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Measuring RNase Activity — A Real-time Kinetic Analysis
The RNaseAlert® QC System, when used in conjunction with a fluorometer capable of kinetic analysis, provides a rapid and ultra-sensitive assay for determining RNase A specific activity. The RNaseAlert QC System also detects a variety of other nucleases, including RNase A, T1, RNase I, micrococcal nuclease, S1 nuclease, mung bean nuclease, and Benzonase®. In addition to detecting nuclease activity, this kit is useful for standardizing activities of these enzymes.
Sources of RNase
Ribonucleases (RNases) are ubiquitous in the environment, and they are present in relatively high concentrations in many biological materials. For example, the pancreas is rich in RNase (>1 mg RNase/1 g tissue) and is the source for most commercially produced RNase A (and its glycosylated derivative, RNase B).
RNase A is an endonuclease that cleaves 3' of C and U residues. It is commonly used in molecular biology applications such as the removal of contaminating RNA from DNA preparations and ribonuclease protection assays (RPA). Ambion sells an RPA grade RNase A (Cat # 2272) that is equivalent to molecular biology grade RNase A from other suppliers, as well as affinity-purified RNase A (Cat # 2270 and # 2271), which is recommended when the absence of DNase and other nonspecific nuclease activities is essential.
RNase A is an endonuclease that cleaves 3' of C and U residues. It is commonly used in molecular biology applications such as the removal of contaminating RNA from DNA preparations and ribonuclease protection assays (RPA). Ambion sells an RPA grade RNase A (Cat # 2272) that is equivalent to molecular biology grade RNase A from other suppliers, as well as affinity-purified RNase A (Cat # 2270 and # 2271), which is recommended when the absence of DNase and other nonspecific nuclease activities is essential.
Measuring Specific Activity
Recently, Ambion introduced the RNaseAlert® QC System, a quantitative fluorometric high-throughput assay for the detection of contaminating RNases. The assay consists of an oligonucleotide substrate that contains a fluorophore on one end and a quencher on the other. Once cleaved by RNase, the fluor and quencher are separated. When excited with 490 nm light, the fluor emits a bright green signal that can be quantitated with a fluorometer (Figure 1).
Figure 1. Schematic of the RNaseAlert® QC System.
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 37°C 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. 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.
Figure 1. Schematic of the RNaseAlert® QC System.
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 37°C 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. 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
- Kunitz, M (1950) Crystalline Deoxyribonuclease I. Isolation and general properties, spectrophotometric method for the measurement of desoxyribonuclease activity. J. Gen. Physiol. 33: 349-362.
- Crook, E, Mathias, A Rabin, B (1960) Spectrophotometric assay of bovine pancreatic ribonuclease by the use of cytidine 2',3'-phosphate. Biochem. J. 74: 234.
Are Your Enzyme Units Uniform?During routine protein purification some proportion of the enzyme population is inevitably inactivated. Therefore, the proportion of active enzyme is routinely quantitated by measuring the specific activity, the amount of active enzyme per mg of the total protein. In 1950, M. Kunitz developed the first spectrophotometric assay for measuring DNase I activity. The assay was based on the increase in A260 at pH 5.0 and 25°C caused by the degradation of a DNA substrate. Kunitz also recognized that the rate of increase of A260 under the proper conditions, is both linear and constant and that the linear rates were proportional to the concentration of the active enzyme.
The Kunitz unit has since become the standard unit of measurement for enzymatic activity. However, the original Kunitz assay was performed under conditions that are not optimal for many enzymes, so most manufacturers have developed their own unit assays. Therefore, it is important to look closely at both the 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, 25°C or 1 unit hydrolyzes a substrate at a rate constant of K = 1 at pH 5.0, 25°C.
The Kunitz unit has since become the standard unit of measurement for enzymatic activity. However, the original Kunitz assay was performed under conditions that are not optimal for many enzymes, so most manufacturers have developed their own unit assays. Therefore, it is important to look closely at both the 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, 25°C or 1 unit hydrolyzes a substrate at a rate constant of K = 1 at pH 5.0, 25°C.