2University of Waikato, Private Bag 3105, Hamilton, New Zealand
Traditional colorimetric protein assays such as Biuret, Lowry, and modified Lowry (U-1988) are unsuitable for colored biological samples. Here we describe an improved Lowry protein assay (U-2012), which utilizes stable reagents and offers enhanced sensitivity over the U-1988 assay. U-2012 circumvents interference from colored pigments and other substances (for example sugars) bound to perchloric acid (PCA) precipitated proteins by hydrogen peroxide (H2O2) induced oxidation at 50°C. Unused hydrogen peroxide is neutralized with sodium pyruvate before protein estimation for a stable end color. The U-2012 assay is carried out on the PCA precipitated protein pellet after neutralization (with Na2CO3 plus NaOH), solubilization (in Triton-NaCl), decolorization (by H2O2) and pyruvate treatment. Protein contents in red wine and homogenates of beetroot and blueberry are calculated from standard curves established for various proteins and generated using a rectangular hyperbola with parameters estimated with Microsoft Excel's Solver add-in. The U-2012 protein assay represents an improvement over U-1988 and gives a more accurate estimation of protein content.
Quantitative measures of physiological traits such as enzyme activity are often expressed as units of activity per milligram protein. Although numerous assays have been developed to measure protein content, including the colorimetric assays of Amido Black (1), Biuret (2), Bicinchoninic Acid (3) and Coomassie Blue (4,5), the Lowry assay (6) or its modifications (7,8) are more commonly used than other assays (9). The Lowry assay is simple, sensitive and precise, and is the most cited (10) procedure for quantitative protein determination.
A wide variety of compounds that react with Folin-Ciocalteu phenol (Folin's) reagent (11) are a source of potential interference in Lowry and modified Lowry protein assays. Fortunately, corrections through an appropriate blank is sufficient for most compounds (6,7) except lipids (12), detergents (13) and colored substances (14). Difficulties in assaying proteins in presence of lipids and detergents (used in the solubilization of adipose tissue, myelin and skeletal muscles) were overcome by the modified Lowry assay (15; referred to in this paper as the U-1988 assay, 16). Color interference in determining the protein content in red wine (14,17,18) was overcome by employing extensive chromatography. The above approach is cumbersome and not very practical for handling large numbers of samples. None of the known protein assays were suitable for measuring proteins in colored biological samples e.g., colored fruits and vegetables, red wine, pigmented microbes and ruminant bile.
Our development of the U-2012 assay from its predecessors the U-1988 and the Lowry assay has achieved three major advantages (i) convenience through stability of the reagent formulations, (ii) measurement of protein in both colorless and colored biological samples without compromising the sensitivity, and (iii) assaying proteins at very low concentrations. This novel assay will be applicable to quantitative determination of protein in both colorless and colored biological sample homogenates, including those rich in lipids (e.g., avocado) and those difficult to homogenize.
Materials and methods
Biological samples – beetroot, blueberry and red wine
Beetroot and blueberry homogenates were prepared as described in the Supplementary Material. Red wine did not require protein extraction prior to the U-2012 assay.
All chemical reagents, except sodium hypochlorite and perchloric acid (PCA), were obtained from Sigma or Sigma-Aldrich (St Louis, MO. USA). Sodium hypochlorite was from Acros Organic, New Jersey, USA. PCA was obtained from BDH (England).
Improvements to U-1988 assay
Switching from carbonate to phosphate buffer at pH 12.0 improved reagent stability and gave a small increase in sensitivity. Acetonitrile was introduced to avoid detergent-induced bubbles. NaOH replaced KOH to avoid precipitation in the protein assay. In addition, efficiency was enhanced by combining various components of the Lowry reagent into one reagent mix.
The U-2012 assay
Full details of the U-2012 assay are provided in Supplementary Material. The protocol, briefly summarized in Figure 1, describes the processing of red wine and the homogenates of beetroot and blueberry, and includes the improvements to the U-1988 assay. The U-2012 assay was employed for unprocessed, processed [treated with either trichloroacetic acid (TCA) or PCA followed by hydrogen peroxide (H2O2)] and reverse processed (H2O2 treatment followed by TCA or PCA precipitation) proteins. Assays were carried out on BSA, carbonic anhydrase, cytochrome C, isocitrate dehydrogenase, lysozyme and trypsin for the development of standard curves and in colored biological samples. The determination of proteins in the biological samples was carried out by calibrating to appropriate standard curves.
Estimation of color interferences in the U-2012 assay
Color interference was determined by comparing absorbency from processed and unprocessed beetroot, blueberry and red wine samples both with and without the use of Folin's reagent as described in Figure 1. The ratio [(Abs1 – Abs2) / (Abs3 – Abs4)] was used to establish the extent of interference, where Abs1 is absorbance of unprocessed samples with Folin's reagent; Abs2 is absorbance of unprocessed samples without Folin's reagent; Abs3 is absorbance of processed samples with Folin's reagent; and Abs4 is absorbance of processed samples without Folin's reagent.
The standard curve and its parameters
Solution-1B and 1C described in the Recipes section of the attached Supplementary Material were used for the development of the standard curves. The concentration of BSA and the corresponding absorbance values were plotted using an X-Y scatter graph. The form of this graph (Figure 2) shows a saturating response at higher concentrations with a very limited initial linear response. This was a preferred curve form reported previously (20). Initially this was modeled using an exponential form (19) but later studies showed that a rectangular hyperbola gave an improved alignment with the response, particularly at lower concentrations. This latter form has now been standardized and the following three-parameter equation was used to describe the absorbance-protein concentration relationship: