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Many biochemistry courses incorporate a laboratory project published as experiment 3 in Modern Experimental Biochemistry (1). This is a fine lab that combines equilibrium binding analyses with gel filtration chromatography. Students mix the dye phenol red with the protein bovine serum albumin (BSA), and separate free and bound dye chromatographically. The protocol calls for alkalinization of eluted dye in order to increase absorbance. However, because the maximum molar absorptivity of the pink alkaline form (ε557 = 41,000 M–1 cm–1) is less than three times greater than that for the yellow acidic form (ε430 = 15,000M–1cm–1, this is not really necessary. Omitting the alkalinization step makes the procedure easier, faster, and safer. If you do choose to alkalinize, note that the λmax for deprotonated phenol red is 557 nm. Absorbance measurements should therefore be made around 560 nm, and not at 520 nm, as specified by Boyer (pp 252–253). In analyzing the data, Boyer recommends that students calculate the fraction of bound dye using the areas of the two eluted peaks in the absorbance versus fraction number plot (bound dye elutes first, then free dye). This will only work if the dye’s absorbance is identical for both the bound and the free form. It turns out that this is not the case. I titrated 25 nmol of dye with different amounts of protein, up to about 45 nmol. Although the wavelength of maximum absorbance remained unchanged at 560 nm, the absorbance at λmax decreased from 1.06 to 0.79. This means that binding to the protein quenches dye absorbance, and bound dye has a lower molar absorptivity than free dye. The solution to this problem could be fairly straightforward. If students do a standard curve with different concentrations of dye in buffer, they can determine the molar absorptivity of the free dye in buffer. Then they can use this value along with the absorbance and volume of each fraction containing free dye to determine the moles of free dye. Subtracting this from the total dye added initially gives the amount of dye bound to protein. The ratio of bound dye molecules to total protein, [L·M]eq/[M]tot = ν (or r in some texts) can then be calculated, and plotted against [L]free on the x axis to give the hyperbolic saturation curve. Two typographical errors in Boyer’s theoretical discussion preceding the phenol red—BSA lab protocol are worth noting. On page 247, after Equation E3.7, the text should read “To further simplify Equation E3.7, let the term  ⁄ (n – ) represent the ratio…,” and not ⁄ n – ν. In the equation that follows this sentence, the final term on the right should be (Kf [L]+1)/n, and not (Kf [L]+1)/ν. Literature Cited- Boyer, R. F. Modern Experimental Biochemistry, 3rd ed.; Benjamin-Cummings: San Francisco, CA, 2000; p 243.
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