Conc = Protein concentration,
A = Absorbance at Conc,
A0 = Absorbance at zero concentration,
AM= Absorbance at max concentration,
C50= Concentration giving absorbance
(AM + A0)/2.
Parameter A0 was experimentally determined while AM and C50 were estimated using Microsoft Excel's Toolbox add-in Solver Function. A trial set of parameters was used to calculate the modeled absorbance at each of the standard concentrations (Conc) using equation . Solver was then commanded to minimize the residual standard deviation between the measured and modeled absorbance for the standard set by adjusting AM and C50.
We observed the relationship between absorbance and concentration to exhibit a non linear curve over the entire concentration range, which is likely to be due to a component of light scattering which increases as the concentration of protein increases when measuring absorbance. A poor linear fit at low absorbance was also reported by Coakley and James (20).
Calculation of protein content in the homogenates
Assays were carried out on processed and unprocessed red wine and homogenates of beetroot and blueberry. BSA and other protein samples were treated identically for appropriate standard curves to determine A0, AM and C50 values. These parameters were then used to convert sample absorbance (A) to protein concentration in each homogenate using:
Because the equation has a saturating form, the sensitivity reduces as absorbance (A), and hence concentration, increases. Errors in protein estimations may be minimized by adjusting the concentrations of homogenates in the assay so that they do not excessively exceed the C50 value.
The Homogenate Conc value was then converted to tissue protein concentration (Tissue Conc in mg/g of tissue) using the following formula:
where Homogenate Conc (in mg protein/mL) has been corrected for any pre-concentration or dilution during the assay. Homogenate percentage was 100 g of tissue homogenized to a total volume of 200 mL (in our case 50%).
In a separate study, a rectangular hyperbola model was fitted using the non linear mixed effects (NLME) package (21) in R (22) (Figure 2). Each BSA solution, made independently in the laboratory, was modeled as a random effect, with a common A0 but different AM and C50 coefficients. This models the hierarchy of biological sample replicates and technical assay replicates.
Results and discussion
Improvements in the U-1988 assay
The limitation of the U-1988 and the Lowry assay is the instability of the carbonate-based reagent. The carbonate buffer (pH 11.4 at 2% = 188.7 mM) in U-1988 was replaced with 40 mM phosphate at pH values ranging from 11.4 to 12.5. Initial slopes from the standard curves of the protein assay using BSA at 0.5 mg BSA/ mL and 1.0 mg BSA / mL were calculated. The initial slopes with phosphate buffers at pH 11.4 and at its optimal pH 12.0 were 99 x10-6 and 197 x10-6respectively. The slope for the carbonate buffer (pH 11.4) was 162 x10-6.Since the slope value is a direct indication of assay sensitivity, phosphate buffer (pH 12.0) was chosen to replace carbonate buffer, giving a 25% increase in sensitivity.
Greater stability was achieved by increasing concentration of the phosphate buffer to 100 mM. The resulting phosphate/CuSO4/Na-K-tartrate solution was stable at room temperature for two weeks, considerably longer than the carbonate/CuSO4/Na-K-tartrate solution, which must be prepared daily before protein assay. For all future experiments, 100 mM phosphate (pH 12.0) was used to prepare the CuSO4/Na-K-tartrate solution. We believe that replacement of carbonate with phosphate will enhance the convenience of the U-2012 assay.
Detergent induced bubbles become a major source of error in absorbance measurements when using a multi-well plate reader (not an issue with cuvettes). These bubbles were reduced considerably by the addition of a number of polar solvents (e.g., acetone, acetonitrile, ethanol and methanol). Acetonitrile, the most polar of these solvents (23) was chosen for its effectiveness and included in Solution-2 (see Figure 1 caption and Recipes section of Supplementary Material).
Phosphate buffer, CuSO4, Na-K-tartrate, SDS and acetonitrile can be added individually and the order of their addition does not affect the resulting absorbance. However, using a premixed solution further enhances convenience, especially when large numbers of samples are to be assayed. We therefore grouped these components of the assay mix into Solution-2 (Figure 1). Such a premixed solution was not feasible for the original Lowry assay (6) due to instability of the carbonate solution. The attempt to include Solution-3 in Solution-2 resulted in dramatic reduction in the development of blue color and was not considered further.
Protein estimation in colored biological samples
Proteins from beetroot and blueberry were extracted in Triton X-100-NaCl solution with mild homogenization. Such homogenates retain their enzyme activities (15). This extraction was not required for red wine.
Eliminate interfering substances
For colored samples it is necessary to remove the interference due to the inherent sample color and other non-protein substances that react with the protein reagents before colorimetric protein assay. The novelty of U-2012 is in devising a decolorizing protocol compatible with a colorimetric protein assay.
Decolorization of colored pigments by sodium hypochlorite or H2O2 and selective precipitation of proteins by PCA or TCA were considered for removal of interfering substances. Sodium hypochlorite, H2O2, TCA and PCA were evaluated for their compatibility with the U-2012 assay using BSA as the test protein. Between sodium hypochlorite and H2O2, only H2O2 was compatible as a precipitate was formed in the presence of hypochlorite. Proteins precipitated by TCA or PCA can be assayed by U-2012 after adequate neutralization of residual acid in the pellet. The superiority of PCA over TCA for protein precipitation has been reported (24,25). In contrast, in our comparative evaluation, revealed similar C50 values for PCA (1.395) and TCA (1.400). We preferred PCA because it is readily available as a pre-made solution (70% v/v) and therefore easily diluted to the required strength. TCA is a hygroscopic solid that is difficult to weigh precisely due to its variable water content.
There are two possible ways of combining PCA and H2O2. For “processed” proteins PCA treatment was followed by H2O2 treatment and for “reverse-processed” protein, H2O2 treatment preceded PCA precipitation. Advantages of using ‘processed’ protein were the removal of a number of interfering substances in the supernatant and the possible inactivation of proteolytic enzymes during sample preparation. This was confirmed by assaying processed and reverse-processed trypsin and BSA (see Table 1). Only processed samples were used to determine the actual protein content of colored biological samples