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Measuring RNase Activity A Real-time Kinetic Analysis

he manufacturer's unit definition as well as the assay conditions to determine how an enzyme will function in your specific application.

One Kunitz unit (K) produces a change in A260 of 0.001/min/ml at pH 5.0, 25C or 1 unit hydrolyzes a substrate at a rate constant of K = 1 at pH 5.0, 25C.

Here we report the steady-state kinetic analysis of RNase A activity using the RNaseAlert QC System. RNase A (700 U/mg, 10 ng/ml) was serially diluted and incubated with a large excess of substrate such that the amount of input enzyme was proportional to the rate of the reaction. A baseline was established by monitoring substrate alone in RNaseAlert Buffer. After several minutes, experimental samples containing 0.5 pg, 5.0 pg and 50 pg of RNase A were added. The rate of increase in fluorescence was measured in real-time at 37C in 2 minute increments for 1 hour. Figure 2 shows reaction curves plotted for 3 concentrations of enzyme: 50 pg, 5.0 pg, and 0.5 pg. After enzyme addition, the rate of increase in fluorescence was constant for several minutes. Under these conditions, the reaction is at equilibrium and the rate of increase in fluorescence (the slope of the line) can be used to calculate the initial rate (or velocity) of the reaction. As expected, when initial velocities were compared (see legend, Figure 2), there was a 10-fold difference between each 10-fold serial dilution. These data demonstrate that the dilutions maintain the mathematical proportionality predicted for steady-state kinetic analysis.

Figure 2.
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