Both PCA and H2O2 treatments of colored samples were necessary for interference elimination in U-2012 assay. Acid precipitation alone of the colored samples did not remove interference completely. With all the colored samples, some color was discarded in the supernatant, but the pellets were also colored. Color was eliminated from the pellets by H2O2 treatment. Alkaline conditions were required for both effective decolorization by H2O2 (26) and the color development by Folin's reagent to ensure that protein levels are measured correctly. Although both NaOH and KOH could provide the required alkalinity, only NaOH was compatible with the U-2012 assay. A precipitate was formed in the presence of KOH. In pellets, the PCA was neutralized using Na2CO3 and NaOH (27). Additional NaOH was added during the assay; the optimized volume was between 50 to 70 µL (60 µL was routinely used); see Figure 1.
Beetroot, blueberry, and red wine were decolorized with 15 µL of 30% H2O2 taking 0.5 and 2 h at 50°C and room temperature, respectively. Twenty microliters of 30% H2O2 for 1 h at 50°C was used to cope with stronger colored samples. Oxidation of substances like sugars bound to proteins by H2O2 at 50°C seems critical as room temperature processing overestimates protein content. In the case of beetroot, 50°C processing reduced the apparent protein estimate to 14% of the unprocessed, whereas room temperature processing only halved that estimate.
It was evident from the colorimetric assays carried out after hydrogen peroxide treatment that some H2O2 was not utilized in the decolorization. In such a sample, the end color of the Lowry assay was partially destroyed. It was therefore necessary to destroy the remaining hydrogen peroxide before protein assay. There are two sources of H2O2 in the U-2012 assay; H2O2 added for decolorization and H2O2 present as a contaminant in the Triton X-100 (0.22%, Product information: Triton X-100, www.sigmaaldrich.com). Hydrogen peroxide is commonly degraded by the enzyme catalase. However, the high pH of the protein assay would inactivate known catalases. Also, adding catalase would lead to the addition of extra protein. We chose chemical destruction of H2O2 using pyruvate (28). The chemistry of the pyruvate-H2O2 interaction equation  is well established (28,29). Pyruvate destroys H2O2 at room temperature according to the following reaction:
Residual H2O2 in the pellet suspension was destroyed by treating with 0.9 M pyruvate (1.5x concentration of H2O2) for 0.5 h at room temperature. To counteract the contaminating H2O2 in Triton X-100, extra pyruvate was also added in the protein assay (Figure 1). The addition of pyruvate gave lower absorbance for a non-protein blank [0.089 (with pyruvate) versus 0.104 (without pyruvate)]. We suggest that the peroxide contaminant in Triton X-100 reacts with the acetonitrile in Solution-2, giving slightly higher absorbency.
The color interference associated with the colored biological samples cannot simply be taken into account by running a protein assay in the absence of the Folin's reagent. The calculated ratios (Abs1-Abs2)/(Abs3-Abs4) indicated that interference from sample color was the highest for red wine (= 40) and less for blueberry (= 6) and beetroot (= 2). This interference translated into the abnormally high estimates of true protein levels; for example, the concentration of protein using unprocessed and processed beetroot homogenates (20.21 versus 2.89 mg protein / g tissue, respectively). In addition to color interference, red wine and homogenates of beetroot and blueberry are likely to contain substances that will react with Folin's reagent in the U-2012 assay (e.g., small peptides and complex sugars). These were removed by selectively precipitating proteins with ice-cold PCA at a final concentration of 5% (Figure 1).
Standard curves and their parameters
Standard curves for unprocessed and processed BSA are shown in Figure 2. The derived parameters (A0, AM and C50) are also listed in Table 1 for BSA and other proteins.
The results show that the residual standard error in the model is low (0.012 to 0.048) indicating the better fit of the data to the rectangular hyperbola trend. For comparing information between various proteins and their processing, the parameters were converted to the concentration for absorbance = 1.0 at 650 nm (right column in Table 1).
These results show that loss of protein (compared with unprocessed protein) in processed samples was less than the reverse-processed samples. This loss was more apparent in the case of trypsin and it can be explained on the basis of its auto-catalytic activity during the reverse-processing. We recommend that the ‘processed’ protocol (Supplementary Material) should only be followed for biological samples that are likely to contain proteolytic enzymes.
In the original Lowry assay (6) and its modified version U-1988 (15), only the linear portion of the standard curve obtained by plotting the absorbance against the amount of protein was used in the quantitative determination of protein. In the U-2012 assay we use the data more effectively by fitting a rectangular hyperbola equation as described in the Materials and methods section in line with Coakley and James (20).
Protein content of colored homogenate
Protein concentrations in unknown samples were calculated by equation  and  against processed BSA standard and the average of all processed proteins listed in Table 1. The latter will be closer to a true estimate for biological samples that contain a mixture of proteins. We estimated the amounts of protein in blueberry and beetroot relative to red wine as approximately 60 and 230-fold respectively (Table 2).
Like BSA, red wine and 50% homogenates of beetroot and blueberry were processed by PCA precipitation and decolorization by H2O2 (Figure 1). At this stage the biological samples were concentrated 40 times for red wine and 4 times for beetroot and blueberry. Similarly BSA (2 mg/mL) was also concentrated 4 times to 8 mg/mL. The absorbance of the colored samples that was close to the absorbance for C50 (for unprocessed BSA) was used to calculate the protein content, as described by equation  and .
In conclusion, the U-2012 assay has employed stable reagents, provided improved sensitivity (even for colorless biological samples) and overcome color-induced interference for colored biological samples. The U-2012 assay is not constrained to the linear portion of the response between protein concentration and absorbance and makes more efficient use of data in the nonlinear region through a rectangular hyperbolic curve model fitted to the standards using simple procedures within Microsoft Excel.
Authors acknowledge the Foundation for Research, Science and Technology New Zealand for financial support (C06X0809).
The authors declare no competing interests.
Address correspondence to Girish Upreti, Plant & Food Research Ruakura, Private Bag 3230, Hamilton, 3240, New Zealand. Email: [email protected]
1.) Kaplan, R.S., and P.L. Pedersen. 1985. Determination of microgram quantities of protein in the presence of milligram levels of lipid with amido black 10B. Anal. Biochem. 150:97-104.[CrossRef] [PubMed] 2.) Gornall, A.G., C.J. Bardawill, and M.M. David. 1949. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751-766.[CrossRef] [PubMed] 3.) Smith, P.K., R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85.[CrossRef] [PubMed] 4.) Bradford, M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef] [PubMed] 5.) Zor, T., and Z. Selinger. 1996. Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal. Biochem. 236:302-308.[CrossRef] [PubMed] 6.) Lowry, O.H., N.J. Rosbrough, A.L. Farr, and R.J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.[CrossRef] [PubMed] 7.) Peterson, G.L. 1979. Review of the folin phenol protein quantification method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem. 100:201-220.[CrossRef] [PubMed] 8.) Sapan, C.V., R.L. Lundablad, and N.C. Price. 1999. Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 29:99-108.[CrossRef] [PubMed] 9.) Okutucu, B., A. Dınçer, Ö. Habib, and F. Zıhnıoglu. 2007. Comparison of five methods for determination of total plasma protein concentration. J. Biochem. Biophys. Methods 70:709-711.[CrossRef] [PubMed] 10.) Kresge, N., R.D. Simoni, and R.L. Hill. 2005. The most highly cited paper in publishing history: protein determination by Oliver H. Lowry. J. Biol. Chem. 25:280.[CrossRef] 11.) Everette, J.D., Q.M. Bryant, A.M. Green, Y.A. Abbey, G.W. Wangila, and R.B. Walker. 2010. Thorough study of reactivity of various compound classes toward the Folin-Ciocalteu Reagent. J. Agric. Food Chem. 58:8139-8144.[CrossRef] [PubMed] 12.) Eichberg, J., and L.C. Mokrasch. 1969. Interference by oxidized lipids in the determination of protein by the Lowry procedure. Anal. Biochem. 30:386-390.[CrossRef] [PubMed] 13.) Dulley, J.R., and P.A. Grieve. 1975. A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Anal. Biochem. 64:136-141.[CrossRef] [PubMed] 14.) Brillouet, J.-M., M.-P. Belleville, and M. Moutounet. 1991. Possible protein-polysaccharide complexes in red wines. Am. J. Enol. Vitic. 42:150-152.[CrossRef] 15.) Upreti, G.C., R.A. Ratcliff, and P.C. Riches. 1988. Protein estimation in tissues containing high levels of lipid: modifications to Lowry method of protein determination. Anal. Biochem. 168:421-427.[CrossRef] [PubMed] 16.) Upreti, G.C., C. Davis, and J. Oliver. 1991. Preparation of representative homogenates of biological tissues: effect of salt on protein extraction. Anal. Biochem. 198:298-301.[CrossRef] [PubMed] 17.) Smith, M.R., M.H. Penner, S.E. Bennett, and A.T. Bakalinsky. 2011. Quantitative Colorimetric Assay for Total Protein Applied to the Red Wine Pinot Noir. J. Agric. Food Chem. 59:6871-6876.[CrossRef] [PubMed] 18.) Wigand, P., S. Tenzer, H. Schild, and H. Decker. 2009. Analysis of Protein Composition of Red Wine in Comparison with Rosé and White Wines by Electrophoresis and High-Pressure Liquid Chromatography-Mass Spectrometry (HPLC-MS). J. Agric. Food Chem. 57:4328-4333.[CrossRef] [PubMed] 19.) Upreti, G.C., Y. Wang, A. Sharrock, N. Feisst, M. Davy, and B. Jordan. 2009. A stable and sensitive protein assay (U-2009 modified assay) for colored biological samples. ComBiol., New Zealand Final Programme December 2009. University of Canterbury, Christchurch.[CrossRef] [PubMed] 20.) Coakley, W.T., and C.J. James. 1978. A simple linear transform for the Folin-Lowry protein calibration curve to 1.0 mg/mL. Anal. Biochem. 85:90-97.[CrossRef] [PubMed] 21.) Pinheiro, J.C., and D.M. Bates. 2000. Mixed-Effects Models in S and S-PLUS, Statistics and Computing Series. Springer-Verlag, New York, NY.[CrossRef] [PubMed] 22.) R Development Core Team 2009. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria ISBN 3-900051-07-0, URLhttp://www.R-project.org..[CrossRef] [PubMed] 23.) Khachik, F., G.R. Beecher, J.T. Vanderslice, and G. Furrow. 1988. Liquid chromatographic artifacts and peak distortion: Sample-solvent interaction in the separation of carotenoides. Anal. Chem. 60:807-811.[CrossRef] [PubMed] 24.) Cernik, A.A. 1970. Determination of lead chelated with ethylenediaminetetra-acetic acid in blood after precipitation of protein with perchloric acid. Brit. J. Industry Med. 27:40-42.[CrossRef] 25.) Moughan, P.J., A.J. Darragh, W.C. Smith, and C.A. Butts. 1990. Perchloric and trichloroacetic acids as precipitants of protein in endogenous ileal digesta from the rat. J. Sci. Food Agric. 52:13-21.[CrossRef] 26.) Galbács, Z.M., and L.J. Csányi. 1983. Alkali-induced decomposition of hydrogen peroxide. J. Chem. Soc., Dalton Trans. 11:2353-2357.[CrossRef] [PubMed] 27.) Scopes, R.K. 1988. Protein Purification: Principles and Practice, Second Ed. Springer-Verlag New York Inc., New York, NY.[CrossRef] [PubMed] 28.) Upreti, G.C., K. Jensen, R. Munday, D.M. Duganzich, R. Vishwanath, and J.F. Smith. 1998. Studies on aromatic amino acid oxidase activity in ram spermatozoa: role of pyruvate as an antioxidant. Anim. Reprod. Sci. 51:275-287.[CrossRef] [PubMed] 29.) Holleman, M.A.F. 1904. Notice sur l'action de l'eau oxygenee sur les acids α-cetoniques et sur les dicetones 1.2. Recl. Trav. Chim. Pays-Bas Belg. 23:169-172.[CrossRef]