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[63] DNase-I inhibitor protein. Isolation with DNase-agarose
[63]
ISOLATION OF DNase-I INHIBITOR PROTEIN
[63] DNase-I
517
Inhibitor Protein. Isolation with DNase-Agarose By UNO LINDBERG
The presence of a protein DNase I-inhibitor has been demonstrated in many different types of mammalian tissues. The inhibitor protein has been purified by conventional techniques from calf spleen 1 and thymus °and from rat serum, '~ and, as described here, a generally applicable method for the purification of this protein by affinity chromatography on DNaseSepharose has been achieved? It has been found that the DNase I inhibitor is present in the ceils in surprisingly large amounts. For example, this protein constitutes 5 - 1 0 % of the soluble protein of thymus cells. However, nothing is yet known about its biological function or, for that matter, its relation to deoxyribonucleases in the living ceil. Procedure Buffers
Wash buffer I: 4 M guanidine-HCl; 30% glycerol; 0.5 M sodium acetate at pH 6.5 Wash buffer II: 10 mM Tris chloride at pH 7.5; 5 mM CaC12 Extraction buffer: 10 mM Tris chloride at pH 7.5; 10 mM NaC1; 10 mM E D T A Elution buffer I: 0.75 M guanidine-HC1; 0.5 M sodium acetate30% glycerol; adjusted to pH 6.5 Elution buffer II: 3 M guanidine-HC1; 1.0 M sodium acetate; 30% glycerol adjusted to pH 6.5. Coupling of DNase I to Agarose
Activation of Sepharose with cyanogen bromide is performed as described by Kato and Anfinsen. 5 Twenty milliliters of the commercial slurry of Sepharose 4B is washed with 600 ml of deionized water and resuspended in 40 ml of water. Cyanogen bromide solution (20 ml containing 0.6 g of CNBr) is added, and the pH is brought to 11-11.5 by the addition of 2 M ~U. Lindberg, Biochemistry 6, 323 (1967). 2U. Lindberg and L. Skoog, Eur. J. Biochem. 13, 326 (1970). '~G. Berger and P. May, Biochim. Biophys. Acta 139, 148 (1967). 4U. Lindberg and S. Eriksson, Eur. I. Biochem. 18, 474 (1971). 6I. Kato and C. B. Anfinsen, J. Biol. Chem. 244, 5849 (1969).
518
NUCLEIC ACIDS, NUCLEOTIDES, AND DERIVATIVES
[63]
NaOH and maintained by dropwise addition of NaOH. After 5 - 1 0 minutes, the pH remains stable and the activated Sepharose is immediately washed with ice-cold deionized water on a Btichner funnel. Bovine pancreatic DNase (Sigma) ~ 20 mg in 2 ml of 0.1 M NaHCO3, is mixed with the activated Sepharose, which has been resuspended in 25 ml of 0.1 M NaHCO3 at 4 °. The time course of the coupling reaction is followed by the absorbance at 280 nm of aliquots of the reaction mixture after centrifugation. Within 2 hours 90% of the enzyme protein has been fixed to the gel, and essentially all after another 24 hours at 4 °. The DNaseSepharose is then washed on a Btichner funnel with 500 ml each of water, 0.1 M NaHCO3, and again with water. It is packed into a column, and noncovalently bound protein is removed by passage through 50 ml of wash buffer I followed by 500 ml of wash buffer II. The DNase-Sepharose may be stored for several months at 4 ° without loss in effectiveness. After a purification procedure has been performed, the DNase-Sepharose is washed with wash buffers I and II as indicated above and is again ready for use. When incubated with the DNA substrate (calf thymus DNA, Sigma) used in the standard DNase assay, 7 DNase-Sepharose conjugate causes a slow but significant degradation of the DNA. The rate of the reaction is about 1% that obtained with an equal amount of free DNase.
Purification of DNase-Inhibitor ]rom Crude Extracts In the preparation described here calf thymus was used as the source of the inhibitor, although the same procedure has been effective for the purification of the inhibitor from several different tissues and species including tissue-cultured human cells. Frozen thymus tissue, 50 g, is thawed in 250 ml of extraction buffer and homogenized for 30 seconds in a high speed Turmix homogenizer after the addition of 0.1 volume of 5% NP-4(~. s The homogenate is centrifuged for 10 minutes at 12,000 g and the supernatant fluid is centrifuged again for 2 hours at 100,000 g. The supernatant liquid from this step may be stored at - 2 0 ° without loss of inhibitor activity. A sample of the crude extract (22 ml containing 450 mg of protein; a total of 870,000 inhibitor units) is applied to a column of DNaseSepharose (2 × 1.5 cm). After the sample has entered the column, unadsorbed protein is washed out with wash buffer II. Adsorbed proteins are then eluted in two steps with elution buffer I in the first step and elution ° Before use, the DNase (DN-.100, Sigma) is purified by chromatography on Sephadex G-100 in wash buffer II. ~U. Lindberg, Biochim. Biophys. Acta 82, 237 (1964). ~Nonidet P-40 (NP-40) is a nonionic detergent obtained from Shell Chemical Co.
[63]
ISOLATION OF DNase-I INHIBITOR PROTEIN
519
SUMMARY OF THE ]~ESULTS OF THE AFFINITY CHROMATOGRAPHY OF THE CRUDE EXTRACT OF CALF THYMUS a,b
Fraction 1. Crude extract 2. Nonadsorbable protein 3. Lightly adsorbed proteinb 4. Firmly adsorbed proteinb
Activ- ActivSpecific ity ity Pro- activity (k yield tein (units/ units) (C/c) (mg) mg) 870 55 145 71
100 6 17 8
450 415 20 8
1900 130 7200 8800
A26o/A2so
(a)
(b)
DNA (~g)
1.49 1.18 1.65 1.55
--1.45 1.30
--50 7
From U. Lindberg and S. Eriksson, Eur. J. Biochem. 18, 474 (1971). b Fractions 3 and 4 were analyzed before (a) and after (b) concentration by salt precipitation (dialysis against saturated ammonium sulfate overnight). After salt precipitation, the samples were dissolved in 10 mM sodium acetate, pH 6.5, and assayed for inhibitor activity after the addition of 0.75 M guanidine-HC1-0.5 M sodium acetate-3(}~',~,glycerol pH 6.5.
a
buffer II in the second. As much as 28 mg ( 6 % ) of the protein of the crude extract is adsorbed to the column. Of this total, 20 mg are relatively loosely bound and are removed with the first buffer whereas the remaining, more firmly attached protein consisting mainly of the DNase-inhibitor, is displaced by the second buffer. About 20% of the inhibitor activity is found in the first eluate and 10% in the second. The absorbance spectrum of the eluted fraction exhibit a maximum at 260 nm with a ratio of 1.6 between the absorbancies at 260 and 280 nm indicating the presence of nucleic acid material. It has been shown that this is due largely to RNA, but the reason for the adsorption of R N A to DNase-Sepharose has not been investigated. That the protein in the second eluate consists mainly of the inhibitor protein has been demonstrated by polyacrylamide gel electrophoresis and by peptide mapping.' A summary of the affinity chromatography of the calf thymus extract on DNase-Sepharose is given in the table. It should be pointed out that the inhibitor is n o t recovered in a highly active form and that conditions have not been found as yet which increase the activity of the adsorbed and eluted inhibitor protein. From the work on the purification of the DNase-inhibitor by conventional methods, it is known that the protein has a marked tendency to form high molecular weight aggregates with low inhibitory activity. From those studies it is also known that 0.75 M guanidine-HCI-0.5 M sodium acetate30% glycerol is effective in dissociating such aggregates to protein monomers of high specific activity. However, these conditions do not dissociate the enzyme-inhibitor complex. It is possible therefore that the presence
520
NUCLEIC ACIDS, NUCLEOTIDES, AND DERIVATIVES
[64]
of inhibitor activity in the first eluate is due to release of inhibitor molecules from inhibitor aggregates bound to the DNase-Sepharose, rather than to release of active inhibitor from the DNase as such. The situation in 3 M guanidine-HC1 is different. This concentration of guanidine-HCl is known to rapidly inactivate the inhibitor and would lead to dissociation of the DNase-inhibitor complex. 4
[64] Purification of Thymidylate Synthetase 2'-Deoxyuridylate-Agarose
with
B y PETER V. DANENBERG and CHARLES HEIDELBERGER
The partial purification of thymidylate synthetase from a number of bacterial and animal sources has been achieved by multistep sequences of conventional procedures. 1 6 Recently Crusberg e t al.: and Dunlap et al. 8 have reported the complete purification of thymidylate synthetase from antifolate-resistant strains of L a c t o b a c i l l u s casei. However, the purification of the mammalian enzyme has been hampered by the labile nature of this protein and low enzyme levels in cells and tissues. This disadvantage is especially evident in the case of Ehrlich ascites carcinoma cells, ~,9 and if reasonable quantities of enzyme are required for study, the usual procedures employed in purification become quite laborious. It is apparent, therefore, that a single-step procedure based on affinity chromatography should greatly facilitate the purification process as well as enhance the recovery of enzyme activity. The present article describes the preparation of a specific adsorbent for thymidylate synthetase involving attachment of the substrate, 2'-deoxyuridylic acid (dUrd-5'-P) to agarose via an ,o-aminoalkyl "arm." This mate~C. K. Mathews and S. S. Cohen, I. Biol. Chem. 238, 367 (1963). 2R. L. Blakley, J. Biol. Chem. 238, 2113 (1963). ~M. Friedkin, E. J. Crawford, E. Donovan, and E. J. Pastore, J. Biol. Chem. 237, 3811 (1962). 4D. M. Greenberg, R. Nath, and G. K. Humphreys, I. Biol. Chem. 236, 2271 (1961). 5M. Y. Lorenson, G. F. Maley, and F. Maley, J. Biol. Chem. 242, 3332 (1967). ~A. Fridland, R. J. Langenbach, and C, Heidelberger, J. Biol. Chem. 246, 7110 (1971). 7T. C. Crusberg, R. Leary, and R. L. Kisliuk, J. Biol. Chem. 245, 5292 (1970). 8R. B. Dunlap, N. G. L. Harding, and F. M. Huennekens, Biochemistry 10, 88 (1971). 9K.-U. Hartmann and C. Heidelberger, J. Biol. Chem. 236, 3006 (1961).
ISOLATION OF DNase-I INHIBITOR PROTEIN
[63] DNase-I
517
Inhibitor Protein. Isolation with DNase-Agarose By UNO LINDBERG
The presence of a protein DNase I-inhibitor has been demonstrated in many different types of mammalian tissues. The inhibitor protein has been purified by conventional techniques from calf spleen 1 and thymus °and from rat serum, '~ and, as described here, a generally applicable method for the purification of this protein by affinity chromatography on DNaseSepharose has been achieved? It has been found that the DNase I inhibitor is present in the ceils in surprisingly large amounts. For example, this protein constitutes 5 - 1 0 % of the soluble protein of thymus cells. However, nothing is yet known about its biological function or, for that matter, its relation to deoxyribonucleases in the living ceil. Procedure Buffers
Wash buffer I: 4 M guanidine-HCl; 30% glycerol; 0.5 M sodium acetate at pH 6.5 Wash buffer II: 10 mM Tris chloride at pH 7.5; 5 mM CaC12 Extraction buffer: 10 mM Tris chloride at pH 7.5; 10 mM NaC1; 10 mM E D T A Elution buffer I: 0.75 M guanidine-HC1; 0.5 M sodium acetate30% glycerol; adjusted to pH 6.5 Elution buffer II: 3 M guanidine-HC1; 1.0 M sodium acetate; 30% glycerol adjusted to pH 6.5. Coupling of DNase I to Agarose
Activation of Sepharose with cyanogen bromide is performed as described by Kato and Anfinsen. 5 Twenty milliliters of the commercial slurry of Sepharose 4B is washed with 600 ml of deionized water and resuspended in 40 ml of water. Cyanogen bromide solution (20 ml containing 0.6 g of CNBr) is added, and the pH is brought to 11-11.5 by the addition of 2 M ~U. Lindberg, Biochemistry 6, 323 (1967). 2U. Lindberg and L. Skoog, Eur. J. Biochem. 13, 326 (1970). '~G. Berger and P. May, Biochim. Biophys. Acta 139, 148 (1967). 4U. Lindberg and S. Eriksson, Eur. I. Biochem. 18, 474 (1971). 6I. Kato and C. B. Anfinsen, J. Biol. Chem. 244, 5849 (1969).
518
NUCLEIC ACIDS, NUCLEOTIDES, AND DERIVATIVES
[63]
NaOH and maintained by dropwise addition of NaOH. After 5 - 1 0 minutes, the pH remains stable and the activated Sepharose is immediately washed with ice-cold deionized water on a Btichner funnel. Bovine pancreatic DNase (Sigma) ~ 20 mg in 2 ml of 0.1 M NaHCO3, is mixed with the activated Sepharose, which has been resuspended in 25 ml of 0.1 M NaHCO3 at 4 °. The time course of the coupling reaction is followed by the absorbance at 280 nm of aliquots of the reaction mixture after centrifugation. Within 2 hours 90% of the enzyme protein has been fixed to the gel, and essentially all after another 24 hours at 4 °. The DNaseSepharose is then washed on a Btichner funnel with 500 ml each of water, 0.1 M NaHCO3, and again with water. It is packed into a column, and noncovalently bound protein is removed by passage through 50 ml of wash buffer I followed by 500 ml of wash buffer II. The DNase-Sepharose may be stored for several months at 4 ° without loss in effectiveness. After a purification procedure has been performed, the DNase-Sepharose is washed with wash buffers I and II as indicated above and is again ready for use. When incubated with the DNA substrate (calf thymus DNA, Sigma) used in the standard DNase assay, 7 DNase-Sepharose conjugate causes a slow but significant degradation of the DNA. The rate of the reaction is about 1% that obtained with an equal amount of free DNase.
Purification of DNase-Inhibitor ]rom Crude Extracts In the preparation described here calf thymus was used as the source of the inhibitor, although the same procedure has been effective for the purification of the inhibitor from several different tissues and species including tissue-cultured human cells. Frozen thymus tissue, 50 g, is thawed in 250 ml of extraction buffer and homogenized for 30 seconds in a high speed Turmix homogenizer after the addition of 0.1 volume of 5% NP-4(~. s The homogenate is centrifuged for 10 minutes at 12,000 g and the supernatant fluid is centrifuged again for 2 hours at 100,000 g. The supernatant liquid from this step may be stored at - 2 0 ° without loss of inhibitor activity. A sample of the crude extract (22 ml containing 450 mg of protein; a total of 870,000 inhibitor units) is applied to a column of DNaseSepharose (2 × 1.5 cm). After the sample has entered the column, unadsorbed protein is washed out with wash buffer II. Adsorbed proteins are then eluted in two steps with elution buffer I in the first step and elution ° Before use, the DNase (DN-.100, Sigma) is purified by chromatography on Sephadex G-100 in wash buffer II. ~U. Lindberg, Biochim. Biophys. Acta 82, 237 (1964). ~Nonidet P-40 (NP-40) is a nonionic detergent obtained from Shell Chemical Co.
[63]
ISOLATION OF DNase-I INHIBITOR PROTEIN
519
SUMMARY OF THE ]~ESULTS OF THE AFFINITY CHROMATOGRAPHY OF THE CRUDE EXTRACT OF CALF THYMUS a,b
Fraction 1. Crude extract 2. Nonadsorbable protein 3. Lightly adsorbed proteinb 4. Firmly adsorbed proteinb
Activ- ActivSpecific ity ity Pro- activity (k yield tein (units/ units) (C/c) (mg) mg) 870 55 145 71
100 6 17 8
450 415 20 8
1900 130 7200 8800
A26o/A2so
(a)
(b)
DNA (~g)
1.49 1.18 1.65 1.55
--1.45 1.30
--50 7
From U. Lindberg and S. Eriksson, Eur. J. Biochem. 18, 474 (1971). b Fractions 3 and 4 were analyzed before (a) and after (b) concentration by salt precipitation (dialysis against saturated ammonium sulfate overnight). After salt precipitation, the samples were dissolved in 10 mM sodium acetate, pH 6.5, and assayed for inhibitor activity after the addition of 0.75 M guanidine-HC1-0.5 M sodium acetate-3(}~',~,glycerol pH 6.5.
a
buffer II in the second. As much as 28 mg ( 6 % ) of the protein of the crude extract is adsorbed to the column. Of this total, 20 mg are relatively loosely bound and are removed with the first buffer whereas the remaining, more firmly attached protein consisting mainly of the DNase-inhibitor, is displaced by the second buffer. About 20% of the inhibitor activity is found in the first eluate and 10% in the second. The absorbance spectrum of the eluted fraction exhibit a maximum at 260 nm with a ratio of 1.6 between the absorbancies at 260 and 280 nm indicating the presence of nucleic acid material. It has been shown that this is due largely to RNA, but the reason for the adsorption of R N A to DNase-Sepharose has not been investigated. That the protein in the second eluate consists mainly of the inhibitor protein has been demonstrated by polyacrylamide gel electrophoresis and by peptide mapping.' A summary of the affinity chromatography of the calf thymus extract on DNase-Sepharose is given in the table. It should be pointed out that the inhibitor is n o t recovered in a highly active form and that conditions have not been found as yet which increase the activity of the adsorbed and eluted inhibitor protein. From the work on the purification of the DNase-inhibitor by conventional methods, it is known that the protein has a marked tendency to form high molecular weight aggregates with low inhibitory activity. From those studies it is also known that 0.75 M guanidine-HCI-0.5 M sodium acetate30% glycerol is effective in dissociating such aggregates to protein monomers of high specific activity. However, these conditions do not dissociate the enzyme-inhibitor complex. It is possible therefore that the presence
520
NUCLEIC ACIDS, NUCLEOTIDES, AND DERIVATIVES
[64]
of inhibitor activity in the first eluate is due to release of inhibitor molecules from inhibitor aggregates bound to the DNase-Sepharose, rather than to release of active inhibitor from the DNase as such. The situation in 3 M guanidine-HC1 is different. This concentration of guanidine-HCl is known to rapidly inactivate the inhibitor and would lead to dissociation of the DNase-inhibitor complex. 4
[64] Purification of Thymidylate Synthetase 2'-Deoxyuridylate-Agarose
with
B y PETER V. DANENBERG and CHARLES HEIDELBERGER
The partial purification of thymidylate synthetase from a number of bacterial and animal sources has been achieved by multistep sequences of conventional procedures. 1 6 Recently Crusberg e t al.: and Dunlap et al. 8 have reported the complete purification of thymidylate synthetase from antifolate-resistant strains of L a c t o b a c i l l u s casei. However, the purification of the mammalian enzyme has been hampered by the labile nature of this protein and low enzyme levels in cells and tissues. This disadvantage is especially evident in the case of Ehrlich ascites carcinoma cells, ~,9 and if reasonable quantities of enzyme are required for study, the usual procedures employed in purification become quite laborious. It is apparent, therefore, that a single-step procedure based on affinity chromatography should greatly facilitate the purification process as well as enhance the recovery of enzyme activity. The present article describes the preparation of a specific adsorbent for thymidylate synthetase involving attachment of the substrate, 2'-deoxyuridylic acid (dUrd-5'-P) to agarose via an ,o-aminoalkyl "arm." This mate~C. K. Mathews and S. S. Cohen, I. Biol. Chem. 238, 367 (1963). 2R. L. Blakley, J. Biol. Chem. 238, 2113 (1963). ~M. Friedkin, E. J. Crawford, E. Donovan, and E. J. Pastore, J. Biol. Chem. 237, 3811 (1962). 4D. M. Greenberg, R. Nath, and G. K. Humphreys, I. Biol. Chem. 236, 2271 (1961). 5M. Y. Lorenson, G. F. Maley, and F. Maley, J. Biol. Chem. 242, 3332 (1967). ~A. Fridland, R. J. Langenbach, and C, Heidelberger, J. Biol. Chem. 246, 7110 (1971). 7T. C. Crusberg, R. Leary, and R. L. Kisliuk, J. Biol. Chem. 245, 5292 (1970). 8R. B. Dunlap, N. G. L. Harding, and F. M. Huennekens, Biochemistry 10, 88 (1971). 9K.-U. Hartmann and C. Heidelberger, J. Biol. Chem. 236, 3006 (1961).