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U-2012: An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity
 
Girish C. Upreti1, Yanming Wang1, Alona Finn1, Abigail Sharrock1,2, Nicholas Feisst1, Marcus Davy1, and Robert B. Jordan1
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Supplementary Material


Table 1: Three point parameters of various protein standard curves. (Click to enlarge)


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 [4] 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 [2] and [3] 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 [2] and [3].

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.

Acknowledgments

Authors acknowledge the Foundation for Research, Science and Technology New Zealand for financial support (C06X0809).

Competing interests

The authors declare no competing interests.

Correspondence
Address correspondence to Girish Upreti, Plant & Food Research Ruakura, Private Bag 3230, Hamilton, 3240, New Zealand. Email: [email protected]

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