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A simple qualitative method for the detection of nucleases
ANALYTICAL
BIOCHEMISTRY
A Simple
Department
108,
320-324 (1980)
Qualitative
Method
WILLIAM
F. BURKE,
of Botany
and Microbiology,
for the Detection
JR.,’ AND BARBARA Arizona
State
University,
of Nucleases
S. SLINKER Tempe,
Arizona
85281
Received March 3, 1980 A simple and specific qualitative assay was developed for nucleases which degrade nucleic acids to nucleotides or small oligonucleotides. Reaction mixtures containing ethidium bromide complexed with nucleic acid lost fluorescence as the result of nucleolytic activity. The assay is adaptable for use with both deoxyribonucleases and ribonucleases, and is especially useful in detecting nuclease activity in column effluents.
Most procedures used for assaying deoxyribonuclease or ribonuclease activity involve the measurement of formation of acid-soluble products from radioactively labeled nucleic acids, decrease in viscosity of the substrate (l), or increase in absorbance at 260 nm (2). Although extensively used, these assays have serious disadvantages in that they can be time consuming and expensive. The inconveniences of these detection systems are especially noticeable when the number of samples to be assayed is great and the number of samples expected to have nuclease activity is small, as in the case of column chromatography of nuclease-containing extracts. Therefore, we have developed a simple assay based on the observation that the fluorescence of the dye ethidium bromide increases as it complexes with nucleic acid (3). When the nucleic acid is hydrolyzed by a nuclease, the ethidium bromide-nucleic acid complex is destroyed, and the fluorescence is decreased. For convenience this assay technique will be referred to as the microtiter nuclease assay. 1 Author addressed.
to whom
correspondence
0003-2697/80/160320-05$02.00/O Copyright &r 1980 by Academic Press. Inc. AU rights of reproduction in any form reserved.
should
be
320
MATERIALS
AND METHODS
Enzymes
Pancreatic DNase I and RNase A were obtained from Sigma. Calcium-dependent nucleases from Bacillus subtilis 168 were prepared using the following technique. Bacteria were grown in 200-ml cultures at 37°C for 18 h in the nutrient broth sporulation medium of Leighton and Doi (4). Following incubation, the cells were harvested by centrifugation at 10,OOOg and 4°C for 15 min. The supernatant fluid was then brought to 40% saturation by the addition of solid ammonium sulfate. After 4 h at 4°C the suspension was centrifuged at 10,OOOg for 15 min at 4°C. The resultant supernatant fluid was brought to 70% ammonium sulfate saturation. The precipitate was collected by centrifugation at 10,OOOg for 15 min at 4°C after incubation for 4 h at 4°C. The pellet was dissolved in 2 ml of 10 mM Tris-HCI, pH 7.5, and 0.02% sodium azide (Buffer A). A l-ml aliquot of the enzyme solution was then applied to a 2.6 x 90-cm Sephadex G150 column equilibrated with Buffer A. The protein was eluted with the same buffer at a flow rate of 28 ml/h at room temperature. Fractions of 6.3 ml were collected for use
DETECTION
with the microtiter and stored at 4°C.
nuclease assay system
Enzymatic Assays The B. subtilis nucleases were assayed by the method of Burke and Spizizen (5) using [3H]DNA as substrate. A unit of B. subtilis nuclease activity is defined as the amount of enzyme catalyzing the formation of 10 nmol of acid-soluble nucleotides at 37°C in 30 min. DNase I and RNase A activity were determined as described by Kunitz (2). The microtiter nuclease assay mixture employed with the B. subtilis nucleases con(pH 8.0), 10 mM tained 0.1 M Tris-HCl CaCl,, 0.8 mg/ml DNA (Difco), and 0.15 &ml ethidium bromide (Sigma). Alternatively, acridine orange (Eastman) was substituted for ethidium bromide in the reaction mixture also at a concentration of 0.15 pg./ml. The reaction mixture utilized for pancreatic DNase I contained 0.25 mM MgSO, in addi-
OF NUCLEASES
321
tion to the other components. Reaction mixtures were stable for weeks at 4°C when protected from the light. The DNA was prepared as a stock solution of 5 mg/ml in water, sterilized by autoclaving, and stored at 4°C. The ribonuclease reaction mixture was (pH 8.0), 10 composed of 0.1 M Tris-HCl mM CaCl,, 1.6 mg/ml RNA (type VI from Torula yeast, Sigma), and 0.15 E.Lg/mlethidium bromide. The RNA was prepared as a stock solution of 10 mg/ml in water and stored at -20°C. The assay was performed by transferring loo-p1 aliquots of the appropriate reaction mixture to the wells of polyvinyl chloride microtitration plates (Dynatech Laboratories, Inc.). To each well, the sample to be assayed and sufficient sterile water were added to bring the total volume in the well to 200 ~1. The plate was covered securely with Parafilm and incubated at 40°C. Periodically, the plates were examined using either short-wave (254 nm) or long-wave
FIG. 1. Effect of DNase I concentration on fluorescence of microtiter nuclease reaction mixture. Each well contains 100 ~1 of enzyme solution or sterile water and 100 ~1 of reaction mixture. From left to right the wells contain sterile water, 50 units DNase I, 10 units DNase I, 5 units DNase I, 1 unit DNase I, and heat-inactivated sample (originally containing 50 units of DNase I). The microtiter plate was incubated at 40°C for 24 h.
322
BURKE AND SLINKER
FIG. 2. Specificity of the assay. Row A contained DNase reaction mixture and row B contained RNase reaction mixture. In addition to reaction mixture, from left to right the wells contained sterile DNase, 10 units pancreatic RNase A, and sterile water. The microtiter plate water, 10 unitsB. subtilis was incubated at 40°C for 2 h.
(366 nm) ultraviolet light. The plates were placed on a long-wave uv light (UVL-56, Ultraviolet Products, Inc.) and photographed with a Polaroid MP3 system, using type 55P/N film through combined Kodak gel filters 23A and 8.
at which fluorescence was lost was proportional to the quantity of the enzyme present, the position of peak fractions of nuclease activity could be determined in eluates of gel filtration and ion-exchange columns simply by periodic examination of the plates. While the enzyme cannot be quantified with this method, activities of different samples RESULTS AND DISCUSSION can be compared on a relative basis. HeatWhen IOO-~1 aliquots of DNase I solu- inactivated DNase I (lOO°C, 15 min) protions containing from 1 to 50 units of enzyme duced no loss of fluorescence. As little as activity were added to lOO-~1 aliquots of 1 unit of DNase I or 0.5 units of B. subtilis microtiter nuclease reaction mixture, a de- exonuclease could be detected using this crease in fluorescence of the reaction mixtechnique. ture was observed in all DNase containing The assay can also be adapted for RNase wells after 24 h (Fig. 1). The nuclease con- detection by replacing the DNA in the reaction mixture with RNA. The specificity of taining wells could also be identified when viewed with white light. The color of the re- the reaction was demonstrated using purified action mixture changed from a deep pink to a B. subtilis DNase and pancreatic RNase A. light orange as the DNA was degraded. Wells No detectable decrease in fluorescence was containing 50 units of DNase I lost fluoresobserved when RNase A was added to the cence within 15-30 min, while wells with 1 DNase reaction mixture or when B. subtifis unit of the enzyme exhibited loss of fluoresDNase was added to the RNase reaction cence only after 16-24 h. Because the rate mixture as shown in Fig. 2.
DETECTION
Another intercalating dye, acridine orange, can be substituted for ethidium bromide in the reaction mixtures. However, the reduction in fluorescence of the reaction mixture following enzymatic hydrolysis was less pronounced. Reducing the concentration of DNA or RNA resulted in significant decreases in fluorescence of the system. Although the assay was still functional, the detection of nuclease containing samples
w
323
OF NUCLEASES
was more difficult. In order to detect enzymes which are inhibited by ethidium bromide, the dye may be added after a fixed incubation period. The utility and precision of this assay method are clearly demonstrated in Fig. 3. A partially purified B. subtilis exonuclease preparation was chromatographed on a Sephadex G-150 column, and the resulting fractions were assayed both with the micro-
50
35 Fraction
Number
FIG. 3. Correlation of microtiter nuclease assay to [3H]DNA quantitative assay. Partially purified B. subfilis extract was chromatographed on a Sephadex G-150 column as described under Materials and Methods. The resulting fractions were subjected to the [3H]DNA assay system. The elution profiles of the nucleases present in the extract are shown in the graph. The microtiter nuclease assay of the fractions is shown in the photograph of the microtiter plate following 14 h of incubation at 40°C. Well A contained 100 ~1 of reaction mixture and a 100~~1 aliquot of fraction 10; subsequent wells contained samples from consecutive fractions. Well B, the positive control, contained 100 ~1 of reaction mixture and 100 ~1 of cell-free B. subtilis culture fluid. Well C, the negative control, contained sterile water and reaction mixture.
324
BURKE
AND SLINKER
titer assay and the quantitative [3H]DNA nuclease assay system. The positions of the two peaks of DNase activity determined with the microtiter n&ease assay were confirmed using the sensitive isotope assay system. We have utilized this microtiter assay system extensively with gel filtration and ionexchange chromatography to locate and identify fractions containing nuclease activity. The method can also be used for detecting nuclease activity in crude cell lysates and culture supernatants and as a technique for screening extracts of presumptive mutant cells.
ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health Grant GM 25343 and by the Faculty Grant-In-Aid Program, Arizona State University.
REFERENCES 1. Smith, H. O., and Wilcox, K. W. (1970) J. Mol. Biol.
51, 379-391.
2. Kunitz, M. (1950) J. Gen Physiol. 33, 363-377. 3. Le Pecq, J. B., and Paoletti, C. (1966) Anal. Biothem. 17, 100-107. 4. Leighton, T. J., and Doi, R. H. (1971) J. Biol. Chem.
Z&,3189-3195.
5. Burke, W. F., Jr., and Spizizen, J. (1977) Biochemistry 16, 403-410.
BIOCHEMISTRY
A Simple
Department
108,
320-324 (1980)
Qualitative
Method
WILLIAM
F. BURKE,
of Botany
and Microbiology,
for the Detection
JR.,’ AND BARBARA Arizona
State
University,
of Nucleases
S. SLINKER Tempe,
Arizona
85281
Received March 3, 1980 A simple and specific qualitative assay was developed for nucleases which degrade nucleic acids to nucleotides or small oligonucleotides. Reaction mixtures containing ethidium bromide complexed with nucleic acid lost fluorescence as the result of nucleolytic activity. The assay is adaptable for use with both deoxyribonucleases and ribonucleases, and is especially useful in detecting nuclease activity in column effluents.
Most procedures used for assaying deoxyribonuclease or ribonuclease activity involve the measurement of formation of acid-soluble products from radioactively labeled nucleic acids, decrease in viscosity of the substrate (l), or increase in absorbance at 260 nm (2). Although extensively used, these assays have serious disadvantages in that they can be time consuming and expensive. The inconveniences of these detection systems are especially noticeable when the number of samples to be assayed is great and the number of samples expected to have nuclease activity is small, as in the case of column chromatography of nuclease-containing extracts. Therefore, we have developed a simple assay based on the observation that the fluorescence of the dye ethidium bromide increases as it complexes with nucleic acid (3). When the nucleic acid is hydrolyzed by a nuclease, the ethidium bromide-nucleic acid complex is destroyed, and the fluorescence is decreased. For convenience this assay technique will be referred to as the microtiter nuclease assay. 1 Author addressed.
to whom
correspondence
0003-2697/80/160320-05$02.00/O Copyright &r 1980 by Academic Press. Inc. AU rights of reproduction in any form reserved.
should
be
320
MATERIALS
AND METHODS
Enzymes
Pancreatic DNase I and RNase A were obtained from Sigma. Calcium-dependent nucleases from Bacillus subtilis 168 were prepared using the following technique. Bacteria were grown in 200-ml cultures at 37°C for 18 h in the nutrient broth sporulation medium of Leighton and Doi (4). Following incubation, the cells were harvested by centrifugation at 10,OOOg and 4°C for 15 min. The supernatant fluid was then brought to 40% saturation by the addition of solid ammonium sulfate. After 4 h at 4°C the suspension was centrifuged at 10,OOOg for 15 min at 4°C. The resultant supernatant fluid was brought to 70% ammonium sulfate saturation. The precipitate was collected by centrifugation at 10,OOOg for 15 min at 4°C after incubation for 4 h at 4°C. The pellet was dissolved in 2 ml of 10 mM Tris-HCI, pH 7.5, and 0.02% sodium azide (Buffer A). A l-ml aliquot of the enzyme solution was then applied to a 2.6 x 90-cm Sephadex G150 column equilibrated with Buffer A. The protein was eluted with the same buffer at a flow rate of 28 ml/h at room temperature. Fractions of 6.3 ml were collected for use
DETECTION
with the microtiter and stored at 4°C.
nuclease assay system
Enzymatic Assays The B. subtilis nucleases were assayed by the method of Burke and Spizizen (5) using [3H]DNA as substrate. A unit of B. subtilis nuclease activity is defined as the amount of enzyme catalyzing the formation of 10 nmol of acid-soluble nucleotides at 37°C in 30 min. DNase I and RNase A activity were determined as described by Kunitz (2). The microtiter nuclease assay mixture employed with the B. subtilis nucleases con(pH 8.0), 10 mM tained 0.1 M Tris-HCl CaCl,, 0.8 mg/ml DNA (Difco), and 0.15 &ml ethidium bromide (Sigma). Alternatively, acridine orange (Eastman) was substituted for ethidium bromide in the reaction mixture also at a concentration of 0.15 pg./ml. The reaction mixture utilized for pancreatic DNase I contained 0.25 mM MgSO, in addi-
OF NUCLEASES
321
tion to the other components. Reaction mixtures were stable for weeks at 4°C when protected from the light. The DNA was prepared as a stock solution of 5 mg/ml in water, sterilized by autoclaving, and stored at 4°C. The ribonuclease reaction mixture was (pH 8.0), 10 composed of 0.1 M Tris-HCl mM CaCl,, 1.6 mg/ml RNA (type VI from Torula yeast, Sigma), and 0.15 E.Lg/mlethidium bromide. The RNA was prepared as a stock solution of 10 mg/ml in water and stored at -20°C. The assay was performed by transferring loo-p1 aliquots of the appropriate reaction mixture to the wells of polyvinyl chloride microtitration plates (Dynatech Laboratories, Inc.). To each well, the sample to be assayed and sufficient sterile water were added to bring the total volume in the well to 200 ~1. The plate was covered securely with Parafilm and incubated at 40°C. Periodically, the plates were examined using either short-wave (254 nm) or long-wave
FIG. 1. Effect of DNase I concentration on fluorescence of microtiter nuclease reaction mixture. Each well contains 100 ~1 of enzyme solution or sterile water and 100 ~1 of reaction mixture. From left to right the wells contain sterile water, 50 units DNase I, 10 units DNase I, 5 units DNase I, 1 unit DNase I, and heat-inactivated sample (originally containing 50 units of DNase I). The microtiter plate was incubated at 40°C for 24 h.
322
BURKE AND SLINKER
FIG. 2. Specificity of the assay. Row A contained DNase reaction mixture and row B contained RNase reaction mixture. In addition to reaction mixture, from left to right the wells contained sterile DNase, 10 units pancreatic RNase A, and sterile water. The microtiter plate water, 10 unitsB. subtilis was incubated at 40°C for 2 h.
(366 nm) ultraviolet light. The plates were placed on a long-wave uv light (UVL-56, Ultraviolet Products, Inc.) and photographed with a Polaroid MP3 system, using type 55P/N film through combined Kodak gel filters 23A and 8.
at which fluorescence was lost was proportional to the quantity of the enzyme present, the position of peak fractions of nuclease activity could be determined in eluates of gel filtration and ion-exchange columns simply by periodic examination of the plates. While the enzyme cannot be quantified with this method, activities of different samples RESULTS AND DISCUSSION can be compared on a relative basis. HeatWhen IOO-~1 aliquots of DNase I solu- inactivated DNase I (lOO°C, 15 min) protions containing from 1 to 50 units of enzyme duced no loss of fluorescence. As little as activity were added to lOO-~1 aliquots of 1 unit of DNase I or 0.5 units of B. subtilis microtiter nuclease reaction mixture, a de- exonuclease could be detected using this crease in fluorescence of the reaction mixtechnique. ture was observed in all DNase containing The assay can also be adapted for RNase wells after 24 h (Fig. 1). The nuclease con- detection by replacing the DNA in the reaction mixture with RNA. The specificity of taining wells could also be identified when viewed with white light. The color of the re- the reaction was demonstrated using purified action mixture changed from a deep pink to a B. subtilis DNase and pancreatic RNase A. light orange as the DNA was degraded. Wells No detectable decrease in fluorescence was containing 50 units of DNase I lost fluoresobserved when RNase A was added to the cence within 15-30 min, while wells with 1 DNase reaction mixture or when B. subtifis unit of the enzyme exhibited loss of fluoresDNase was added to the RNase reaction cence only after 16-24 h. Because the rate mixture as shown in Fig. 2.
DETECTION
Another intercalating dye, acridine orange, can be substituted for ethidium bromide in the reaction mixtures. However, the reduction in fluorescence of the reaction mixture following enzymatic hydrolysis was less pronounced. Reducing the concentration of DNA or RNA resulted in significant decreases in fluorescence of the system. Although the assay was still functional, the detection of nuclease containing samples
w
323
OF NUCLEASES
was more difficult. In order to detect enzymes which are inhibited by ethidium bromide, the dye may be added after a fixed incubation period. The utility and precision of this assay method are clearly demonstrated in Fig. 3. A partially purified B. subtilis exonuclease preparation was chromatographed on a Sephadex G-150 column, and the resulting fractions were assayed both with the micro-
50
35 Fraction
Number
FIG. 3. Correlation of microtiter nuclease assay to [3H]DNA quantitative assay. Partially purified B. subfilis extract was chromatographed on a Sephadex G-150 column as described under Materials and Methods. The resulting fractions were subjected to the [3H]DNA assay system. The elution profiles of the nucleases present in the extract are shown in the graph. The microtiter nuclease assay of the fractions is shown in the photograph of the microtiter plate following 14 h of incubation at 40°C. Well A contained 100 ~1 of reaction mixture and a 100~~1 aliquot of fraction 10; subsequent wells contained samples from consecutive fractions. Well B, the positive control, contained 100 ~1 of reaction mixture and 100 ~1 of cell-free B. subtilis culture fluid. Well C, the negative control, contained sterile water and reaction mixture.
324
BURKE
AND SLINKER
titer assay and the quantitative [3H]DNA nuclease assay system. The positions of the two peaks of DNase activity determined with the microtiter n&ease assay were confirmed using the sensitive isotope assay system. We have utilized this microtiter assay system extensively with gel filtration and ionexchange chromatography to locate and identify fractions containing nuclease activity. The method can also be used for detecting nuclease activity in crude cell lysates and culture supernatants and as a technique for screening extracts of presumptive mutant cells.
ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health Grant GM 25343 and by the Faculty Grant-In-Aid Program, Arizona State University.
REFERENCES 1. Smith, H. O., and Wilcox, K. W. (1970) J. Mol. Biol.
51, 379-391.
2. Kunitz, M. (1950) J. Gen Physiol. 33, 363-377. 3. Le Pecq, J. B., and Paoletti, C. (1966) Anal. Biothem. 17, 100-107. 4. Leighton, T. J., and Doi, R. H. (1971) J. Biol. Chem.
Z&,3189-3195.
5. Burke, W. F., Jr., and Spizizen, J. (1977) Biochemistry 16, 403-410.