- Email: [email protected]
A simple method for elimination of RNAase contamination from DNAase preparations
Journal o f Biochemical and Biophysical Methods, 1 (1979) 335--339
335
© Elsevier/North-Holland Biomedical Press
A SIMPLE METHOD FOR ELIMINATION OF RNAase CONTAMINATION FROM DNAase P R E P A R A T I O N S
GIORGOS J. DIMITRIADIS National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
(Received 6 May 1979; accepted 26 June 1979)
RNAase which usually contaminates commercial pancreatic DNAase preparations can be removed by affinity chromatography on agarose-coupled anti-RNAase antibodies. RNA treated with purified DNAase can be re-isolated intact, as determined by polyacrylamide gel electrophoresis under denaturing conditions. This method might be applicable to purification of other preparations which are used in RNA research, such as PNPase (polynucleotide phosphorylase) and specific antibodies for polysome immunoprecipitation. The non-specific binding of DNAase in our system is less than 5% and the loss of specific activity of DNAase I is less than 1%. Key words: RNAase; DNAase; purification; immunoadsorbent.
INTRODUCTION T h e presence o f c o n t a m i n a t i n g R N A a s e activity in c o m m e r c i a l l y available ' R N A a s e - f r e e ' e n z y m e p r e p a r a t i o n s used i n R N A research causes partial d e g r a d a t i o n o f the R N A and t h e r e f o r e limits their usefulness. It has been r e p o r t e d t h a t t h e R N A a s e activity m a y be p a r t l y r e m o v e d b y DEAE-cellulose c h r o m a t o g r a p h y , b y i o d o a c e t a t e t r e a t m e n t [1] and b y affinity c h r o m a t o g r a p h y o n agarose-coupled a m i n o p h e n y l p h o s p h o r y l u r i d i n e 2'{3')p h o s p h a t e [ 2 , 3 ] . T h e first t w o m e t h o d s are n o t very e f f i c i e n t and the third has the disadvantage o f high non-specific binding o f a vast n u m b e r o f proteins such as 7-globulins, catalase, PNPase, and thus can n o t be used for t h e p u r i f i c a t i o n o f these p r e p a r a t i o n s f r o m RNAase activity. We describe here a m e t h o d o f a f f i n i t y c h r o m a t o g r a p h y on agarose-coupled anti-RNAase antibodies which very e f f i c i e n t l y removes c o n t a m i n a t i n g RNAase activity f r o m p a n c r e a t i c DNAase I. No loss o f DNAase-specific activity was d e t e c t e d after t h e p u r i f i c a t i o n , which is rapid and simple, and non-specific binding is less t h a n 5%. MATERIALS AND METHODS R a b b i t r e t i c u l o c y t e s , r e t i c u l o c y t e p o l y s o m e s and r i b o s o m a l R N A ( r R N A ) were p r e p a r e d as described previously [ 4 , 5 ] . I o d i n a t i o n o f r R N A was carried
336
out as described in [6]. Calf t h y m u s DNA and electrophoretically purified pancreatic DNAase I (DNAase I, RNAase-free, DPFF) were purchased from Worthington (U.S.A.). Diethylpyrocarbonate (DEPC) was obtained from the Sigma Chemical Co. (Poole, Dorset, U.K.). Formamide (BDH Chemicals Ltd. was deionized by stirring for 2 h with Amberlite MB1 (BDH Chemicals Ltd.). Sepharose-4B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). All other reagents were from BDH Chemicals Ltd. Rabbit antiRNAase antibodies were a gift from Drs. S. Avrameas and T. Ternynck, Institute Pasteur, Paris, France. 5 mg/ml of RNAase were mixed with an equal volume of Freund's complete adjuvant and injected into the rabbit. After 2 mth three more injections (5 mg/ml RNAase in PBS) at intervals of 2 wk were performed. Gamma globulin fractions were prepared from antisera by 40% a m m o n i u m sulphate precipitation. Agarose beads were activated by the CNBr method described by Axen et al. [7] and the coupling of anti-RNAase antibodies to CNBr-activated agarose was carried out as described in ref. 8. The resultant immunoadsorbent (~1.1 mg of protein per ml of resin) was stored at 4--8°C. DNAase activity was determined in an assay mixture containing 0.1 M Tris-HC1 (pH 7.4), 0.1 M NaC1, 5 mM MgC12, 50 pg calf t h y m u s DNA and enzyme in a total volume of 1 ml. The DNAase activity was followed by an increase in absorbance at 260 nm. The concentration of DNAase protein was determined by the Lowry method [9]. RNAase activity in DNAase preparations was assayed by analysing the breakdown of ~:SI-labelled ribosomal RNA by polyacrylamide gel electrophoresis in the presence of formamide [2]. '2SI-labelled reticulocyte ribosomal RNA (~100 000 cpm, spec. act. 10 s cpm/pg) was incubated with either purified or unpurified DNAase preparations, 50 pg/ml, made up to a total volume of 1 ml with 0.1 M Tris-HC1, pH 7.4/0.1 M NaCl/5 wM MgC12 buffer, for 60 min at 37°C. After incubation, the reaction was terminated by the addition of EDTA (10 mM final concentration), SDS (0.5% final concentration), diethylpyrocarbonate (10 pl/ml) and 20 pg unlabelled ribosomal RNA {carrier) were added. RNA was extracted with an equal volume of phenol/chloroform (1 : 1, v/v). The aqueous phase was extracted once more with phenol/chloroform and finally was made 0.2 M in sodium acetate (pH 5.0). The RNA was precipitated by the addition of 2.5 vols. of ethanol at --20°C. Electrophoresis of RNA was carried out on 4% polyacrylamide gels containing 99% formamide buffered with 0.02 M barbitone at pH 9.0 [10]. Gel slicing and measuring of radioactivity were carried out as described elsewhere [ 11 ]. All solutions and glassware were autoclaved before use in order to inactivate any RNAase activity. RESULTS AND DISCUSSION As referred to in the Introduction, the main problem in the affinity chromatography of RNAase is non-specific binding. We have avoided this
337
0"2
0"1
s
I
i
O0
! I' 1
1
1 N acetic acLd
10 20 fractions (0-2ml)
30
Fig. 1. A f f i n i t y c h r o m a t o g r a p h y of D N A a s e I p r e p a r a t i o n o n a c o l u m n of a n t i - R N A a s e - agarose. Bed v o l u m e : 0.5 ml ( ~ 0 . 5 5 m g o f a n t i - R N A a s e ) . T h e c o l u m n was l o a d e d w i t h 2 m g D N A a s e I a n d w a s h e d w i t h b o r a t e - b u f f e r e d saline. T h e b o u n d m a t e r i a l was e l u t e d w i t h 1 N acetic acid.
problem by using as washing buffer, borate-buffered saline (0.075 M NaCI/ 0.1 M boric acid/0.025 M Na2B40~, pH 8.5), containing 1% Tween 80 and 1 M NaCl [12]. As shown in Fig. 1, the non-specific binding is less than 5%. Brison and Chambon [2] also used alkaline buffer to reduce the nonspecific binding, but UMP--agarose is unstable at alkaline pH [13] and the affinity of RNAase for the resin is decreased. RNAase activity in purified DNAase preparations was assayed by incubation of '2SI-labelled ribosomal RNA in the presence of DNAase I and subsequent electrophoretic analysis of the products. Fractions from the column as indicated by the horizontal bar in Fig. 1 were pooled and dialysed against 0.1 M Tris-HCl, pH 7.4/0.1 M NaC1/5 mM MgC12 buffer. The assay for DNAase activity shows no appreciable loss of specific activity of DNAase I (less than 1%) after purification and dialysis. As shown in Fig. 2, [~2sI]rRNA, incubated in the absence of DNAase migrates as two well resolved peaks (28 and 18 s). When [12SI]rRNA was incubated with the commercial preparation of pancreatic DNAase I ('RNAase-free'), degradation of the ribosomal R N A to smaller polynucleotides resulted. However, ['2SI]rRNA incubated with purified DNAase I remains intact. The use of commercially available enzyme preparations in R N A research has been hindered by the presence of RNAase activity. It is difficult to remove completely the DNA from preparations of nuclear RNA, for example, without using DNAase. Applications for polynucleotide phosphorylase include the synthesis of high-molecular-weight ribopolymers and ribonucleotide sequence analysis. The enzymes can be useful in these applications only if they have very low levels of RNAase activity. We believe
338 ~s
~s
~s
4s
5
0
g o
~
• Jee {1ram)
Fig. 2. 4% polyacrylamide, 99% formamide gel electrophoresis of rRNA after incubation. A, without DNAase; B, with non-purified DNAase; C, with purified DNAase. Unlabelled total reticulocyte RNA and yeast tRNA were used as markers. t h at c h r o m a t o g r a p h y on anti-RNAase--agarose would be a useful tool for the purification o f such preparations. The main advantages of our m e t h o d over m e t h o d s published so far for the elimination of RNAase activity from different preparations [1--3] are minimum non-specific binding and minimum loss o f DNAase activity. The m e t h o d may be used for e n z y m e preparations which show very high affinity for UMP-- or UTP--agarose, such as polynucleotide phosphorylase. ACKNOWLEDGMENTS We would like to thank Drs. S Avrameas and T. T e r n y n c k for the generous gift of anti-RNAase antibodies, Dr. J.R. Tata for his hospitality, M. L a y t o n for technical assistance, Mr. I. Hunt er for helping in the preparation o f anti-RNAase--agarose resin and Mrs. R. Harris for typing and drawings. G.J.D. is in receipt of a Wellcome Trust fellowship. SIMPLIFIED D E S C R I P T I O N O F T H E M E T H O D
A N D ITS A P P L I C A T I O N S
By using anti-RNAase immunoadsorbent commercial DNAase can be purified. Nonspecific binding was avoided by using borate-buffered saline (0.075 M NaCl/0.1 M boric
339 acid/0.025 M Na2B407, pH 8.5). The method might be applicable to purification of other preparations which are used in RNA research such as 7-globulins, PNPase. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13
Zimmerman, S.B. and Sandeen, G. (1966) Anal. Biochem. 14,269--277 Brison, O. and Chambon, P. (1976) Anal. Biochem. 75,402--409 Maxwell, I.H., Maxwell, F. and Hahn, W.E. (1977) Nucl. Acids Res. 4,241--246 Dimitriadis, G.J. and Georgatsos, J.G. (1974) FEBS Lett. 46, 96--100 Dimitriadis, G.J. (1978) FEBS Lett. 86, 289--293 Dimitriadis, G.J. and Georgatsos, J.G. (1975) Nucl. Acids Res. 2, 1719--1726 Axen, R., Porath, J. and Ernback, S. (1967) Nature (London) 214, 1302--1304 Pharmacia Fine Chemicals, Uppsala, Sweden. Affinity Chromatography: Principles and Methods, p. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265--275 Staynov, D.Z., Pinder, J.C. and Gratzer, W.B. (1972) Nature New Biol. 235, 108-110 Dimitriadis, G.J. (1978) Nucl. Acids Res. 5, 1381--1386 Smith, J.A., Hurell, J.G.R., and Leach, S.J. (1978) Anal. Biochem. 87,299--305 Smith, G.K., Schray, K.J. and Schaffer, S.W. (1978) Anal. Biochem. 84,406--414
335
© Elsevier/North-Holland Biomedical Press
A SIMPLE METHOD FOR ELIMINATION OF RNAase CONTAMINATION FROM DNAase P R E P A R A T I O N S
GIORGOS J. DIMITRIADIS National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
(Received 6 May 1979; accepted 26 June 1979)
RNAase which usually contaminates commercial pancreatic DNAase preparations can be removed by affinity chromatography on agarose-coupled anti-RNAase antibodies. RNA treated with purified DNAase can be re-isolated intact, as determined by polyacrylamide gel electrophoresis under denaturing conditions. This method might be applicable to purification of other preparations which are used in RNA research, such as PNPase (polynucleotide phosphorylase) and specific antibodies for polysome immunoprecipitation. The non-specific binding of DNAase in our system is less than 5% and the loss of specific activity of DNAase I is less than 1%. Key words: RNAase; DNAase; purification; immunoadsorbent.
INTRODUCTION T h e presence o f c o n t a m i n a t i n g R N A a s e activity in c o m m e r c i a l l y available ' R N A a s e - f r e e ' e n z y m e p r e p a r a t i o n s used i n R N A research causes partial d e g r a d a t i o n o f the R N A and t h e r e f o r e limits their usefulness. It has been r e p o r t e d t h a t t h e R N A a s e activity m a y be p a r t l y r e m o v e d b y DEAE-cellulose c h r o m a t o g r a p h y , b y i o d o a c e t a t e t r e a t m e n t [1] and b y affinity c h r o m a t o g r a p h y o n agarose-coupled a m i n o p h e n y l p h o s p h o r y l u r i d i n e 2'{3')p h o s p h a t e [ 2 , 3 ] . T h e first t w o m e t h o d s are n o t very e f f i c i e n t and the third has the disadvantage o f high non-specific binding o f a vast n u m b e r o f proteins such as 7-globulins, catalase, PNPase, and thus can n o t be used for t h e p u r i f i c a t i o n o f these p r e p a r a t i o n s f r o m RNAase activity. We describe here a m e t h o d o f a f f i n i t y c h r o m a t o g r a p h y on agarose-coupled anti-RNAase antibodies which very e f f i c i e n t l y removes c o n t a m i n a t i n g RNAase activity f r o m p a n c r e a t i c DNAase I. No loss o f DNAase-specific activity was d e t e c t e d after t h e p u r i f i c a t i o n , which is rapid and simple, and non-specific binding is less t h a n 5%. MATERIALS AND METHODS R a b b i t r e t i c u l o c y t e s , r e t i c u l o c y t e p o l y s o m e s and r i b o s o m a l R N A ( r R N A ) were p r e p a r e d as described previously [ 4 , 5 ] . I o d i n a t i o n o f r R N A was carried
336
out as described in [6]. Calf t h y m u s DNA and electrophoretically purified pancreatic DNAase I (DNAase I, RNAase-free, DPFF) were purchased from Worthington (U.S.A.). Diethylpyrocarbonate (DEPC) was obtained from the Sigma Chemical Co. (Poole, Dorset, U.K.). Formamide (BDH Chemicals Ltd. was deionized by stirring for 2 h with Amberlite MB1 (BDH Chemicals Ltd.). Sepharose-4B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). All other reagents were from BDH Chemicals Ltd. Rabbit antiRNAase antibodies were a gift from Drs. S. Avrameas and T. Ternynck, Institute Pasteur, Paris, France. 5 mg/ml of RNAase were mixed with an equal volume of Freund's complete adjuvant and injected into the rabbit. After 2 mth three more injections (5 mg/ml RNAase in PBS) at intervals of 2 wk were performed. Gamma globulin fractions were prepared from antisera by 40% a m m o n i u m sulphate precipitation. Agarose beads were activated by the CNBr method described by Axen et al. [7] and the coupling of anti-RNAase antibodies to CNBr-activated agarose was carried out as described in ref. 8. The resultant immunoadsorbent (~1.1 mg of protein per ml of resin) was stored at 4--8°C. DNAase activity was determined in an assay mixture containing 0.1 M Tris-HC1 (pH 7.4), 0.1 M NaC1, 5 mM MgC12, 50 pg calf t h y m u s DNA and enzyme in a total volume of 1 ml. The DNAase activity was followed by an increase in absorbance at 260 nm. The concentration of DNAase protein was determined by the Lowry method [9]. RNAase activity in DNAase preparations was assayed by analysing the breakdown of ~:SI-labelled ribosomal RNA by polyacrylamide gel electrophoresis in the presence of formamide [2]. '2SI-labelled reticulocyte ribosomal RNA (~100 000 cpm, spec. act. 10 s cpm/pg) was incubated with either purified or unpurified DNAase preparations, 50 pg/ml, made up to a total volume of 1 ml with 0.1 M Tris-HC1, pH 7.4/0.1 M NaCl/5 wM MgC12 buffer, for 60 min at 37°C. After incubation, the reaction was terminated by the addition of EDTA (10 mM final concentration), SDS (0.5% final concentration), diethylpyrocarbonate (10 pl/ml) and 20 pg unlabelled ribosomal RNA {carrier) were added. RNA was extracted with an equal volume of phenol/chloroform (1 : 1, v/v). The aqueous phase was extracted once more with phenol/chloroform and finally was made 0.2 M in sodium acetate (pH 5.0). The RNA was precipitated by the addition of 2.5 vols. of ethanol at --20°C. Electrophoresis of RNA was carried out on 4% polyacrylamide gels containing 99% formamide buffered with 0.02 M barbitone at pH 9.0 [10]. Gel slicing and measuring of radioactivity were carried out as described elsewhere [ 11 ]. All solutions and glassware were autoclaved before use in order to inactivate any RNAase activity. RESULTS AND DISCUSSION As referred to in the Introduction, the main problem in the affinity chromatography of RNAase is non-specific binding. We have avoided this
337
0"2
0"1
s
I
i
O0
! I' 1
1
1 N acetic acLd
10 20 fractions (0-2ml)
30
Fig. 1. A f f i n i t y c h r o m a t o g r a p h y of D N A a s e I p r e p a r a t i o n o n a c o l u m n of a n t i - R N A a s e - agarose. Bed v o l u m e : 0.5 ml ( ~ 0 . 5 5 m g o f a n t i - R N A a s e ) . T h e c o l u m n was l o a d e d w i t h 2 m g D N A a s e I a n d w a s h e d w i t h b o r a t e - b u f f e r e d saline. T h e b o u n d m a t e r i a l was e l u t e d w i t h 1 N acetic acid.
problem by using as washing buffer, borate-buffered saline (0.075 M NaCI/ 0.1 M boric acid/0.025 M Na2B40~, pH 8.5), containing 1% Tween 80 and 1 M NaCl [12]. As shown in Fig. 1, the non-specific binding is less than 5%. Brison and Chambon [2] also used alkaline buffer to reduce the nonspecific binding, but UMP--agarose is unstable at alkaline pH [13] and the affinity of RNAase for the resin is decreased. RNAase activity in purified DNAase preparations was assayed by incubation of '2SI-labelled ribosomal RNA in the presence of DNAase I and subsequent electrophoretic analysis of the products. Fractions from the column as indicated by the horizontal bar in Fig. 1 were pooled and dialysed against 0.1 M Tris-HCl, pH 7.4/0.1 M NaC1/5 mM MgC12 buffer. The assay for DNAase activity shows no appreciable loss of specific activity of DNAase I (less than 1%) after purification and dialysis. As shown in Fig. 2, [~2sI]rRNA, incubated in the absence of DNAase migrates as two well resolved peaks (28 and 18 s). When [12SI]rRNA was incubated with the commercial preparation of pancreatic DNAase I ('RNAase-free'), degradation of the ribosomal R N A to smaller polynucleotides resulted. However, ['2SI]rRNA incubated with purified DNAase I remains intact. The use of commercially available enzyme preparations in R N A research has been hindered by the presence of RNAase activity. It is difficult to remove completely the DNA from preparations of nuclear RNA, for example, without using DNAase. Applications for polynucleotide phosphorylase include the synthesis of high-molecular-weight ribopolymers and ribonucleotide sequence analysis. The enzymes can be useful in these applications only if they have very low levels of RNAase activity. We believe
338 ~s
~s
~s
4s
5
0
g o
~
• Jee {1ram)
Fig. 2. 4% polyacrylamide, 99% formamide gel electrophoresis of rRNA after incubation. A, without DNAase; B, with non-purified DNAase; C, with purified DNAase. Unlabelled total reticulocyte RNA and yeast tRNA were used as markers. t h at c h r o m a t o g r a p h y on anti-RNAase--agarose would be a useful tool for the purification o f such preparations. The main advantages of our m e t h o d over m e t h o d s published so far for the elimination of RNAase activity from different preparations [1--3] are minimum non-specific binding and minimum loss o f DNAase activity. The m e t h o d may be used for e n z y m e preparations which show very high affinity for UMP-- or UTP--agarose, such as polynucleotide phosphorylase. ACKNOWLEDGMENTS We would like to thank Drs. S Avrameas and T. T e r n y n c k for the generous gift of anti-RNAase antibodies, Dr. J.R. Tata for his hospitality, M. L a y t o n for technical assistance, Mr. I. Hunt er for helping in the preparation o f anti-RNAase--agarose resin and Mrs. R. Harris for typing and drawings. G.J.D. is in receipt of a Wellcome Trust fellowship. SIMPLIFIED D E S C R I P T I O N O F T H E M E T H O D
A N D ITS A P P L I C A T I O N S
By using anti-RNAase immunoadsorbent commercial DNAase can be purified. Nonspecific binding was avoided by using borate-buffered saline (0.075 M NaCl/0.1 M boric
339 acid/0.025 M Na2B407, pH 8.5). The method might be applicable to purification of other preparations which are used in RNA research such as 7-globulins, PNPase. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13
Zimmerman, S.B. and Sandeen, G. (1966) Anal. Biochem. 14,269--277 Brison, O. and Chambon, P. (1976) Anal. Biochem. 75,402--409 Maxwell, I.H., Maxwell, F. and Hahn, W.E. (1977) Nucl. Acids Res. 4,241--246 Dimitriadis, G.J. and Georgatsos, J.G. (1974) FEBS Lett. 46, 96--100 Dimitriadis, G.J. (1978) FEBS Lett. 86, 289--293 Dimitriadis, G.J. and Georgatsos, J.G. (1975) Nucl. Acids Res. 2, 1719--1726 Axen, R., Porath, J. and Ernback, S. (1967) Nature (London) 214, 1302--1304 Pharmacia Fine Chemicals, Uppsala, Sweden. Affinity Chromatography: Principles and Methods, p. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265--275 Staynov, D.Z., Pinder, J.C. and Gratzer, W.B. (1972) Nature New Biol. 235, 108-110 Dimitriadis, G.J. (1978) Nucl. Acids Res. 5, 1381--1386 Smith, J.A., Hurell, J.G.R., and Leach, S.J. (1978) Anal. Biochem. 87,299--305 Smith, G.K., Schray, K.J. and Schaffer, S.W. (1978) Anal. Biochem. 84,406--414