A

Because spectrophotometry-based total protein analyses produce calibration plots of Ax versus concentration (c), the slopes of the plots are either directly equal to molar absorption coefficients (if c is in units of molarity) or directly proportional to molar absorption coefficients (if other units are used for the horizontal axis). A well-characterized pure protein (e.g., bovine serum albumin, BSA) used for calibration should produce a calibration curve with a slope (absorption coefficient) that is reasonable based on the composition of the protein for the chromogen or prochromogen. BSA has close to 18 phenolic (tyrosine) groups per protein molecule. Calibration of a procedure such as the Folin assay that depends on phenolic side chains should respond at least semiquantitative^ to the protein's composition as well as its concentration variation. Folin assays for phenolic (and cresol) compounds produce e700 values in the range 7500 to 8500 M-1 cm-1; the Folin "color yield" for BSA should be within ±5% of the value for an equivalent number of cresol side chains.

Careful performance of total protein assays and recording of the results are helpful for two reasons. First of all, information from the calibration curve can be compared to known absorption coefficients for purified or partially purified proteins to evaluate the accuracy of the assay. Second, the results can be used as a guide in planning additional experiments to minimize the amount of sample protein required for analysis. Even approximate calculations of the amount of protein, numbers of tyrosine side chains in individual proteins, useful dilution ranges, or amounts of lyophilized protein to use can be helpful in the planning stages and initial benchwork.

It is not necessary for the horizontal axis to be plotted in units of molarity. However, it is desirable both for planning and for crosschecking results that units include a clear statement of volume so they can be readily converted to other units as needed for reporting and planning. One appropriate unit is |g or mg/ml of final assay volume (per ml f.a.v.), which specifies the concentration of the analyzed protein in the operational volume of a solution for spectrophotometric reading after the sample, reagents, and diluents have all been combined.

Table 3.4.2 lists the slopes of calibration curves, equivalent to absorption coefficients and assay sensitivities, for proteins and sugars in some commonly used assays. For some total protein assays, it is a good strategy to monitor sugars in parallel to evaluate removal of interfering compounds. Details for total sugar and reducing sugar assays are outlined in Lovrien et al. (1987) and references therein.

Proteins are frequently investigated and put into large-scale production and use before their molecular weights are known. However, their dry weights may be available. Hence the relationship between molar absorption coefficients (e^) and weight absorption coefficients (E1%) at a stated wavelength is useful for converting data using Equation 3.4.2. The molar absorption coefficient and the weight absorption coefficient are related to absorbance readings at

Table 3.4.2 Slopes of Calibration Plots for Spectrophotometry Assays"

Assay

Calibrating compound

Measured slope

Dinitrosalicylate (DNS)

Glucose

5.50 A575 (mg sugar/ml f.a.v.)-1 cm-1

Nelson-Somogyi reducing

Glucose

6.3 X 10-3 A520 (nmol glucose/ml f.a.v.)-1 cm-1

sugar

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