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Measuring RNase Activity A Real-time Kinetic Analysis

Kinetics of RNaseAlert Cleavage by RNase A. Using conditions of substracte excess, the rate of increase in fluorescence (the slope of the line) was used to calculate the initial reaction rates for three concentrations of RNase A. The initial velocities were 0.005 RFU/min, 0.054 RFU/min, and 0.5 RFU/min for 0.5 pg, 5.0 pg and 50 pg Rnase A, respectively.


In the Kunitz assay, the linear rate of increase of A260 is proportional to the concentration of the enzyme in solution. The activity of the enzyme is expressed in terms of the slope of the plotted curve of absorbance versus time. Likewise, when tested with the RNaseAlert QC System, the early, linear phase of the reaction yields an increase in relative fluorescence units (RFU) that is proportional to the concentration of enzyme in solution. Therefore, the RNaseAlert QC System, when used in conjunction with a fluorometer capable of kinetic analysis, provides a rapid and ultra-sensitive assay for quantitative measurements of RNase A activity. Since this assay also detects a variety of other nucleuses (RNase T1, RNase I, micrococcal nuclease, S1 nuclease, mung bean nuclease, and Benzonase), it should also be useful for standardizing their specific activity.


References

  1. Kunitz, M (1950) Crystalline Deoxyribonuclease I. Isolation and general properties, spectrophotometric method for the measurement of desoxyribonuclease activity. J. Gen. Physiol. 33: 349-362.
  2. 2. Crook, E, Mathias, A Rabin, B (1960) Spectrophotometric assay of bovine pancreatic
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