Some biochemistry courses feature a laboratory project derived from Sohl and Splittgerber’s paper (1) on the binding of Coomassie Blue to bovine serum albumin. A version of this project was also published as experiment 5 in Modern Experimental Biochemistry (2). The original paper has a few errors that bear correcting. First, the dye concentration used in Figure 3 (p 264) is actually 60 µM, not 6 µM as stated in the text. Thus the reported values for n and K_b are a factor of 10 too low.

Second, the authors’ interpretation of the thermodynamic parameters for the two types of binding sites, loose versus tight, is incorrect. In commenting on the results plotted in Figure 3 on page 264, the authors state (correctly) that at low protein concentrations (i.e., excess dye), both tight and loose binding sites are occupied. “In this region [of protein concentration], the straight line… represents binding to both primary (tight) and secondary (loose) classes of binding site.” However, the changes in absorbance in this region are due to binding changes at the loose sites only, since the tight sites remain completely saturated. Hence the thermodynamic parameters derived from the straight line in Figure 3, n and K_d, represent the loose class of sites alone, and not the sum (or average) of both types of sites.

Finally, the analysis of the y intercept for the loose binding at low protein concentrations is flawed. The authors state that “because the minimum value of the function 1/(1-Y) is 1, rather than 0, the Y intercept of the plot is found at an x value of 1” (1). It does not follow that the y value at x = 1 can be arbitrarily defined as the y intercept simply because
x = 1 is the lowest value “allowed” for this function. The y intercept is still the y value when x = 0, even though this x value is one that is not attainable experimentally. The fact that “the linear section of the plot at low protein levels has a negative intercept, rendering the data meaningless” is noted by the authors. What we must conclude from this is not that the y intercept should be found at some value other than
x = 0, but that there is some problem with either the experimental system or the theoretical data analysis framework in this domain of low protein concentration.

Literature Cited

1. Sohl, J. L.; Splittgerber, A. G. J. Chem. Educ. 1991, 68, 262-264.
2. Boyer, R. F. Modern Experimental Biochemistry, 2nd ed.; Benjamin-Cummings: Redwood City, CA, 1993; p 285.