mixing equal sample and reagent volumes, typical for a usual microassay). This would require adjustment of the sample ph to 0.4 (besides adjusting it to 1 N HCl and 2% Fig. 7 a Effect of reagent CBB HCl TCA ph on BSA standard curve slope (ΔA 610nm /Δμg BSA) and on CBB neutral species concentration expressed as the maximum absorbance at 650 nm of difference spectra between the spectra of the ph-adjusted CBB HCl TCA reagent (hydrophobic CBB TCA reagent) and its corresponding spectra after addition of 1 N HCl. The insert shows CBB HCl TCA reagent difference spectra a and b with its ph unadjusted and adjusted to ph 0.4, respectively). b Absorption spectra (and their peaks) of the protein CBB dye complex resulting from the reaction of the CBB HCl TCA reagent at ph 0.4 without BSA and with 50 μg BSA (in 1 ml reaction volume) used for the standard and microplate assay (spectrum 1) and the AS-based CBB HCl reagent used for the microassay (spectrum 2). The insert shows the effect of incubation time on the BSA standard curve slope using hydrophobic CBB TCA reagent in the standard assay version (1) and the hydrophobic CBB AS reagent used in the microassay version (2) Fig. 8 Linear standard curve of the standard version and the microplate version (see the insert) of the hydrophobic CBB TCA assay. Error bars designate standard deviation

11 Anal Bioanal Chem (2008) 391: Table 1 Protein sensitivity of the Coomassie brilliant blue G-250 (CBB) ammonium sulfate (AS)/trichloroacetic acid (TCA) assay in comparison with the Sigma and Pierce Bradford and bicinchoninic acid (BCA) assays Assay sensitivity Hydrophobic CBB TCA/AS assay Sigma assays Pierce assays Bradford BCA Coomassie plus BCA enhanced Standard assay Detection limit (μg) 0.1 a Linear standard curve lower and 2 60 a 100 1, , , upper limits (μg ml 1 ) Microplate assay Detection limit (μg) 0.05 a Linear standard curve lower and a 100 1, , upper limits (μg ml 1 ) Microassay Detection limit (μg) 0.15 b 1 NA NA 0.5 Linear standard curve lower and upper limits (μg ml 1 ) b 1 10 NA The sensitivity limits of the Sigma and Pierce assays are those stated in the technical bulletins of the corresponding products [11 13] NA not available a With the hydrophobic CBB TCA assay reagent b With the hydrophobic CBB AS assay reagent TCA), which is quite cumbersome and impractical for testing large sample numbers. Nor can the CBB HCl AS reagent be used unmodified for the microassay version because its 2 times dilution by the protein sample will decrease its neutral species concentration (besides decreasing 2 times its AS concentration and increasing its ph to levels causing neutral species precipitation). To avoid all these problems, we initially dissolved CBB in 2 N HCl (resulting in 2.4-fold dissolved dye over the 1 N HCl solvent, data not shown). We then tested various dilutions Table 2 Interference of various substances on the CBB TCA assay in comparison with the corresponding Sigma and Pierce assays Substance Hydrophobic CBB TCA assay Sigma Bradford assay Pierce Coomassie plus assay Acetone 10% 10% 10% Acetonitrile 10% 10% 10% Methanol 20% 10% 10% Ethanol 15% 10% 10% Dimethyl sulfoxide 20% 10% 10% NaOH 0.15 M 0.1 M 0.1 M 3-(N-Morpholino)propanesulfonic acid 0.1 M 0.1 M 0.1 M Guanidine hydrochloride 2 M 3.5 M 2 M Tris(hydroxymethyl)aminomethane 0.3 M 2 M 0.25 M hydrochloride Ammonium sulfate 0.7 M 1 M 1 mm EDTA 0.1 M 0.1 M 0.1 mm Ascorbic acid 0.02 M 0.05 M 0.05 M Urea 1 M 3 M 3 M 3-[(3-Cholamidopropyl)dimethylammonio]- 0.1% 5% 5% 1-propanesulfonate Triton X % 0.125% 0.062% Tween % 0.062% 0.031% Sodium dodecyl sulfate 0.004% 0.125% 0.016% Concentrations of the interfering reagents were determined with the standard version of the assays. For the hydrophobic CBB TCA assay, the concentrations of the interfering reagents were determined at assay BSA standard curve limit of 2 μg ml 1 (Table 1). Interference values for the Sigma Bradford assay and for the Pierce Coomassie plus assay are those reported in the corresponding assay technical bulletins [11, 13]

12 402 Anal Bioanal Chem (2008) 391: Fig. 9 Linear standard curve of the hydrophobic CBB AS microassay. Error bars designate standard deviation (with 2 N HCl) of this new CBB 2HCl stock in the presence of 1.36 M AS (that is, 2 times the 0.68 M in the CBB HCl AS reagent) in order to keep dissolved the neutral species in the resulting (CBB 2HCl) x(dilution factor) 1.36AS reagents (the undissolved neutral species was removed by centrifugation at 12,000 g for 5 min). The appropriate reagent was the (CBB 2HCl) 2x 1.36AS reagent (resulting from 2 times dilution of the CBB 2HCl stock) since mixing equal volumes of this reagent and a protein sample made in 0 50 mm phosphate buffer (ph 7.0) keeps the ph of the resulting assay mixture below 0.3 (where the neutral species does not precipitate under the conditions tested). It should be noted that the CBB 2HCl stock previously diluted 2 times with double-distilledh 2 O cannot be used for preparing the hydrophobic CBB TCA reagent as well, because the amount of neutral species resulting after TCA and ph adjustment to 2% and 0.4 (with Na 3 PO 4 ), respectively, is 17% less than that in the same reagent made from the CBB stock (in 1 N HCl). This is attributed to coprecipitation effects due to the higher neutral species concentration in the 2 times dilution of the CBB 2HCl stock. The protein neutral species complex formed by the (CBB 2HCl) 2x 1.36AS reagent, designated as hydrophobic CBB AS reagent, has an absorbance peak at 610 nm, the value of which remains constant at least for 20 min (Fig. 7 panel b, spectrum 2, insert, slope 2). In this study we have presented a new hydrophobic CBB TCA/AS assay which uses the hydrophobic CBB TCA reagent for the standard assay and microplate assay and the hydrophobic CBB AS reagent for the microassay. The hydrophobic CBB TCA standard assay uses protein samples up to 50 μl with a minimum of 100 ng (or 2 μgml 1 )protein. The BSA standard curve is linear up to 3 μg (or60μg ml 1 ) (Fig. 8). Slopes similar to those for BSA were obtained with other proteins, such as lysozyme, cytochrome c, hemoglobin and pepsin (data not shown). Standard assay sensitivity (the minimum detected protein quantity) is 100- and 40-fold higher than those of the most sensitive Bradford assay versions of Sigma and Pierce, respectively, and this sensitivity is even higher for the Pierce assay when expressed as protein concentration (micrograms or nanograms per milliliter). It should be noted that the claimed linear range of 100 1,400 μg ml 1 of the Sigma standard Bradford assay is not confirmed by the data presented in the technical bulletin for the assay [11]. Furthermore, the hydrophobic CBB TCA standard assay is 200-fold and fivefold more sensitive than the Sigma and Pierce BCA assays, respectively (Table 1). The standard assay version was used for the determination of the interference of some substances (Table 2). The assay shows a degree of interference similar to those of the Sigma and Pierce Bradford assays for the same substances tested, with the exception of certain detergents where the interference in the hydrophobic CBB TCA assay is slightly higher. Nevertheless, because of the higher sensitivity of this assay for proteins, if the maximum concentrations of the interfering detergents (and the other interfering substances) for this assay are scaled up to the protein concentration of the BSA stock solution used for the linear standard curve detection limit of the Sigma and Pierce assays (100 and 125 μg ml 1, respectively, Table 1), it can be concluded that the hydrophobic CBB TCA assay is proportionally less sensitive to these interfering substances. The hydrophobic CBB TCA microplate assay version detects very low quantities of protein (at the nanogram level) in samples of very low volume (up to 5 μl), with the lowest detectable quantity being 50 ng (10 μg ml 1 )(Fig.8, insert),makingthemicroplate assay tenfold and 76-fold more sensitive than the Sigma Bradford assay and the Coomassie dry plate protein assay, respectively (Table 1). Sensitivities similar to those obtained with BSA were obtained with other proteins, such as lysozyme, cytochrome c, hemoglobin and pepsin (data not shown). The assay is 100-fold and tenfold more sensitive than the Sigma and Pierce, respectively, BCA microplate assays (Table 1). The hydrophobic CBB AS microassay is used for samples up to 0.5 ml with very low protein concentration. The linear BSA standard curve is depicted in Fig. 9. Slopes similar to those for BSA were obtained with other proteins, such as lysozyme, cytochrome c, hemoglobin and pepsin (data not shown). The sensitivity of the microassay is 6.6-fold higher than that of the Sigma Bradford assay and 3.3-fold higher of the Pierce BCA assay (Table 1). Conclusions In this study we have shown that not only the anionic species (as previously suggested [1, 5]) but also the neutral species bind to proteins among the three ionic species of CBB. This protein neutral species binding is enhanced by AS and TCA,

13 Anal Bioanal Chem (2008) 391: both known to increase the hydrophobic interactions. Our hydrophobic CBB AS/TCA assay is sensitive for proteins at the nanogram level, and is and fold more sensitive than the most sensitive Bradford assay modifications by Sigma and Pierce, respectively, because unlike them it uses hydrophobic reagents (AS and TCA) to increase the binding of more CBB molecules per protein molecule. Acknowledgements This work was supported financially by the Greek Ministry of Education, University of Patras, Greece. K.G. thanks the Cultural Institute of Moral and Social Education, Athens, Greece, for supporting him financially. References 1. Bradford MM (1976) Anal Biochem 72: Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) J Biol Chem 193: Sedmak JJ, Grossberg ES (1977) Anal Biochem 79: Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Anal Biochem 150: Compton SJ, Jones CG (1985) Anal Biochem 151: Yvon M, Chabanet C, Pélissier JP (1989) Int J Pept Protein Res 34: Bramanti E, Ferri F, Sortino C, Onor M, Raspi G, Venturini M (2003) Biopolymers 69: Englard S, Seifter S (1990) In: Deutscher PM (ed) Guide to protein purification, vol 182. Academic, New York, pp Yaacobi M, Ben-Naim A (1973) J Solution Chem 2: Castronuovo G, Elia V, Moniello V, Velleca F, Perez-Casas S (1999) Phys Chem Chem Phys 1: Sigma-Aldrich (2004) Technical bulletin of Sigma Bradford assay (product no B6916). b6916bul.pdf. Accessed 9 Dec Sigma-Aldrich (2006) Technical bulletin of Sigma BCA assay (product no B9643). b9643bul.pdf. Accessed 9 Dec Pierce (2006) Pierce protein assay technical handbook. piercenet.com/files/ %20proteinassay.pdf. Accessed 9 Dec 2007