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Inhibition of a nuclease contaminant in the commercial preparations of escherichia coli alkaline phosphatase
ANALYTICAL
BIOCHEMISTRY
Inhibition
95,458-464
(1979)
of a Nuclease Contaminant in the Commercial of Escherichia co/i Alkaline Phosphatasel MORIKAZU SHINAGAWAAND Department
Preparations
R. PADMANABHAN~
of Biological Chemistry, University of Maryland 660 West Redwood Street, Baltimore, Maryland
School 21201
of Medicine,
Received August 24, 1978 Escherichia co/i alkaline phosphatase is a valuable reagent for removal of terminal phosphate from both ribo- and deoxyribo-oligonucleotides or from restriction enzyme fragments of DNA. Some commercial preparations of this enzyme were found to be contaminated with nucleases which could degrade both DNA and RNA. These contaminating nucleases can be completely eliminated by carrying out the enzymic reaction in the presence of 0. l- 1% sodium dodecyl sulfate without any loss of phosphatase activity. This report has immediate application in the sequence analysis of DNA or RNA.
Escherichia coli alkaline phosphatase [EC 3.1.3. l] is a heat-stable metalloenzyme whose active form is dimer and is known to be localized in the periplasmic space of the cell (1). The procedure for the purification of the enzyme has been described (2,3) and the enzyme has also been crystallized (4). Alkaline phosphatase is a valuable reagent used for the analysis of the primary structure of RNA and DNA. It is used widely to remove the terminal phosphate from ribo- or deoxyribo-oligonucleotides at their 3’ or 5’ termini. Since the discovery of restriction enzymes [see Ref. (5) for a review] which cleave DNA at specific nucleotide sequences, structural as well as genetic analyses of DNA have been progressing extremely rapidly. Recently, rapid methods have become available for the nucleotide sequence analysis of both DNA and RNA (6-9). These methods are dependent on successful labeling techniques involving 32P of very high specific activities at a L This work was supported by U. S. Public Health Service Grant CA 20369. R. P. gratefully acknowledges the support from Research Career Development Award from National Institutes of Health (5 K04 CA 00235). * To whom reprint requests should be sent. 0003-2697/79/080458-07$02.00/O Copynght All rights
8 1979 by Academic Press, Inc. of reproduction in any form reserved.
unique terminus. A restriction enzyme fragment of DNA can be labeled at its 5’ termini by treatment with alkaline phosphatase to remove the terminal phosphate residues, followed by phosphorylation using polynucleotide kinase and Y-[~~P]ATP (10,ll). The alkaline phosphatase is now commercially available. Yet commercial preparations are often contaminated with small amounts of nucleases which could degrade the DNA or the RNA and thus reduce the yield of labeled fragment. We wish to report here that the presence of 0.1% sodium dodecyl sulfate (SDS)3 in the incubation mixture completely inhibits the nuclease activity without altering the phosphatase activity. A DNA fragment obtained by cleaving human adenovirus type 2 DNA with restriction enzyme SmaI (isolated from Serratia marcescens)4 was dephosphorylated by treating with alkaline phosphatase in the 3 Abbreviations used: SDS, sodium dodecyl sulfate; Ad, adenovirus; TEN buffer, 0.01 M Tris-HCl (pH 7.4), 1 mtvfEDTA, 0.1 M NaCI; DEAE, diethylaminoethyl; dpC, deoxycytidine Y-phosphate. 4 The cleavage map of adenovirus type 2 DNA was established by Mulder et al. (C. Mulder, M. Greene, and H. Delius, personal communication). 458
ALKALINE
PHOSPHATASE
presence of 0.1% SDS. This fragment could then be labeled at its 5’ termini using polynucleotide kinase and -Y-[~~P]ATP. We have demonstrated that a DNA fragment labeled at the 5’ termini in this manner could be successfully used for sequence analysis following the procedure of Maxam and Gilbert (7). MATERIALS
AND METHODS
Enzymes and chemicals. Alkaline phosphatase from E. coli was purchased from Worthington Biochemical Corporation (BAPF grade). The enzyme, suspended in 3.2 M (NH,),SO, at a concentration of 20 units/ mg, was centrifuged at 10,000 t-pm for 5 min. The precipitate was dissolved in 0.1 M Tris-HCl, pH 8.0, and 10 PM ZnSO, and dialyzed against the same buffer for 12 h at 4°C. The concentration of enzyme after dialysis was 48 units/ml. Enzyme dilutions were made in 0.1 M Tris-HCl (pH 8.0). Polynucleotide kinase was obtained from P. L. Biochemicals. Yeast RNA which contains a mixture of 5.8 S and 5 S ribosomal RNA as well as tRNA was a gift from Mr. Guy Adami. SmaI and iYaeII1 restriction enzymes were kindly provided by Dr. Werner Buettner. Sodium p-nitrophenyl phosphate, SDS, and ethidium bromide were from Sigma Chemical Company. SDS was recrystallized from aqueous ethanol before use. SeaKem agarose was obtained from Microbiological Associates (Rockville, Md.) +X174 DNA (RFI) was prepared as described previously (12). Spectrophotometric measurement of alkaline phosphatase activity. Hydrolysis of p-nitrophenyl phosphate was measured colorimetrically as described by Garen and Levinthal (2). When the SDS was used in the incubation mixture, the concentration was either 0.1 or 1% as indicated. The amount of enzyme used per assay was 0.006 U in a total volume of 1 ml. Alkaline phosphatase treatment of +X174 DNA. A reaction mixture containing 1.9 pg
FROM
E. co/i
459
of DNA, 0.1 M Tris-HCl buffer, pH 8.0, and 0.24 units of the enzyme was incubated in a total volume of 60 ~1 in the absence or presence of 0.1% SDS. After incubation at 37°C for 2 h, 40 ~1 of a mixture containing 50% sucrose, 25 mM EDTA, and 0.02% bromophenol blue was added. An aliquot of 50 ~1 was applied to an agarose gel (1.4%) for electrophoresis. Agarose gel electrophoresis. DNA treated with alkaline phosphatase was analyzed by electrophoresis on an agarose gel (1.4%) and detected by its fluorescence with ethidium bromide (13). The buffer system used for electrophoresis was: 36 mM Tris, 17 mM N&HP04, 13 mM NaH,PO,, 1 mM EDTA (pH 7.8). Phosphorylation by polynucleotide kinase of Smal-M fragment after treatment with alkalinephosphatase. SmaI cleaves Ad 2 DNA into 13 fragments (C. Mulder et al., personal communication). The fragments were separated by a 1.4% agarose gel and visualized by their ethidium bromide fluorescence. The smallest fragment M (about 140 base pairs long) was isolated from the gel using hydroxyapatite according to the procedure described by Wu et al. (14). The M fragment was dephosphorylated either in the presence or absence of 0.1% SDS by incubating a mixture containing 22 pmol (3 pg) of M fragment, 0.1 M Tris-HCl, pH 8.0, and 0.24 units of alkaline phosphatase for 60 min at 37°C. After the incubation, the reaction mixture was diluted to 200 pl with 0.01 M Tris-HCI (pH 7.4), 1 mM EDTA, and 0.1 M NaCl (TEN buffer). It was then extracted with 200 pl of redistilled phenol saturated with TEN buffer. The phenol layer was washed with 100 ~1 of TEN buffer. The pooled aqueous layer was extracted with an equal volume of ether four times. The DNA was precipitated with ethanol and stored at -70°C for 3 h. After centrifugation at 10,000 rpm for 20 min, the DNA pellet was dissolved in 200 ~1 of 0.3 M sodium acetate and the DNA was reprecipitated
460
SHINAGAWA
AND PADMANABHAN
with 3 vol of ethanol. The DNA pellet was then dried and dissolved in 20 ~1 of sterile distilled H,O. Polynucleotide kinase reaction of M fragment was carried out in a total volume of 55 ~1 containing 11 pmol(l.5 pg) of dephosphorylated DNA, 5.5 ~1 of 10 x kinase buffer pH 8.0, 0.1 M MgC&, (0.5 M Tris-HCl, 4.5 ~1 of 1 mM and 0.05 M dithiothreitol), spermidine-HCl (15), 35 ~1 of H20, 150 pmol of y-[32P]ATP (specific activity 414 mCi/pmol) and 2 units of polynucleotide kinase. The incubation was carried out at 37°C. Aliquots of 3-~1 samples were taken in duplicate at 0 (before the addition of enzyme), 15, and 30 min. Acid-insoluble radioactivity was determined as described (16).
1
2
3
OFORM
II-
FORM IllFORM
I-
RESULTS
Figure 1 shows the activity of the enzyme in the absence or presence of 1% SDS. The enzyme is not inhibited by SDS. Urea (7.5 M), however, inhibits the enzyme activity to 79%, very close to the value reported
i o.?l-0 t
C
0.5
I.0 TIME
I.5
2.0
2.5
FIG. 2. Agarose gel electrophoresis of phosphatasetreated +X174 DNA RFI. $X174 DNA (Form I) was treated with phosphatase in the presence (lane 2) or absence (lane 3) of 0.1% SDS. The untreated DNA is shown in lane 1. Phosphatase treatment was carried out as described under Materials and Methods. Electrophoresis was carried out at 100 V (6.6 V/cm) on a 1.4% agarose gel for 9 h and the ethidium bromide fluorescence of the DNA was photographed (13). Under these conditions of ionic and electric field strengths, the mobilities of the three different conformational forms of 4x174 DNA are in the following decreasing order: Form I > Form III > Form II, as expected (25-27).
IN MINUTES
FIG. 1. The enzymic activity of alkaline phosphatase in the presence or absence of SDS. The activity was measured as the rate of hydrolysis of sodium p-nitrophenyl phosphate as described previously (2), using a Cary 15 spectrophotometer equipped with a recorder. (a) No SDS (control) or 0.1% or 1% SDS; the rates in all three cases were identical. (b) 4 M guanidine-HCI was present in the incubation mixture. (c) 7.5 M urea was present in the reaction.
by Heppel et al. (17). When 4 M guanidine hydrochloride was present in the incubation mixture, the activity was 57% of the initial value (Fig. lb). Sarkosyl, like SDS, does not show any inhibition of phosphatase activity. Commercial preparations of alkaline phosphatase (even the BAPF grade from
ALKALINE
PHOSPHATASE
FROM E.
coli
461
Table 1, the fraction of M fragment which by the phosphatase PHOSPHORYLATION OF SnzaI-M FRAGMENT OF Ad 2 was dephosphorylated DNA BY POLYNUCLEOTIDE KINASE in the presence of SDS served as a substrate AND y[32P]ATPz with as much efficiency as the fraction which was treated with phosphatase in the Acid-insoluble radioabsence of SDS. The small decrease in the Duration of activity incorporated incorporation of radioactivity in the fraction polynucleotide into M fragment which was treated with alkaline phosphakinase reaction (min) A B tase in the absence of SDS may be attributable to a slight reduction in the amount of 0 1,299 812 substrate due to the degradation of the frag15 51,079 42,654 ment by the contaminant nuclease present 30 57,730 45,015 in the phosphatase. U The SlnuI-M fragment of Ad 2 DNA was isolated We have tested the suitability of a DNA as described under Materials and Methods. Dephosfragment labeled in this manner for sephorylation of M fragment was carried out using alkaof line phosphatase either in the presence of 0.1% SDS quence analysis using the procedure Maxam and Gilbert (7). Figure 3 shows a (A) or in its absence (B). Dephosphorylated M fragment partial sequence of a region about 42 nucleowas then labeled at its 5’ termini using polynucleotide kinase and Y[~~P]ATP as described under Materials and tides away from the 5’ terminus of Hue111 Methods, and acid-insoluble radioactivity incorporated recognition site at the right-hand end of Ad was measured ( 16). The values represent an average of 2 DNA. The sequence can be read unamsamples in duplicate. biguously from the 5’-labeled terminus of the terminal Hue111 fragment of Ad 2 DNA. Worthington) were found to contain small Moreover, we digested Ad 2 DNA with amounts of deoxyribonuclease which could TuqI (Bethesda Research Laboratory, Inc.) convert $X 174 Form I to II (nicked circular) and dephosphorylated the DNA fragments and III (linear DNA), which was then fur- using the alkaline phosphatase in the presther degraded upon incubation with the en- ence of 0.1% SDS. After the fragments were zyme at 37°C for 2 h (Fig. 2, lane 3). How- labeled at their 5’ termini with szP using ever, this contaminating nuclease activity polynucleotide kinase and y-[32P]ATP as could be completely inhibited by carrying described under Materials and Methods the out the phosphatase reaction in the presence 5’ end-group analysis of the labeled fragof 0.1% SDS (Fig. 2, lane 2). ments showed 89% as dpC residue. The In order to test whether treatment of a remaining 11% was distributed among the restriction enzyme fragment of DNA with other three nucleotides. The DNA sequence alkaline phosphatase in the presence of recognized by TuqI at the cleavage site is SDS, followed by the removal of SDS, leaves any inhibitor to the polynucleotide 1 5’-T-C-G-A-3’ kinase reaction, the SmaI-M fragment of Ad 2 DNA (about 140 base pairs long) was 3’-A-G-C-T-5’ used for labeling the termini. Alkaline phos? phatase treatment of M fragment was carried out either in the presence or absence and hence dpC residues are expected at the of 0.1% SDS as described under Materials 5’ termini of the cleavage products (18). and Methods. The dephosphorylated M These data show that there is very little, fragment was then treated with polynucleoif any, 5’ -+ 3’ exonuclease activity present tide kinase and y-[“*P]ATP. As shown in under these conditions and the labeled fragTABLE
1
462
SHINAGAWA
AND PADMANABHAN
ments can be used for DNA sequence analysis. We also tested whether there is any contamination of a nuclease that could degrade RNA. Yeast RNA, extracted following the procedure of Rubin (19), was used as a substrate for the phosphatase in the presence or absence of SDS. The data presented in Fig. 4 show that a contaminating nuclease which caused degradation of 5.8 S and 5 S ribosomal RNAs (bands 1 and 2 in lane 2) could be inhibited by the addition of 0.1% SDS to the reaction mixture. DISCUSSION
Bacterial alkaline phosphatase is a remarkably stable enzyme. Heppel et al. (17) reported that the recovery of the activity of the enzyme after exposure to 925°C for 5 min is very fast at room temperature. Within 100 min more than 80% of the activity was restored. Urea (7.5 M) inhibited the hydrolysis ofp-nitrophenyl phosphate to the extent of 75% but this is completely reversible after 4 h of incubation (17). The presence of nucleases in the commercial preparations of the phosphatase has been a problem, interfering with the specific labeling of the termini after dephosphorylation of the DNA (or RNA) fragment. Smith and Birnstiel(20) described a procedure to inactivate the con-
FIG. 3. Partial DNA sequence analysis of terminal Hue111 fragment of Ad 2 DNA. SmaI-K fragment of Ad 2 DNA is at the right-hand terminus3 and about 590 base pairs in length. SmaI-K fragment (6.7 pmol) was digested with 5 units of the restriction enzyme Hue111 [for a review, see Ref. (S)] in 50 ~1 of reaction mixture containing 6 mM each of Tris-HCI, pH 7.6, MgCI*, dithiothreitol, and NaCl. Incubation was car-
tied out at 37°C for 80 min. Subsequently, the reaction mixture was adjusted to 0.1 M Tris-HCl, pH 8.0, 10 PM ZnSO,, and 0.1% SDS. Dephosphorylation was carried out at 37°C for 90 min by adding 10 ~1 of the phosphatase (0.85 units) in 0.1 M Tris-HCI, pH 8.0, and 10 PM ZnSO,. The reaction mixture was terminated by extracting with phenol. Procedures involved in the recovery of the DNA fragments, labeling at their 5’ termini, isolation of the labeled fragments, and their sequence analysis were according to Maxam and Gilbert (7). We used an acrylamide concentration of 12% for the fractionation of the labeled products generated by the chemical degradation (7). Only a partial DNA sequence of a region which is about 42 nucleotides away from the 5’ terminus of the Hue111 recognition site at the right-hand end of Ad 2 DNA is lettered in this figure.
ALKALINE
PHOSPHATASE
FROM
E. co/i
463
We report in this communication an alternate method useful for the complete inhibiO-0 tion of the contaminating nucleases present in the commercial phosphatase enzyme. The amount of the contaminating nucleases present in the commercial preparations varies from batch to batch. One batch (Lot No. 54M423 of BAPF grade from Worthing-Band 1 ton) was found to contain appreciable amounts of nuclease activity compared to -Band 2 another batch (Lot No. 58H339 of BAPF grade). The specific activity of the enzyme in the former lot was 20 U/mg compared to 67 U/mg present in the latter batch. The type of the endonuclease activity degrading the double-stranded DNA is not known. It is quite possible that E. cofi endonuclease I is the likely contaminant. In addiFIG. 4. Polyacrylamide gel electrophoresis of phostion, we find that a nuclease which could phatase-treated yeast RNA. Yeast RNA was isolated degrade RNA was also present in the batch by phenol extraction as described by Rubin (19). Treat(Lot No. 54M423 BAPF; see Fig. 4) which ment with the phosphatase was carried out in a reaction mixture containing 21 pg of RNA as described under we tested. These contaminating nucleases Materials and Methods except that the duration of the could be inhibited by carrying out the deincubation period was 3 h. A 10% polyacrylamide slab phosphorylation in the presence of 0.1% gel system (bisacrylamide:acrylamide ratio, 1:29) was SDS (Figs. 2 and 4). In our studies, we found used in which the electrode buffer contained 50 mM that there was no difference in phosphatase Tris-borate, pH 8.3, and 1 mM EDTA and the gel contained, in addition, 7 M urea. Electrophoresis was activity between 0.1% and 1% SDS present carried out at 600 V for 2 h. The gel was stained with in the incubation mixture. Single-strand 0.2% methylene blue in 0.4 M acetate buffer (28) and DNA-specific S, nuclease from Aspergillus destained with water for 8 h. The mobilities of the commyzae is another example of an enzyme ponents of yeast RNA are shown without any treatment (lane I), or after treatment with phosphatase in the which is active in the presence of SDS up to a concentration of 0.3% (23). presence (lane 3) or absence (lane 2) of 0.1% SDS. Bands I and 2 represent the 5.8 S and 5 S ribosomal Several properties of the subunit of E. cofi RNA, and the heavily stained region is due to tRNA alkaline phosphatase have been studied in (19). detail (24). In contrast to the dimer, the subunit is more labile, easily denatured by peritaminating nucleases by heating the phos- odate and ionic detergents. Our present phatase to 95°C for 10 min. Weiss et al. communication, that the nuclease activity (21) reported a procedure for the further present as a contaminant in commercial purification of the commercial alkaline preparations of E. coli alkaline phosphatase phosphatase (BAPC grade from Worthingcan be selectively destroyed, will be useful ton) using a column of DEAE-cellulose for in the rapid methodology of sequence analythe removal of endonuclease activity. Cha- sis of DNA (or RNA). conas et al. (22) utilized the property of reversal of T4 polynucleotide kinase activity REFERENCES in the presence of a phosphate acceptor 1. Malamy, M. H., and Horecker. B. L. (1964) Kc>(ADP) in 5’ end-group labeling of RNA and chemistry 3, 18891892. double-stranded DNA without using the Bi+ 2. Garen, A., and Levinthal, C. (1960) Biochim. phys. Acta 38, 470-483. phosphatase treatment.
1
2
3
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SHINAGAWA
AND PADMANABHAN
3. Schwartz, J. H., and Lipmann, F. (1961) Proc. Nat. Acad. Sci. USA 47, 1996-2005. 4. Malamy, M. H., and Horecker, B. L. (1964) Biochemistry 3, 1893-1897. 5. Roberts, R. J. (1976) CRC Crit. Rev. Biochem. 4, 123-164. 6. Sanger, F., and Coulson, A. R. (1975) J. Mol. Biol. 94,441-448. 7. Maxam, A. M., and Gilbert, W. (1977) Proc. Nat. Acad. Sci. USA 74, 560-564. 8. Keller, H. D., Maxam, A. M., and Gilbert, W. (1977) Nucl. Acids Res. 4, 2527-2537. 9. Simonesits, A., Brownlee, G. G., Brown, R. S., Rubin, 3. R., and Guilley, H. (1977) Nature (London) 269, 833-836. 10. Richardson, C. C. (1965) Proc. Nat. Acad. Sci. USA 54, 158-165. 11. Novogradosky, A., and Hurwitz, J. (1966)J. Biol. Chem. 241, 2923-2932. 12. Johnson, P. H., and Sinsheimer, R. L. (1974) J. Mol. Biol. 83, 47-61. 13. Sharp, P. A., Sugden, B., and Sambrook, J. (1973) Biochemistry 12, 3055-3063. 14. Wu, R., Jay, E., and Roychundhury, R. (1976) in Methods in Cancer Research (Busch, H., ed.), Vol. 12, pp. 87- 176, Academic Press, New York. 15. Lillehaug, J. R., and Kleppe, K. (1975) Biochemistry 14, 1225-1229. 16. Wu, R., Padmanabhan, R., and Bambara, R. (1974)
17. 18. 19.
20. 21. 22.
23. 24. 25. 26. 27. 28.
in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 29E, pp. 23 l-254, Academic Press, New York. Heppel, L. A., Harkness, D. R., and Hilmoe, R. 3. (1962) J. Biol. Chem. 237, 841-846. Sato, S., Hutchison, C. A., and Harris, J. 1. (1977) Proc. Nat. Acad. Sci. USA 74, 542-546. Rubin, G. M. (1975) in “Methods in Cell Biology” (Prescott, D. M., ed.), Vol. 12, pp. 45-63, Academic Press, New York. Smith, H. O., and Birnstiel, M. L. (1976) Nucl. Acids Res. 3, 2387-2398. Weiss, B., Live, T. R., and Richardson, C. C. (1968) J. Biol. Chem. 243, 4530-4542. Chaconas, G., van de Sande, J. H., and Church, R. B. (1975) Biochem. Biophys. Res. Commun. 66, %2-969. Vogt, V. M. (1973) Eur. J. Biochem. 33,192-200. Schlesinger, M. J. (1965) J. Biol. Chem. 240,42934298. Aaij, C., and Borst, P. (1972) Biochim. Biophys. Acta 269, 192-200. Dingman, C. W., Fisher, M. P., and Kakefuda, T. (1972) Biochemistry 11, 1242-1250. Johnson, P. H., and Grossman, L. I. (1977) Biochemistry 16,4217-4224. Peacock, A. C., and Dingman, C. W. (1967) Biochemistry 6, 1818-1827.
BIOCHEMISTRY
Inhibition
95,458-464
(1979)
of a Nuclease Contaminant in the Commercial of Escherichia co/i Alkaline Phosphatasel MORIKAZU SHINAGAWAAND Department
Preparations
R. PADMANABHAN~
of Biological Chemistry, University of Maryland 660 West Redwood Street, Baltimore, Maryland
School 21201
of Medicine,
Received August 24, 1978 Escherichia co/i alkaline phosphatase is a valuable reagent for removal of terminal phosphate from both ribo- and deoxyribo-oligonucleotides or from restriction enzyme fragments of DNA. Some commercial preparations of this enzyme were found to be contaminated with nucleases which could degrade both DNA and RNA. These contaminating nucleases can be completely eliminated by carrying out the enzymic reaction in the presence of 0. l- 1% sodium dodecyl sulfate without any loss of phosphatase activity. This report has immediate application in the sequence analysis of DNA or RNA.
Escherichia coli alkaline phosphatase [EC 3.1.3. l] is a heat-stable metalloenzyme whose active form is dimer and is known to be localized in the periplasmic space of the cell (1). The procedure for the purification of the enzyme has been described (2,3) and the enzyme has also been crystallized (4). Alkaline phosphatase is a valuable reagent used for the analysis of the primary structure of RNA and DNA. It is used widely to remove the terminal phosphate from ribo- or deoxyribo-oligonucleotides at their 3’ or 5’ termini. Since the discovery of restriction enzymes [see Ref. (5) for a review] which cleave DNA at specific nucleotide sequences, structural as well as genetic analyses of DNA have been progressing extremely rapidly. Recently, rapid methods have become available for the nucleotide sequence analysis of both DNA and RNA (6-9). These methods are dependent on successful labeling techniques involving 32P of very high specific activities at a L This work was supported by U. S. Public Health Service Grant CA 20369. R. P. gratefully acknowledges the support from Research Career Development Award from National Institutes of Health (5 K04 CA 00235). * To whom reprint requests should be sent. 0003-2697/79/080458-07$02.00/O Copynght All rights
8 1979 by Academic Press, Inc. of reproduction in any form reserved.
unique terminus. A restriction enzyme fragment of DNA can be labeled at its 5’ termini by treatment with alkaline phosphatase to remove the terminal phosphate residues, followed by phosphorylation using polynucleotide kinase and Y-[~~P]ATP (10,ll). The alkaline phosphatase is now commercially available. Yet commercial preparations are often contaminated with small amounts of nucleases which could degrade the DNA or the RNA and thus reduce the yield of labeled fragment. We wish to report here that the presence of 0.1% sodium dodecyl sulfate (SDS)3 in the incubation mixture completely inhibits the nuclease activity without altering the phosphatase activity. A DNA fragment obtained by cleaving human adenovirus type 2 DNA with restriction enzyme SmaI (isolated from Serratia marcescens)4 was dephosphorylated by treating with alkaline phosphatase in the 3 Abbreviations used: SDS, sodium dodecyl sulfate; Ad, adenovirus; TEN buffer, 0.01 M Tris-HCl (pH 7.4), 1 mtvfEDTA, 0.1 M NaCI; DEAE, diethylaminoethyl; dpC, deoxycytidine Y-phosphate. 4 The cleavage map of adenovirus type 2 DNA was established by Mulder et al. (C. Mulder, M. Greene, and H. Delius, personal communication). 458
ALKALINE
PHOSPHATASE
presence of 0.1% SDS. This fragment could then be labeled at its 5’ termini using polynucleotide kinase and -Y-[~~P]ATP. We have demonstrated that a DNA fragment labeled at the 5’ termini in this manner could be successfully used for sequence analysis following the procedure of Maxam and Gilbert (7). MATERIALS
AND METHODS
Enzymes and chemicals. Alkaline phosphatase from E. coli was purchased from Worthington Biochemical Corporation (BAPF grade). The enzyme, suspended in 3.2 M (NH,),SO, at a concentration of 20 units/ mg, was centrifuged at 10,000 t-pm for 5 min. The precipitate was dissolved in 0.1 M Tris-HCl, pH 8.0, and 10 PM ZnSO, and dialyzed against the same buffer for 12 h at 4°C. The concentration of enzyme after dialysis was 48 units/ml. Enzyme dilutions were made in 0.1 M Tris-HCl (pH 8.0). Polynucleotide kinase was obtained from P. L. Biochemicals. Yeast RNA which contains a mixture of 5.8 S and 5 S ribosomal RNA as well as tRNA was a gift from Mr. Guy Adami. SmaI and iYaeII1 restriction enzymes were kindly provided by Dr. Werner Buettner. Sodium p-nitrophenyl phosphate, SDS, and ethidium bromide were from Sigma Chemical Company. SDS was recrystallized from aqueous ethanol before use. SeaKem agarose was obtained from Microbiological Associates (Rockville, Md.) +X174 DNA (RFI) was prepared as described previously (12). Spectrophotometric measurement of alkaline phosphatase activity. Hydrolysis of p-nitrophenyl phosphate was measured colorimetrically as described by Garen and Levinthal (2). When the SDS was used in the incubation mixture, the concentration was either 0.1 or 1% as indicated. The amount of enzyme used per assay was 0.006 U in a total volume of 1 ml. Alkaline phosphatase treatment of +X174 DNA. A reaction mixture containing 1.9 pg
FROM
E. co/i
459
of DNA, 0.1 M Tris-HCl buffer, pH 8.0, and 0.24 units of the enzyme was incubated in a total volume of 60 ~1 in the absence or presence of 0.1% SDS. After incubation at 37°C for 2 h, 40 ~1 of a mixture containing 50% sucrose, 25 mM EDTA, and 0.02% bromophenol blue was added. An aliquot of 50 ~1 was applied to an agarose gel (1.4%) for electrophoresis. Agarose gel electrophoresis. DNA treated with alkaline phosphatase was analyzed by electrophoresis on an agarose gel (1.4%) and detected by its fluorescence with ethidium bromide (13). The buffer system used for electrophoresis was: 36 mM Tris, 17 mM N&HP04, 13 mM NaH,PO,, 1 mM EDTA (pH 7.8). Phosphorylation by polynucleotide kinase of Smal-M fragment after treatment with alkalinephosphatase. SmaI cleaves Ad 2 DNA into 13 fragments (C. Mulder et al., personal communication). The fragments were separated by a 1.4% agarose gel and visualized by their ethidium bromide fluorescence. The smallest fragment M (about 140 base pairs long) was isolated from the gel using hydroxyapatite according to the procedure described by Wu et al. (14). The M fragment was dephosphorylated either in the presence or absence of 0.1% SDS by incubating a mixture containing 22 pmol (3 pg) of M fragment, 0.1 M Tris-HCl, pH 8.0, and 0.24 units of alkaline phosphatase for 60 min at 37°C. After the incubation, the reaction mixture was diluted to 200 pl with 0.01 M Tris-HCI (pH 7.4), 1 mM EDTA, and 0.1 M NaCl (TEN buffer). It was then extracted with 200 pl of redistilled phenol saturated with TEN buffer. The phenol layer was washed with 100 ~1 of TEN buffer. The pooled aqueous layer was extracted with an equal volume of ether four times. The DNA was precipitated with ethanol and stored at -70°C for 3 h. After centrifugation at 10,000 rpm for 20 min, the DNA pellet was dissolved in 200 ~1 of 0.3 M sodium acetate and the DNA was reprecipitated
460
SHINAGAWA
AND PADMANABHAN
with 3 vol of ethanol. The DNA pellet was then dried and dissolved in 20 ~1 of sterile distilled H,O. Polynucleotide kinase reaction of M fragment was carried out in a total volume of 55 ~1 containing 11 pmol(l.5 pg) of dephosphorylated DNA, 5.5 ~1 of 10 x kinase buffer pH 8.0, 0.1 M MgC&, (0.5 M Tris-HCl, 4.5 ~1 of 1 mM and 0.05 M dithiothreitol), spermidine-HCl (15), 35 ~1 of H20, 150 pmol of y-[32P]ATP (specific activity 414 mCi/pmol) and 2 units of polynucleotide kinase. The incubation was carried out at 37°C. Aliquots of 3-~1 samples were taken in duplicate at 0 (before the addition of enzyme), 15, and 30 min. Acid-insoluble radioactivity was determined as described (16).
1
2
3
OFORM
II-
FORM IllFORM
I-
RESULTS
Figure 1 shows the activity of the enzyme in the absence or presence of 1% SDS. The enzyme is not inhibited by SDS. Urea (7.5 M), however, inhibits the enzyme activity to 79%, very close to the value reported
i o.?l-0 t
C
0.5
I.0 TIME
I.5
2.0
2.5
FIG. 2. Agarose gel electrophoresis of phosphatasetreated +X174 DNA RFI. $X174 DNA (Form I) was treated with phosphatase in the presence (lane 2) or absence (lane 3) of 0.1% SDS. The untreated DNA is shown in lane 1. Phosphatase treatment was carried out as described under Materials and Methods. Electrophoresis was carried out at 100 V (6.6 V/cm) on a 1.4% agarose gel for 9 h and the ethidium bromide fluorescence of the DNA was photographed (13). Under these conditions of ionic and electric field strengths, the mobilities of the three different conformational forms of 4x174 DNA are in the following decreasing order: Form I > Form III > Form II, as expected (25-27).
IN MINUTES
FIG. 1. The enzymic activity of alkaline phosphatase in the presence or absence of SDS. The activity was measured as the rate of hydrolysis of sodium p-nitrophenyl phosphate as described previously (2), using a Cary 15 spectrophotometer equipped with a recorder. (a) No SDS (control) or 0.1% or 1% SDS; the rates in all three cases were identical. (b) 4 M guanidine-HCI was present in the incubation mixture. (c) 7.5 M urea was present in the reaction.
by Heppel et al. (17). When 4 M guanidine hydrochloride was present in the incubation mixture, the activity was 57% of the initial value (Fig. lb). Sarkosyl, like SDS, does not show any inhibition of phosphatase activity. Commercial preparations of alkaline phosphatase (even the BAPF grade from
ALKALINE
PHOSPHATASE
FROM E.
coli
461
Table 1, the fraction of M fragment which by the phosphatase PHOSPHORYLATION OF SnzaI-M FRAGMENT OF Ad 2 was dephosphorylated DNA BY POLYNUCLEOTIDE KINASE in the presence of SDS served as a substrate AND y[32P]ATPz with as much efficiency as the fraction which was treated with phosphatase in the Acid-insoluble radioabsence of SDS. The small decrease in the Duration of activity incorporated incorporation of radioactivity in the fraction polynucleotide into M fragment which was treated with alkaline phosphakinase reaction (min) A B tase in the absence of SDS may be attributable to a slight reduction in the amount of 0 1,299 812 substrate due to the degradation of the frag15 51,079 42,654 ment by the contaminant nuclease present 30 57,730 45,015 in the phosphatase. U The SlnuI-M fragment of Ad 2 DNA was isolated We have tested the suitability of a DNA as described under Materials and Methods. Dephosfragment labeled in this manner for sephorylation of M fragment was carried out using alkaof line phosphatase either in the presence of 0.1% SDS quence analysis using the procedure Maxam and Gilbert (7). Figure 3 shows a (A) or in its absence (B). Dephosphorylated M fragment partial sequence of a region about 42 nucleowas then labeled at its 5’ termini using polynucleotide kinase and Y[~~P]ATP as described under Materials and tides away from the 5’ terminus of Hue111 Methods, and acid-insoluble radioactivity incorporated recognition site at the right-hand end of Ad was measured ( 16). The values represent an average of 2 DNA. The sequence can be read unamsamples in duplicate. biguously from the 5’-labeled terminus of the terminal Hue111 fragment of Ad 2 DNA. Worthington) were found to contain small Moreover, we digested Ad 2 DNA with amounts of deoxyribonuclease which could TuqI (Bethesda Research Laboratory, Inc.) convert $X 174 Form I to II (nicked circular) and dephosphorylated the DNA fragments and III (linear DNA), which was then fur- using the alkaline phosphatase in the presther degraded upon incubation with the en- ence of 0.1% SDS. After the fragments were zyme at 37°C for 2 h (Fig. 2, lane 3). How- labeled at their 5’ termini with szP using ever, this contaminating nuclease activity polynucleotide kinase and y-[32P]ATP as could be completely inhibited by carrying described under Materials and Methods the out the phosphatase reaction in the presence 5’ end-group analysis of the labeled fragof 0.1% SDS (Fig. 2, lane 2). ments showed 89% as dpC residue. The In order to test whether treatment of a remaining 11% was distributed among the restriction enzyme fragment of DNA with other three nucleotides. The DNA sequence alkaline phosphatase in the presence of recognized by TuqI at the cleavage site is SDS, followed by the removal of SDS, leaves any inhibitor to the polynucleotide 1 5’-T-C-G-A-3’ kinase reaction, the SmaI-M fragment of Ad 2 DNA (about 140 base pairs long) was 3’-A-G-C-T-5’ used for labeling the termini. Alkaline phos? phatase treatment of M fragment was carried out either in the presence or absence and hence dpC residues are expected at the of 0.1% SDS as described under Materials 5’ termini of the cleavage products (18). and Methods. The dephosphorylated M These data show that there is very little, fragment was then treated with polynucleoif any, 5’ -+ 3’ exonuclease activity present tide kinase and y-[“*P]ATP. As shown in under these conditions and the labeled fragTABLE
1
462
SHINAGAWA
AND PADMANABHAN
ments can be used for DNA sequence analysis. We also tested whether there is any contamination of a nuclease that could degrade RNA. Yeast RNA, extracted following the procedure of Rubin (19), was used as a substrate for the phosphatase in the presence or absence of SDS. The data presented in Fig. 4 show that a contaminating nuclease which caused degradation of 5.8 S and 5 S ribosomal RNAs (bands 1 and 2 in lane 2) could be inhibited by the addition of 0.1% SDS to the reaction mixture. DISCUSSION
Bacterial alkaline phosphatase is a remarkably stable enzyme. Heppel et al. (17) reported that the recovery of the activity of the enzyme after exposure to 925°C for 5 min is very fast at room temperature. Within 100 min more than 80% of the activity was restored. Urea (7.5 M) inhibited the hydrolysis ofp-nitrophenyl phosphate to the extent of 75% but this is completely reversible after 4 h of incubation (17). The presence of nucleases in the commercial preparations of the phosphatase has been a problem, interfering with the specific labeling of the termini after dephosphorylation of the DNA (or RNA) fragment. Smith and Birnstiel(20) described a procedure to inactivate the con-
FIG. 3. Partial DNA sequence analysis of terminal Hue111 fragment of Ad 2 DNA. SmaI-K fragment of Ad 2 DNA is at the right-hand terminus3 and about 590 base pairs in length. SmaI-K fragment (6.7 pmol) was digested with 5 units of the restriction enzyme Hue111 [for a review, see Ref. (S)] in 50 ~1 of reaction mixture containing 6 mM each of Tris-HCI, pH 7.6, MgCI*, dithiothreitol, and NaCl. Incubation was car-
tied out at 37°C for 80 min. Subsequently, the reaction mixture was adjusted to 0.1 M Tris-HCl, pH 8.0, 10 PM ZnSO,, and 0.1% SDS. Dephosphorylation was carried out at 37°C for 90 min by adding 10 ~1 of the phosphatase (0.85 units) in 0.1 M Tris-HCI, pH 8.0, and 10 PM ZnSO,. The reaction mixture was terminated by extracting with phenol. Procedures involved in the recovery of the DNA fragments, labeling at their 5’ termini, isolation of the labeled fragments, and their sequence analysis were according to Maxam and Gilbert (7). We used an acrylamide concentration of 12% for the fractionation of the labeled products generated by the chemical degradation (7). Only a partial DNA sequence of a region which is about 42 nucleotides away from the 5’ terminus of the Hue111 recognition site at the right-hand end of Ad 2 DNA is lettered in this figure.
ALKALINE
PHOSPHATASE
FROM
E. co/i
463
We report in this communication an alternate method useful for the complete inhibiO-0 tion of the contaminating nucleases present in the commercial phosphatase enzyme. The amount of the contaminating nucleases present in the commercial preparations varies from batch to batch. One batch (Lot No. 54M423 of BAPF grade from Worthing-Band 1 ton) was found to contain appreciable amounts of nuclease activity compared to -Band 2 another batch (Lot No. 58H339 of BAPF grade). The specific activity of the enzyme in the former lot was 20 U/mg compared to 67 U/mg present in the latter batch. The type of the endonuclease activity degrading the double-stranded DNA is not known. It is quite possible that E. cofi endonuclease I is the likely contaminant. In addiFIG. 4. Polyacrylamide gel electrophoresis of phostion, we find that a nuclease which could phatase-treated yeast RNA. Yeast RNA was isolated degrade RNA was also present in the batch by phenol extraction as described by Rubin (19). Treat(Lot No. 54M423 BAPF; see Fig. 4) which ment with the phosphatase was carried out in a reaction mixture containing 21 pg of RNA as described under we tested. These contaminating nucleases Materials and Methods except that the duration of the could be inhibited by carrying out the deincubation period was 3 h. A 10% polyacrylamide slab phosphorylation in the presence of 0.1% gel system (bisacrylamide:acrylamide ratio, 1:29) was SDS (Figs. 2 and 4). In our studies, we found used in which the electrode buffer contained 50 mM that there was no difference in phosphatase Tris-borate, pH 8.3, and 1 mM EDTA and the gel contained, in addition, 7 M urea. Electrophoresis was activity between 0.1% and 1% SDS present carried out at 600 V for 2 h. The gel was stained with in the incubation mixture. Single-strand 0.2% methylene blue in 0.4 M acetate buffer (28) and DNA-specific S, nuclease from Aspergillus destained with water for 8 h. The mobilities of the commyzae is another example of an enzyme ponents of yeast RNA are shown without any treatment (lane I), or after treatment with phosphatase in the which is active in the presence of SDS up to a concentration of 0.3% (23). presence (lane 3) or absence (lane 2) of 0.1% SDS. Bands I and 2 represent the 5.8 S and 5 S ribosomal Several properties of the subunit of E. cofi RNA, and the heavily stained region is due to tRNA alkaline phosphatase have been studied in (19). detail (24). In contrast to the dimer, the subunit is more labile, easily denatured by peritaminating nucleases by heating the phos- odate and ionic detergents. Our present phatase to 95°C for 10 min. Weiss et al. communication, that the nuclease activity (21) reported a procedure for the further present as a contaminant in commercial purification of the commercial alkaline preparations of E. coli alkaline phosphatase phosphatase (BAPC grade from Worthingcan be selectively destroyed, will be useful ton) using a column of DEAE-cellulose for in the rapid methodology of sequence analythe removal of endonuclease activity. Cha- sis of DNA (or RNA). conas et al. (22) utilized the property of reversal of T4 polynucleotide kinase activity REFERENCES in the presence of a phosphate acceptor 1. Malamy, M. H., and Horecker. B. L. (1964) Kc>(ADP) in 5’ end-group labeling of RNA and chemistry 3, 18891892. double-stranded DNA without using the Bi+ 2. Garen, A., and Levinthal, C. (1960) Biochim. phys. Acta 38, 470-483. phosphatase treatment.
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3
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3. Schwartz, J. H., and Lipmann, F. (1961) Proc. Nat. Acad. Sci. USA 47, 1996-2005. 4. Malamy, M. H., and Horecker, B. L. (1964) Biochemistry 3, 1893-1897. 5. Roberts, R. J. (1976) CRC Crit. Rev. Biochem. 4, 123-164. 6. Sanger, F., and Coulson, A. R. (1975) J. Mol. Biol. 94,441-448. 7. Maxam, A. M., and Gilbert, W. (1977) Proc. Nat. Acad. Sci. USA 74, 560-564. 8. Keller, H. D., Maxam, A. M., and Gilbert, W. (1977) Nucl. Acids Res. 4, 2527-2537. 9. Simonesits, A., Brownlee, G. G., Brown, R. S., Rubin, 3. R., and Guilley, H. (1977) Nature (London) 269, 833-836. 10. Richardson, C. C. (1965) Proc. Nat. Acad. Sci. USA 54, 158-165. 11. Novogradosky, A., and Hurwitz, J. (1966)J. Biol. Chem. 241, 2923-2932. 12. Johnson, P. H., and Sinsheimer, R. L. (1974) J. Mol. Biol. 83, 47-61. 13. Sharp, P. A., Sugden, B., and Sambrook, J. (1973) Biochemistry 12, 3055-3063. 14. Wu, R., Jay, E., and Roychundhury, R. (1976) in Methods in Cancer Research (Busch, H., ed.), Vol. 12, pp. 87- 176, Academic Press, New York. 15. Lillehaug, J. R., and Kleppe, K. (1975) Biochemistry 14, 1225-1229. 16. Wu, R., Padmanabhan, R., and Bambara, R. (1974)
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in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 29E, pp. 23 l-254, Academic Press, New York. Heppel, L. A., Harkness, D. R., and Hilmoe, R. 3. (1962) J. Biol. Chem. 237, 841-846. Sato, S., Hutchison, C. A., and Harris, J. 1. (1977) Proc. Nat. Acad. Sci. USA 74, 542-546. Rubin, G. M. (1975) in “Methods in Cell Biology” (Prescott, D. M., ed.), Vol. 12, pp. 45-63, Academic Press, New York. Smith, H. O., and Birnstiel, M. L. (1976) Nucl. Acids Res. 3, 2387-2398. Weiss, B., Live, T. R., and Richardson, C. C. (1968) J. Biol. Chem. 243, 4530-4542. Chaconas, G., van de Sande, J. H., and Church, R. B. (1975) Biochem. Biophys. Res. Commun. 66, %2-969. Vogt, V. M. (1973) Eur. J. Biochem. 33,192-200. Schlesinger, M. J. (1965) J. Biol. Chem. 240,42934298. Aaij, C., and Borst, P. (1972) Biochim. Biophys. Acta 269, 192-200. Dingman, C. W., Fisher, M. P., and Kakefuda, T. (1972) Biochemistry 11, 1242-1250. Johnson, P. H., and Grossman, L. I. (1977) Biochemistry 16,4217-4224. Peacock, A. C., and Dingman, C. W. (1967) Biochemistry 6, 1818-1827.