- Email: [email protected]
A simple and efficient method to remove ribonuclease contamination from pancreatic deoxyribonuclease preparations
ANALYTICAL
BIOCHEMISTRY
75, 402-409 (1976)
A Simple and Efficient Method to Remove Ribonuclease Contamination from Pancreatic Deoxyribonuclease Preparations OLIVIER BRISON AND PIERRE CHAMBON Znstitut
de Chimie Biologique, UnitP de Recherche sur le Cancer de I’ZNSERM et Centre Neurochimie du CNRS, Facultk de Midecine. 67085 Strasbourg, France
de
Received February 17, 1976; accepted May 5, 1976 An affinity chromatography method which efficiently removes the contaminating RNase activity from commercial pancreatic DNase preparations is described. The new method is compared with the iodoacetate treatment previously used for the preferential inactivation of RNase.
The presence of contaminating RNase activity in commercially available pancreatic DNase preparations limits their use in the purification of RNA, since RNA should remain intact for further structural and/or functional studies (1). We describe here a method of affinity chromatography which very efficiently removes contaminating RNase from pancreatic DNase preparations. The yield of DNase is high and RNase activity cannot be detected in the purified DNase by using an assay which can detect 20.3 pg/ml of enzyme. MATERIALS
AND METHODS
pH]Uridine-labelled 28s rRNA (20,000 cpm x pg-l) was phenolextracted at 65°C (2) from HeLa cells and was purified by sedimentation through a linear 1%30% sucrose gradient. HeLa cells were grown in suspension as previously described (3), in the presence of 2 &i of pH]uridine (CEA, France) per milliliter [3H]Thymidine-labelled DNA was purified from HeLa cells grown in the presence of [3H]thymidine (0.2 @/ml; CEA, France). Calf thymus DNA (Worthington, U. S. A.) was purified as described previously (4) and yeast RNA was purchased from Worthington. Electrophoretically purified pancreatic DNase I was obtained from Worthington (DNase I RNasefree, DPFF). Chromatographically purified DNase I (DN-CL) and pancreatic RNase (RSOOO) were purchased from the Sigma Chemical Co. (St. Louis, MO.). Agarose 5’(4’aminophenyl)uridine2’(3’) phosphate (Agarose-UMP) was purchased from MilesYeda Ltd. (Great Britain). Bovine serum albumin was obtained from Sigma (A-4378). Formamide (Merck & Co., Inc., Rahway, N.J.) was deionized by stirring for 2 hr with an amberlite-mixed bed resin (AG 501 XSD, Bio402 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction in any form reserved.
RNase-FREE
DNase
PREPARATION
403
Rad) (2 g/60 ml) and by extraction with 0.3 vol ofether. It was then stored at -20°C. All reagents for acrylamide gel electrophoresis were as previously described (5). Polyvinylsulfate was obtained from Eastman Kodak Co. (Rochester, N.Y.). All other reagents were obtained from Merck. Bidistilled water was used, and all solutions and glassware were autoclaved before use in order to inactivate any ambient RNase. DNase activity was determined in an assay mixture containing 100 mM sodium acetate (pH 5.0), 5 mM MgCl,, 58 pg of calf thymus DNA, and enzyme (about 5 pg) in a total volume of 1 ml. The DNase activity was followed by an increase in absorbance at 260 nm (6). A unit of DNase is the amount of enzyme producing an optical density increase of 1.O in 1 min at 260 nm in the above l-ml assay. RNase activity in DNase preparations was assayed by analysing the breakdown of SH-labelled 28s rRNA by polyacrylamide gel electrophoresis in the presence of formamide. 3H-labelled 28s RNA (0.5 to 1.5 pg) was incubated in 1 ml of 50 mM Tris-HCl, pH 7.9, in the presence of 50 ,ug of DNase. After the incubation period (1 hr at 37”(Z), sodium dodecyl sulfate (0.5% final concentration) and yeast RNA (50 pg) were added. RNA was extracted with 1 vol of water-saturated phenol. After centrifugation, the aqueous phase was made 0.2 M sodium acetate (pH 5.0) and RNA was precipitated by the addition of 2 vol of cold ethanol (-20°C). After centrifugation the RNA pellet was dried by lyophilization and redissolved in 20 ~1 of 9% formamide containing 10 mM sodium phosphate, pH 6.0. After heating for 5 min at 37”C, polyvinyl sulfate (final concentration, 10 ,!&ml), glycerol (50% final concentration), and bromophenol-blue (used as a tracking dye) were added. Three and four-tenths percent polyacrylamide gels (10 cm in length, 0.5 in diameter) containing 98% formamide were prepared by a modification of the methods of Reijnders et al. (7) and Haines et al. (8). Acrylamide (850 mg), N,N’-methylene-bisacrylamide (150 mg), N,N,N’,N’-tetramethylethylene diamine (60 PI), and 0.25 ml of 1 M sodium phosphate, pH 6.0, were dissolved in 20 ml of deionized formamide. Two-tenths milliliter of 18% ammonium persulfate was added, the volume was made up to 25 ml with formamide, and the gels were poured and overlaid with 70% formamide in water. After 30 min, the 70% formamide overlay was replaced with 0.3 ml of 9% formamide buffered with 10 mM sodium phosphate, pH 6.0, and the gels were stored overnight at room temperature before use. The RNA sample was then loaded on the gel by underlaying the formamide. The electrophoresis buffer was 10 mM sodium phosphate, pH 6.0, in water. Electrophoresis was carried out at room temperature at 300 V (1.5-2 mA/gel) until the bromophenol-blue had reached the bottom of the gel (about 2.5 hr). After electrophoresis the gels were frozen and 2.0 mm fractions were cut with a multiple razor blade slicer. Each slice was then soaked overnight at 37°C in 0.5 ml of 0.3 N ammonium hydroxyde and counted in a scintillation mixture containing
404
BRISON AND CHAMBON
600
,
400
I--J 0
. 1 y-e 10
20
30
40
SLICES
FIG. 1. Detection of RNase activity by analysing the breakdown of 3H-labelled 28s r-RNA. One-half of one microgram of 3H-labelled 28s r-RNA was incubated for 1 hr at 37°C in a mixture containing 50 mM Tris-HCI (pH 8.0). 0.5 mg/ml of RNase-free bovine serum albumin, and 3 pg/ml (panel a) or 0.3 pgiml (panel b) of pancreatic RNase. After the incubation period the RNA was extracted and electrophoresed on polyacrylamide gels in the presence of formamide as described in Methods. Panel c: 28s r-RNA incubated under the same conditions, but in the absence of RNase. The arrow indicates the position of 4S RNA electrophoresed as a marker in a parallel gel.
25% Triton X-114, 75% Xylene, and Omnifluor (New England Nuclear Corp., Boston, Mass.), 4 g/l. The concentration of DNase protein was determined by measuring the absorbance at 280 nm. One unit of absorbance at 280 nm corresponds to 1 .l mg DNase/ml as determined by the Lowry method (9) when using bovine serum albumin as a standard. RESULTS Sensitivity of a Pancreatic RNase Assay Based on the Breakdown of 28s r-RNA
The most sensitive RNase assays previously used to detect a contaminating RNase activity in pancreatic DNase preparations measure 2 lo-30 pg/ml of enzyme (for a review, see Ref. 10). The results presented in Fig. I show that it is possible to detect as little as 0.3 pg/ml of pancreatic RNase by first incubating an intact labelled 2&S r-RNA with the enzyme
RNase-FREE
TABLE CHROMATOGRAPHY
405
DNase PREPARATION
OF
I
DNASE ON AGAROSE-UMP’
DNase concentration (mg/mll
Specific activity of DNase (U/mg of protein)
Experiment
pH of the buffer
Resin bed volume (mlimg of DNase)
Before chromatography
After chromatography
% Yield of protein
Before chromatography
After chromatography
1 2 3 4
8.0 7.0 6.0 6.0
0.50 0.25 0.25 0.25
12.5 12.3 11.6 10.0
2.10 1.35 0.74 0.63
95 85 70 76
22 22 20 38
26 22 20 40
a Columns were prepared as indicated in the table. at 4°C in Pasteur pipettes and equilibrated with a 20 mM sodium phosphate buffer (pH 6.0 or pH 7.0) or a 20 mM Tris- HCI buffer (pH 8.0). Each column was loaded with about 3 mg of DNase and washed with the appropriate buffer (50 &min). Fractions (OS-ml) were collected and an aliquot (3 ~1) of each fraction was incubated for 5 min at 37°C in 0.25 ml of a mixture containing 0.3 pg of pH]thymidine-labelled DNA (6200 cpm x @g-l). 50 pg of calf thymus DNA, IO mM MgC&. and 50 mM Tris-HCI, pH 8.0. The reaction was stopped by adding 3 ml of ice cold 10% TCA. The precipitate was collected on a Whatman GFC filter, washed with 3% TCA. and counted in toluene containing 4 g/l of Omnifluor (NEN). Fractions containing DNase activity were pooled. The DNase concentration and the DNase activity of the pooled fractions were determined and DNase was assayed for the presence of RNase activity (see Methods and Fig. 2). Experiments 1. 2, and 3 were performed with “RNase-free” DNase from Worthington (DPFF). Experiment 4 was performed with chromatographically purified DNase from Sigma (DN-CL).
and then analysing its breakdown by polyacrylamide gel electrophoresis in the presence of formamide. Under these conditions the secondary structure of the 28s r-RNA is destroyed and any break is revealed. As shown in Fig. 1, the RNA profile is altered even when the labeled 28s RNA is incubated with as little as 0.3 pg/ml of RNase for 1 hr at 37°C. No change in the RNA profile was observed in the presence of 0.03 pg/ml of enzyme (not shown), indicating that the sensitivity of the assay is ~0.3 pgiml of pancreatic RNase under our incubation conditions. No attempt was made to increase the sensitivity of the assay by increasing the incubation period. RNase Activity is Removed from DNase Preparations Chromatography
by Affinity
Pancreatic RNase has a high affinity for Agarose-UMP at pH 5.0 in the presence of a 20 mM sodium acetate buffer. As much as 3 mg of enzyme can be retained by 1 ml of resin under these conditions (Miles analysis report). Unfortunately, pancreatic DNase is also retained under these conditions
406
BRISON AND CHAMBON
Y i
v, 10000
0
10
20
30
40
SLICES
FIG. 2. Removal of RNase contamination by affinity chromatography. One and one-half micrograms of 3H-labelled 28s r-RNA was incubated in the presence of 50 &ml of DNase, extracted, and electrophoresed on polyacrylamide gels in the presence of formamide as described in Methods. Panel a: (-O-), DNase from Worthington (DPFF) before affinity chromatography; (-A-), DNase from Sigma (DN-CL) before affinity chromatography. Panel b: DNase from Worthington (DPFF) or from Sigma (DN-CL) after affinity chromatography (Table 1, Expts. 1 and 4). Panel c: 28s r-RNA incubated under the same conditions, but in the absence of DNase. The arrow indicates the position of 4S RNA, electrophoresed as a marker in a parallel gel.
(result not shown). The results presented in Table 1 show that it is possible to circumvent this problem by increasing the pH of the buffer, which results in a decrease of the affinity of DNase for the resin, Since the affinity of RNase for the resin also decreases when the pH is raised (result not shown), a higher resin/DNase ratio was required at pH 8.0 in order to retain all of the contaminating RNase activity on the column. The DNase recovery ranges from about 70% at pH 6.0 to almost 100% at pH 8.0. However, since even at these higher pHs, DNase has some affinity for the resin, the chromatography results in a dilution (about 6-fold at pH 8.0) of the enzyme. Attempts to increase the yield of DNase at low pHs by increasing the ionic strength of the buffer failed because the affinity of RNase for the resin decreased sharply when the salt concentration was raised. Figure 2 shows that affinity chromatography was indeed very efficient in removing the contaminatine RNase activity from two commercial prepara-
RNase-FREE
407
DNase PREPARATION
3000 2000
z; 1000 v)
: s T =
10000
5000
0
10
20
30
40
SLICES
FIG. 3. Inactivation of RNase contamination by iodoacetate treatment. One microgram of 3H-labelled 28 S r-RNA was incubated with 50 &ml of DNase, extracted, and electrophoresed on polyacrylamide gels in the presence offormamide as described in Methods. Panel a: DNase from Sigma (DN-CL) treated with iodoacetate. Panel b: DNase from Worthington (DPFF) treated with iodoacetate. Panel c: 28s r-RNA incubated under the same conditions but in the absence of DNase. The arrow indicates the position of 4S RNA electrophoresed as a marker in a parallel gel.
tions of pancreatic DNase: a highly purified “RNase-free” DNase preparation from Worthington, and a chromatographically purified preparation from Sigma. No modification of the RNA profile was observed when either of the two DNase preparations purified by affinity chromatography was incubated with labelled 28s r-RNA (Fig. 2, panels B and C). In contrast, none of the 28s r-RNA molecules remained intact after their incubation with the original Worthington “RNase-free” DNase preparation (Fig. 2, panel A), whereas the original Sigma DNase preparation hydrolysed the labelled RNA down to oligonucleotides migrating faster than bromophenol-blue. Comparison of Iodoacetate Treatment and Afjkity for Removing Contaminating RNase Activity
Chromatography
Using an RNase assay which can measure of the order of 2 lo-30 pg/ml of enzyme, Zimmerman and Sandeen (11) have previously shown that iodoacetate treatment can be used to preferentially inactivate the contaminating RNase activity, yielding DNase preparations which appear to be RNase-free. However, about 40% of the DNase activity was lost during this treatment. In order to compare the efficiency of affinity chroma-
408
BRISONANDCHAMBON
tography and of iodoacetate treatment, an “RNase-free” DNase preparation (Worthington) and a chromatographically purified DNase preparation (Sigma) were treated with iodoacetate as described by Zimmerman and Sandeen (11). The yields of DNase activity were 64% and 40%, respectively. Figure 3 shows that, using our RNase assay, the Worthington iodoacetate-treated DNase preparation was free of RNase (Fig. 3b), whereas a low, but definite, level of RNase activity still contaminated the treated Sigma DNase preparation. These results indicate that, in contrast to affinity chromatography, iodoacetate treatment is completely effective only with highly purified DNase preparations which are not heavily contaminated with RNase (compare Figs. 2a, 2b, 3a, and 3b). DISCUSSION
The presence of contaminating RNase in commercial crystalline pancreatic DNase preparations, which was first recognized by Polatnick and Bachrach (I), limits their usefulness during the purification of RNA. Iodoacetate treatment (11) and continuous electrophoresis (Worthington) have been used to remove the RNase contamination from DNase preparations. Our present assay for RNase activity which is more sensitive by at least 30-fold than those previously used (10) for estimating the level of RNase contamination reveals in fact that such “RNase-free” DNase preparations are still contaminated with RNase. Only the iodoacetate treatDNase preparation results in a ment of the Worthington “RNase-free” DNase preparation which appears to be free of RNase as judged from our assay. Affinity chromatography on Agarose-UMP as described in this paper appears to be the method of choice for eliminating RNase from commercially available pancreatic DNase preparations. First, the method is fast and simple and the contaminating RNase is very efficiently removed, since no RNase activity could be detected after the chromatography step when using an assay which can reveal as little as 0.3 pg/ml of enzyme. Second, the DNase recovery is almost 100% and the method can be successfully applied to commercial DNase preparations which are not highly purified. In contrast, the iodoacetate treatment is fully effective only on highly purified DNase preparations, and even then the yield is only about 60%. Garett et al. (12) have published a bentonite adsorption procedure for removing RNase from DNase. However, we cannot validly compare the efficiency of their method with ours, since their assay for contaminating RNase activity in the purified DNase appears to be much less sensitive than the assay used in the present study. It is worth mentioning that the same method can be used to eliminate the RNase activity which could contaminate other protein preparations, provided they are not retained on Agarose-UMP. For instance, we have
RNase-FREE
DNase PREPARATION
409
successfully used an Agarose- UMP affinity column at pH 5 to remove the RNase activity which contaminates all commercially available bovine serum albumin preparations (13,14). On the contrary, this method cannot be used with y-globulins which are strongly retained by the affinity resin. ACKNOWLEDGMENTS Excellent technical assistance was provided by Mrs. C. Hauss. This investigation was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale (ATP-virus oncogenes No. 73.1.427.18). the Fondation pour la Recherche Medicale Francaise. and the Commissariat a I’Energie Atomique.
REFERENCES I. Polatnick. J., and Bachrach, H. L. (1961) Anal. Biochem. 2, ]6]- 168. 2. Scherrer, K. (1969) in Fundamental Techniques in Virology (Habel, K., and Salzman, N. P., eds.), pp. 413-432, Academic Press, New York. 3. Hossenlopp, P.. Wells, D.. and Chambon. P. (1975) Eur. J. Biochem. 58, 237-251. 4. Kedinger, C., Gissinger, F., Gniazdowski. M., Mandel, J. L., and Chambon. P. (1972) Eur.
J. Biochem.
28,269-276.
5. Kedinger, C., Gissinger, F.. and Chambon, P. (1974) Eur. J. Biochem. 44, 421-436. 6. Kunitz, M. (1950) J. Gen. Physiol. 33, 349-357. 7. Reijnders, L., Sloof, P.. Sival, J.. and Borst, P. (1973) Biochim. Biophys. Acfa 324, 320-333. 8. Haines, M. E., Carey, N. H., and Palmiter, R. D. (1974) Eur. J. Biochem. 43,549-560. 9. Lowry, 0. H., Rosebrough. N. J., Farr, A. L., and Randall. R. J. (1951) J. Biol. Chem. 193, 265-275.
IO. Zimmerman. S. B.. and Sandeen, G. (1965) Anal. Biochem. 10, 444-449. 11. Zimmerman, S. B.. and Sandeen, G. (1966) Anal. B&hem. 14, 269-277. 12. Garrett, C. T., Wilkinson. D. S., and Pitot, H. C. (1973)Anal. Biochem. 52, 342-348. 13. Mandel, J. L., and Chambon, P. (1974) Eur. J. Biochem. 41, 379-395. 14. Yarus, M., and Rashbaum, S. (1972) Biochemistry 11, 2043-2049.
BIOCHEMISTRY
75, 402-409 (1976)
A Simple and Efficient Method to Remove Ribonuclease Contamination from Pancreatic Deoxyribonuclease Preparations OLIVIER BRISON AND PIERRE CHAMBON Znstitut
de Chimie Biologique, UnitP de Recherche sur le Cancer de I’ZNSERM et Centre Neurochimie du CNRS, Facultk de Midecine. 67085 Strasbourg, France
de
Received February 17, 1976; accepted May 5, 1976 An affinity chromatography method which efficiently removes the contaminating RNase activity from commercial pancreatic DNase preparations is described. The new method is compared with the iodoacetate treatment previously used for the preferential inactivation of RNase.
The presence of contaminating RNase activity in commercially available pancreatic DNase preparations limits their use in the purification of RNA, since RNA should remain intact for further structural and/or functional studies (1). We describe here a method of affinity chromatography which very efficiently removes contaminating RNase from pancreatic DNase preparations. The yield of DNase is high and RNase activity cannot be detected in the purified DNase by using an assay which can detect 20.3 pg/ml of enzyme. MATERIALS
AND METHODS
pH]Uridine-labelled 28s rRNA (20,000 cpm x pg-l) was phenolextracted at 65°C (2) from HeLa cells and was purified by sedimentation through a linear 1%30% sucrose gradient. HeLa cells were grown in suspension as previously described (3), in the presence of 2 &i of pH]uridine (CEA, France) per milliliter [3H]Thymidine-labelled DNA was purified from HeLa cells grown in the presence of [3H]thymidine (0.2 @/ml; CEA, France). Calf thymus DNA (Worthington, U. S. A.) was purified as described previously (4) and yeast RNA was purchased from Worthington. Electrophoretically purified pancreatic DNase I was obtained from Worthington (DNase I RNasefree, DPFF). Chromatographically purified DNase I (DN-CL) and pancreatic RNase (RSOOO) were purchased from the Sigma Chemical Co. (St. Louis, MO.). Agarose 5’(4’aminophenyl)uridine2’(3’) phosphate (Agarose-UMP) was purchased from MilesYeda Ltd. (Great Britain). Bovine serum albumin was obtained from Sigma (A-4378). Formamide (Merck & Co., Inc., Rahway, N.J.) was deionized by stirring for 2 hr with an amberlite-mixed bed resin (AG 501 XSD, Bio402 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction in any form reserved.
RNase-FREE
DNase
PREPARATION
403
Rad) (2 g/60 ml) and by extraction with 0.3 vol ofether. It was then stored at -20°C. All reagents for acrylamide gel electrophoresis were as previously described (5). Polyvinylsulfate was obtained from Eastman Kodak Co. (Rochester, N.Y.). All other reagents were obtained from Merck. Bidistilled water was used, and all solutions and glassware were autoclaved before use in order to inactivate any ambient RNase. DNase activity was determined in an assay mixture containing 100 mM sodium acetate (pH 5.0), 5 mM MgCl,, 58 pg of calf thymus DNA, and enzyme (about 5 pg) in a total volume of 1 ml. The DNase activity was followed by an increase in absorbance at 260 nm (6). A unit of DNase is the amount of enzyme producing an optical density increase of 1.O in 1 min at 260 nm in the above l-ml assay. RNase activity in DNase preparations was assayed by analysing the breakdown of SH-labelled 28s rRNA by polyacrylamide gel electrophoresis in the presence of formamide. 3H-labelled 28s RNA (0.5 to 1.5 pg) was incubated in 1 ml of 50 mM Tris-HCl, pH 7.9, in the presence of 50 ,ug of DNase. After the incubation period (1 hr at 37”(Z), sodium dodecyl sulfate (0.5% final concentration) and yeast RNA (50 pg) were added. RNA was extracted with 1 vol of water-saturated phenol. After centrifugation, the aqueous phase was made 0.2 M sodium acetate (pH 5.0) and RNA was precipitated by the addition of 2 vol of cold ethanol (-20°C). After centrifugation the RNA pellet was dried by lyophilization and redissolved in 20 ~1 of 9% formamide containing 10 mM sodium phosphate, pH 6.0. After heating for 5 min at 37”C, polyvinyl sulfate (final concentration, 10 ,!&ml), glycerol (50% final concentration), and bromophenol-blue (used as a tracking dye) were added. Three and four-tenths percent polyacrylamide gels (10 cm in length, 0.5 in diameter) containing 98% formamide were prepared by a modification of the methods of Reijnders et al. (7) and Haines et al. (8). Acrylamide (850 mg), N,N’-methylene-bisacrylamide (150 mg), N,N,N’,N’-tetramethylethylene diamine (60 PI), and 0.25 ml of 1 M sodium phosphate, pH 6.0, were dissolved in 20 ml of deionized formamide. Two-tenths milliliter of 18% ammonium persulfate was added, the volume was made up to 25 ml with formamide, and the gels were poured and overlaid with 70% formamide in water. After 30 min, the 70% formamide overlay was replaced with 0.3 ml of 9% formamide buffered with 10 mM sodium phosphate, pH 6.0, and the gels were stored overnight at room temperature before use. The RNA sample was then loaded on the gel by underlaying the formamide. The electrophoresis buffer was 10 mM sodium phosphate, pH 6.0, in water. Electrophoresis was carried out at room temperature at 300 V (1.5-2 mA/gel) until the bromophenol-blue had reached the bottom of the gel (about 2.5 hr). After electrophoresis the gels were frozen and 2.0 mm fractions were cut with a multiple razor blade slicer. Each slice was then soaked overnight at 37°C in 0.5 ml of 0.3 N ammonium hydroxyde and counted in a scintillation mixture containing
404
BRISON AND CHAMBON
600
,
400
I--J 0
. 1 y-e 10
20
30
40
SLICES
FIG. 1. Detection of RNase activity by analysing the breakdown of 3H-labelled 28s r-RNA. One-half of one microgram of 3H-labelled 28s r-RNA was incubated for 1 hr at 37°C in a mixture containing 50 mM Tris-HCI (pH 8.0). 0.5 mg/ml of RNase-free bovine serum albumin, and 3 pg/ml (panel a) or 0.3 pgiml (panel b) of pancreatic RNase. After the incubation period the RNA was extracted and electrophoresed on polyacrylamide gels in the presence of formamide as described in Methods. Panel c: 28s r-RNA incubated under the same conditions, but in the absence of RNase. The arrow indicates the position of 4S RNA electrophoresed as a marker in a parallel gel.
25% Triton X-114, 75% Xylene, and Omnifluor (New England Nuclear Corp., Boston, Mass.), 4 g/l. The concentration of DNase protein was determined by measuring the absorbance at 280 nm. One unit of absorbance at 280 nm corresponds to 1 .l mg DNase/ml as determined by the Lowry method (9) when using bovine serum albumin as a standard. RESULTS Sensitivity of a Pancreatic RNase Assay Based on the Breakdown of 28s r-RNA
The most sensitive RNase assays previously used to detect a contaminating RNase activity in pancreatic DNase preparations measure 2 lo-30 pg/ml of enzyme (for a review, see Ref. 10). The results presented in Fig. I show that it is possible to detect as little as 0.3 pg/ml of pancreatic RNase by first incubating an intact labelled 2&S r-RNA with the enzyme
RNase-FREE
TABLE CHROMATOGRAPHY
405
DNase PREPARATION
OF
I
DNASE ON AGAROSE-UMP’
DNase concentration (mg/mll
Specific activity of DNase (U/mg of protein)
Experiment
pH of the buffer
Resin bed volume (mlimg of DNase)
Before chromatography
After chromatography
% Yield of protein
Before chromatography
After chromatography
1 2 3 4
8.0 7.0 6.0 6.0
0.50 0.25 0.25 0.25
12.5 12.3 11.6 10.0
2.10 1.35 0.74 0.63
95 85 70 76
22 22 20 38
26 22 20 40
a Columns were prepared as indicated in the table. at 4°C in Pasteur pipettes and equilibrated with a 20 mM sodium phosphate buffer (pH 6.0 or pH 7.0) or a 20 mM Tris- HCI buffer (pH 8.0). Each column was loaded with about 3 mg of DNase and washed with the appropriate buffer (50 &min). Fractions (OS-ml) were collected and an aliquot (3 ~1) of each fraction was incubated for 5 min at 37°C in 0.25 ml of a mixture containing 0.3 pg of pH]thymidine-labelled DNA (6200 cpm x @g-l). 50 pg of calf thymus DNA, IO mM MgC&. and 50 mM Tris-HCI, pH 8.0. The reaction was stopped by adding 3 ml of ice cold 10% TCA. The precipitate was collected on a Whatman GFC filter, washed with 3% TCA. and counted in toluene containing 4 g/l of Omnifluor (NEN). Fractions containing DNase activity were pooled. The DNase concentration and the DNase activity of the pooled fractions were determined and DNase was assayed for the presence of RNase activity (see Methods and Fig. 2). Experiments 1. 2, and 3 were performed with “RNase-free” DNase from Worthington (DPFF). Experiment 4 was performed with chromatographically purified DNase from Sigma (DN-CL).
and then analysing its breakdown by polyacrylamide gel electrophoresis in the presence of formamide. Under these conditions the secondary structure of the 28s r-RNA is destroyed and any break is revealed. As shown in Fig. 1, the RNA profile is altered even when the labeled 28s RNA is incubated with as little as 0.3 pg/ml of RNase for 1 hr at 37°C. No change in the RNA profile was observed in the presence of 0.03 pg/ml of enzyme (not shown), indicating that the sensitivity of the assay is ~0.3 pgiml of pancreatic RNase under our incubation conditions. No attempt was made to increase the sensitivity of the assay by increasing the incubation period. RNase Activity is Removed from DNase Preparations Chromatography
by Affinity
Pancreatic RNase has a high affinity for Agarose-UMP at pH 5.0 in the presence of a 20 mM sodium acetate buffer. As much as 3 mg of enzyme can be retained by 1 ml of resin under these conditions (Miles analysis report). Unfortunately, pancreatic DNase is also retained under these conditions
406
BRISON AND CHAMBON
Y i
v, 10000
0
10
20
30
40
SLICES
FIG. 2. Removal of RNase contamination by affinity chromatography. One and one-half micrograms of 3H-labelled 28s r-RNA was incubated in the presence of 50 &ml of DNase, extracted, and electrophoresed on polyacrylamide gels in the presence of formamide as described in Methods. Panel a: (-O-), DNase from Worthington (DPFF) before affinity chromatography; (-A-), DNase from Sigma (DN-CL) before affinity chromatography. Panel b: DNase from Worthington (DPFF) or from Sigma (DN-CL) after affinity chromatography (Table 1, Expts. 1 and 4). Panel c: 28s r-RNA incubated under the same conditions, but in the absence of DNase. The arrow indicates the position of 4S RNA, electrophoresed as a marker in a parallel gel.
(result not shown). The results presented in Table 1 show that it is possible to circumvent this problem by increasing the pH of the buffer, which results in a decrease of the affinity of DNase for the resin, Since the affinity of RNase for the resin also decreases when the pH is raised (result not shown), a higher resin/DNase ratio was required at pH 8.0 in order to retain all of the contaminating RNase activity on the column. The DNase recovery ranges from about 70% at pH 6.0 to almost 100% at pH 8.0. However, since even at these higher pHs, DNase has some affinity for the resin, the chromatography results in a dilution (about 6-fold at pH 8.0) of the enzyme. Attempts to increase the yield of DNase at low pHs by increasing the ionic strength of the buffer failed because the affinity of RNase for the resin decreased sharply when the salt concentration was raised. Figure 2 shows that affinity chromatography was indeed very efficient in removing the contaminatine RNase activity from two commercial prepara-
RNase-FREE
407
DNase PREPARATION
3000 2000
z; 1000 v)
: s T =
10000
5000
0
10
20
30
40
SLICES
FIG. 3. Inactivation of RNase contamination by iodoacetate treatment. One microgram of 3H-labelled 28 S r-RNA was incubated with 50 &ml of DNase, extracted, and electrophoresed on polyacrylamide gels in the presence offormamide as described in Methods. Panel a: DNase from Sigma (DN-CL) treated with iodoacetate. Panel b: DNase from Worthington (DPFF) treated with iodoacetate. Panel c: 28s r-RNA incubated under the same conditions but in the absence of DNase. The arrow indicates the position of 4S RNA electrophoresed as a marker in a parallel gel.
tions of pancreatic DNase: a highly purified “RNase-free” DNase preparation from Worthington, and a chromatographically purified preparation from Sigma. No modification of the RNA profile was observed when either of the two DNase preparations purified by affinity chromatography was incubated with labelled 28s r-RNA (Fig. 2, panels B and C). In contrast, none of the 28s r-RNA molecules remained intact after their incubation with the original Worthington “RNase-free” DNase preparation (Fig. 2, panel A), whereas the original Sigma DNase preparation hydrolysed the labelled RNA down to oligonucleotides migrating faster than bromophenol-blue. Comparison of Iodoacetate Treatment and Afjkity for Removing Contaminating RNase Activity
Chromatography
Using an RNase assay which can measure of the order of 2 lo-30 pg/ml of enzyme, Zimmerman and Sandeen (11) have previously shown that iodoacetate treatment can be used to preferentially inactivate the contaminating RNase activity, yielding DNase preparations which appear to be RNase-free. However, about 40% of the DNase activity was lost during this treatment. In order to compare the efficiency of affinity chroma-
408
BRISONANDCHAMBON
tography and of iodoacetate treatment, an “RNase-free” DNase preparation (Worthington) and a chromatographically purified DNase preparation (Sigma) were treated with iodoacetate as described by Zimmerman and Sandeen (11). The yields of DNase activity were 64% and 40%, respectively. Figure 3 shows that, using our RNase assay, the Worthington iodoacetate-treated DNase preparation was free of RNase (Fig. 3b), whereas a low, but definite, level of RNase activity still contaminated the treated Sigma DNase preparation. These results indicate that, in contrast to affinity chromatography, iodoacetate treatment is completely effective only with highly purified DNase preparations which are not heavily contaminated with RNase (compare Figs. 2a, 2b, 3a, and 3b). DISCUSSION
The presence of contaminating RNase in commercial crystalline pancreatic DNase preparations, which was first recognized by Polatnick and Bachrach (I), limits their usefulness during the purification of RNA. Iodoacetate treatment (11) and continuous electrophoresis (Worthington) have been used to remove the RNase contamination from DNase preparations. Our present assay for RNase activity which is more sensitive by at least 30-fold than those previously used (10) for estimating the level of RNase contamination reveals in fact that such “RNase-free” DNase preparations are still contaminated with RNase. Only the iodoacetate treatDNase preparation results in a ment of the Worthington “RNase-free” DNase preparation which appears to be free of RNase as judged from our assay. Affinity chromatography on Agarose-UMP as described in this paper appears to be the method of choice for eliminating RNase from commercially available pancreatic DNase preparations. First, the method is fast and simple and the contaminating RNase is very efficiently removed, since no RNase activity could be detected after the chromatography step when using an assay which can reveal as little as 0.3 pg/ml of enzyme. Second, the DNase recovery is almost 100% and the method can be successfully applied to commercial DNase preparations which are not highly purified. In contrast, the iodoacetate treatment is fully effective only on highly purified DNase preparations, and even then the yield is only about 60%. Garett et al. (12) have published a bentonite adsorption procedure for removing RNase from DNase. However, we cannot validly compare the efficiency of their method with ours, since their assay for contaminating RNase activity in the purified DNase appears to be much less sensitive than the assay used in the present study. It is worth mentioning that the same method can be used to eliminate the RNase activity which could contaminate other protein preparations, provided they are not retained on Agarose-UMP. For instance, we have
RNase-FREE
DNase PREPARATION
409
successfully used an Agarose- UMP affinity column at pH 5 to remove the RNase activity which contaminates all commercially available bovine serum albumin preparations (13,14). On the contrary, this method cannot be used with y-globulins which are strongly retained by the affinity resin. ACKNOWLEDGMENTS Excellent technical assistance was provided by Mrs. C. Hauss. This investigation was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale (ATP-virus oncogenes No. 73.1.427.18). the Fondation pour la Recherche Medicale Francaise. and the Commissariat a I’Energie Atomique.
REFERENCES I. Polatnick. J., and Bachrach, H. L. (1961) Anal. Biochem. 2, ]6]- 168. 2. Scherrer, K. (1969) in Fundamental Techniques in Virology (Habel, K., and Salzman, N. P., eds.), pp. 413-432, Academic Press, New York. 3. Hossenlopp, P.. Wells, D.. and Chambon. P. (1975) Eur. J. Biochem. 58, 237-251. 4. Kedinger, C., Gissinger, F., Gniazdowski. M., Mandel, J. L., and Chambon. P. (1972) Eur.
J. Biochem.
28,269-276.
5. Kedinger, C., Gissinger, F.. and Chambon, P. (1974) Eur. J. Biochem. 44, 421-436. 6. Kunitz, M. (1950) J. Gen. Physiol. 33, 349-357. 7. Reijnders, L., Sloof, P.. Sival, J.. and Borst, P. (1973) Biochim. Biophys. Acfa 324, 320-333. 8. Haines, M. E., Carey, N. H., and Palmiter, R. D. (1974) Eur. J. Biochem. 43,549-560. 9. Lowry, 0. H., Rosebrough. N. J., Farr, A. L., and Randall. R. J. (1951) J. Biol. Chem. 193, 265-275.
IO. Zimmerman. S. B.. and Sandeen, G. (1965) Anal. Biochem. 10, 444-449. 11. Zimmerman, S. B.. and Sandeen, G. (1966) Anal. B&hem. 14, 269-277. 12. Garrett, C. T., Wilkinson. D. S., and Pitot, H. C. (1973)Anal. Biochem. 52, 342-348. 13. Mandel, J. L., and Chambon, P. (1974) Eur. J. Biochem. 41, 379-395. 14. Yarus, M., and Rashbaum, S. (1972) Biochemistry 11, 2043-2049.