How does the data collected on day 1 validate the assay?
Since we are using our ability to measure the activity of the enzyme to quantitate the concentration of enzyme, there must be a linear relationship between the measured activity and the quantity of enzyme present. Logically, if that is true, then one may expect that if the amount of enzyme present in the reaction is reduced by half, then the measured rate would also reduce by half. Likewise, if enyme is diluted 10-fold, the rate should decrease by the same factor. The following example illustrates the reasonable validity of our assay. Deviations from linearity are due to pipetting error at low enzyme volumes (more than the intended volume of enzyme was added) or, at high volumes, are due to the consumption of substrate becoming rate limiting. As the enzyme volume was decreased by a factor of 2, 5, 10, and 20, the rate decreased, respectively, by factors of 2, 4, 7, and 13.
