[Methods in Enzymology] Volume 2 || [63] Deoxyribonucleases

[63] DEOXYRIBONUCLEASES 437

[63] D e o x y r i b o n u c l e a s e s

B y MARGARET R. MCDONALD

Several enzymes (deoxyribonucleases) capable of hydrolyzing highly polymerized DNA occur in various cells. Although some of their properties are similar, others are markedly dissimilar. Only pancreatic deoxyribonuclease (DNase) has been highly purified and crystallized. Its preparation and properties will be discussed first. Methods for the partial purification of thymus, spleen, yeast, and streptococcal DNase will then be given, followed by a comparison of their properties.

Assay Method

When solutions of DNA are hydrolyzed by DNase, their viscosity decreases and their specific absorption of ultraviolet light increases. Titrat- able acid groups are liberated without the formation of free phosphoric acid. The split products are not precipitable by mineral acids, proteins, or alcohol; they diffuse through collodion or cellophane membranes. Methods of assaying DNase based on all these phenomena have been extensively used; their relative merits have been discussed. L,2 Kunitz's spectrophotometric procedure 3 is probably the most convenient for rou- tine measurements of purified DNase. The procedure described here has been found by the author to be the most generally useful in studies on DNase, applicable to both tissue homogenates and purified preparations. I t is essentially Allfrey and Mirsky's modification 2 of Laskowski's acid- soluble method. 4

Principle. The method is based on the colorimetric determination of the acid-soluble deoxypentose compounds released in the course of enzyme action.

Reagents

Substrate. 200 mg. of Na-DNA 5 in 100 ml. of H20 or 0.05 M MgSO4, depending on the Mg ++ requirement of the DNase being assayed.

1 N. B. Kurnick, Arch. Biochem. 29, 41 (1950). 2 V. Allfrey and A. E. Mirsky, J. Gen. Physiol. 36, 227 (1952). 3 M. Kunitz, J. Gen. Physiol. 33, 349 (1950). 4 M. Laskowski, Arch. Biochem. 11, 41 (1946). 5 Highly polymerized Na-DNA is obtainable from the Worthington Biochemical Sales Co., Freehold, New Jersey. For methods of isolation and purification of this compound, see Vol. III [103].

438 ENZYMES OF NUCLEIC ACID METABOLISM [63]

0.2 M buffer. The composition and pH of the buffer is determined by the DNase being assayed.

3.0 M trichloroacetic acid.

Procedure. One milliliter of substrate plus 1 ml. of buffer is incubated with 1 ml. of enzyme solution at 35 ° for various times, after which 1 ml. of 3.0 M trichloroacetic acid is added. The mixtures are left in an ice- water bath for 15 minutes, then filtered through 7-cm. Whatman No. 42 paper. Aliquots of the filtrate are analyzed by Dische's diphenylamine procedure, 6 and the optical densities obtained are converted to deoxypentose-P equivalents by comparison with those obtained from a standard solution of DNA.

Definition of Unit and Specific Activity. The DNase activity unit is defined as that quantity of enzyme which catalyzes the formation of 1 "~ of acid-soluble deoxypentose-P per hour under the conditions described above. Specific activity is expressed as DNase units per milligram of protein. Protein is determined by the method of Lowry et alJ

I. Crystalline Pancreatic Deoxyribonuclease

Purification Prccedure

The procedure described here is essentially that of Kunitz2 The yield of crystalline DNase is low, owing partly to the fact that at pH 2.8, which is most favorable for crystallization, the enzyme is gradually denatured. From 3 to 5 mg. of dry first crystals are usually obtained for each kilo- gram of ground pancreas extracted. The method has been found to be reproducible in several laboratories.

The saturated (NH4)2S04 solution is prepared at 20 to 25 ° (760 g. of salt per liter of H20). All filtrations, unless otherwise specified, are done with suction.

Step 1. Preliminary Purification. 8 Fresh *~ beef pancreases are collected in ice-cold 0.25 N H2SO4. The glands are drained, cleaned of fat and connective tissue, then minced in a meat grinder. The minced pancreas is suspended in an equal volume of ice-cold H20, and ice-cold 0.25 N H2SO4 is added with stirring until the pH of the suspension is approximately 3.0 (tested with 0.01% methyl orange on a test plate); a volume of acid equal to half that of the H20 added is generally required. The suspension

e Z. Dische, Mikrochemie 8, 4 (1930); see also Vol. I I I [99]. 7 O. H. Lowry, N. J. Rosebrough, A. L. Farr , and R. J. Randall , J. Biol. Chem. 193,

265 (1951); see Vol. I I I [73]. 8 Based on the procedure of M. McCar ty , J. Gen. Physiol. 29, 123 (1946). 9 Frozen pancreas, obta inable from any of the large slaughter-houses, can also be used

if only deoxyribonuclease (or ribonuclease) is to be prepared. They should be thawed by leaving them immersed in 0.25 N H~S04 a t 5 °.


is left at 2 to 5 ° for 18 to 20 hours. I t is then strained through cheesecloth. The residue is resuspended in 1 vol. of ice-cold H20 and again strained. The residue is then discarded, and the combined filtrates are brought to 0.2 saturat ion of (NH4)2SO4 by the addition of 114 g. of salt per liter of filtrate. The precipitate formed is filtered through a rapid filtering paper (such as Ea ton-Dikeman No. 617) with the aid of 10 g. of Celite No. 503 10 and 10 g. of Standard Super-Cel ~° per liter of solution. The filter cake is discarded. The clear filtrate is brought to 0.4 saturation of (NH4)2SO4 by the addition of 121 g. of salt per liter and refiltered with the aid of 3 g. of Celite No. 503 per liter through double paper, E-D No. 612 on top of No. 617. The residue H is suspended in five times its weight of water, the suspension is brought to 0.3 saturation of (NH4)2S04 by the addition of 176 g. of salt per liter of H~O used and refiltered on E-D No. 617 paper; the filtrate is discarded.

Step 2. Incubation at 37 ° Followed by Fraetionation with Ammon ium Sulfate. The residue is suspended in ten times its weight of H20, and the suspension is brought to 0.15 saturation of (NH4)2SO4 by the addition of 83.7 g. of salt per liter of H20. The solution is t i t ra ted to pH 3.2 (glass electrode) with about 2 ml. of 5 N H2SO4 per liter. I t is heated to 37 ° and left for 1 hour at tha t temperature. I t is then cooled to 20 ° and filtered through E-D No. 617 paper with the aid of an additional 5 g. of Celite No. 503 per liter of suspension. The residue is discarded. The filtrate is t i t ra ted to pH 5.3 (glass electrode) with 5 N N aO H (about 2 ml./1.) and brought to 0.5 saturation of (NH4)2S04 by the addition of 220 g. of salt per liter. The precipitate formed, designated 0.5 precipitate, is filtered on E-D No. 617 paper with the aid of 5 g. of Celite No. 503 per liter of solution. The clear filtrate is t i t rated with a few drops of 5 N H2SO4 to pH 4.0 (tested with bromocresol green on a spot plate) and brought to 0.7 saturation of (NH4)2S04 by the addition of 135 g. of salt per liter. The scant precipitate formed, designated 0.7 precipitate, is filtered on E-D No. 612 paper with the aid of 2 g. of Standard Super-Cel per liter and stored. The filtrate is discarded.

The 0.5 precipitate is resuspended in ten times its weight of water and step 2, including the incubation at 37 ° , is repeated several times until no appreciable 0.7 precipitate is formed.

lo Supplied by Johns-Manville, 22 East 40th Street, New York. 11 The filtrate, when adjusted to 0.25 N H2SO4 by the addition of 7 ml. of concen-

trated H2SO~ per liter of H20 used in the extraction and washing of the ground pancreas, can be utilized for the preparation of chymotrypsinogen, trypsinogen, trypsin, trypsin-inhibitor compound (M. Kunitz and J. H. Northrop, J. Gen. Physiol. 19, 1002 (1936) ; see also Vol. II [2, 3, 4]) and for ribonuclease (M. Kunitz, J. Gen. Physiol. 24, 15 (1940); see also Vol. II [62]).

4 4 0 ENZYMES OF NUCLEIC ACID METABOLISM [63]

The 0.7 precipitates are combined and suspended in ten times their weight of H~O and filtered through E-D No. 612 paper. The residue is washed with H20 until the washing is water clear.

Step 3. Fractionation with Ethanol. The combined filtrate and wash- ings are diluted with H~O to a concentration of approximately 1% protein (the approximate concentration of protein can be determined spec- trophotometrically at 280 m~, the optical density being 1.2 per milligram of protein per milliliter). The pH of the solution is adjusted with 5 N H2SO4 to pH 3.8 (tested with methyl orange on a spot plate), and 2 ml. of saturated (NH4)2SQ is added per 100 ml. of solution. The mixture is cooled in an ice-salt bath to 2 °, and one-quarter of its volume of ice-cold 95% ethanol is added slowly, with stirring, keeping the temperature of the solution between 2 and 5 °. The mixture is stored for 24 hours at 2 to 5 ° and is then centrifuged at the same temperature. The residue is discarded, and the clear supernatant is left at - 10 ° for 24 hours, after which it is centrifuged at the same temperature. The supernatant is discarded.

Step 4. Crystallization. The precipitate is dissolved in approximately ten times its volume of ice-cold H20, after which it is brought to 0.38 saturation by the addition of 60 ml. of saturated (NH4)2S04 per 100 ml. of solution. The precipitate formed is filtered with suction on hardened paper (such as Schleicher and Schuell No. 576) at 5 to 10 °. I t is then suspended in three times its weight of ice-cold H20 and dissolved by the slow addition of several drops of 0.25 N NaOH, keeping the pH of the solution below 4.8. If the solution is turbid it is centrifuged clear at about 5 °, then adjusted to pH 2.8 (glass electrode) with several drops of 0.2 N H2SOt. The heavy precipitate, which usually forms at approximately pH 3.5, dissolves readily as the pH of the solution reaches 3.0 or lower. The clear solution is left at 5 ° overnight and then at approximately 20 ° for 6 to 8 hours. Crystals appear during the latter step.

Step 5. Recrystallization. The suspension of crystals is centrifuged. The residue is snspended in approximately 3 vol. of 0.02 saturated (NH4)2SO4 and dissolved with the aid of a few drops of 0.2 N NaOH at a pH of about 4.6. The solution is centrifuged if turbid, titrated to pH 2.8 (glass electrode), and left at 20 °. Crystals of DNase form within an hour. They are filtered on hardened paper at 5 ° , then washed, first with ice-cold acidified 30% ethanol (1 drop of 5 N H2SO4 per 100 ml.), then with ice-cold acetone, and dried at room temperature for several hours.

The mother liquors in steps 4 and 5 yield additional crystals when treated as follows: The solution is diluted threefold with ice-cold H20 and titrated with 0.2 N NaOH to pH 4.6 (tested with bromocresol green on a spot plate). Any insoluble material formed is removed by centrifugation. The clear supernatant is titrated with 0.2 N H2SO~ to ptI 4.0


and then b r o u g h t to 0.38 sa tu ra t ion of (NH4)~S04, as described in step 4, which is then followed th rough in every detail.

TABLE I SUMMARY OF PURIFICATION PROCEDURE a OF PANCREATIC DEOXYRIBONUCLEASE

Specific Fraction activity b Yield'

1. Precipitate from 0.3 saturated (NH4)~S04 2. Filtrate, after 1 hour of incubation at 37 ° 3. Precipitate from 20% alcohol at - 1 0 ° 4. First crystals

First mother liquor 5. Second crystals

Second mother liquor

0.2 100 3-5 30 5-6 15 8-10 5 5-6 10 8-10 8-10

M. Kunitz~ J. Gen. Physiol. 33, 349 (1950). b The specific activity, i.e., activity per milligram of protein, is expressed in terms

of that of the best preparations, which is taken as equal to 10. c The yield is given in per cent of the activity of the first fraction, precipitated in

0.3 saturated (NH4)~SO4.

Properties

Specificity. Crystal l ine pancrea t ic D N a s e hydro lyzes h ighly polymerized D N A and D N A which has been dena tu red so t h a t its physica l propert ies , bu t no t its chemical composi t ion, are altered. 12 D e t a c h m e n t of a por t ion of the purines 12,13 inhibi ts the hydrolys is ; apurinic acid 12 is no t hydro lyzed by DNase . it (Mg ++ alone causes d is in tegra t ion of apurinic acid. 12) D N a s e does no t hydro lyze R N A . 8,15

Analysis of the pyr imidine and pur ine con ten t of the p roduc t s of D N a s e hydrolys is of D N A shows the pyr imid ine-pur ine rat io of the dialyzable p r o d u c t s to be higher t han the pa ren t subs t ra te ; t h a t of the non- dialyzable " c o r e , " lower. ~6,17 This does no t necessari ly mean t h a t D N a s e preferent ia l ly hydro lyzes pyr imidine nucleot ide groupings, ~6 and unti l more is known abou t the composi t ion and s t ruc ture of the nucleot ides formed, little can be said abou t the bond specificity of DNase .

Kinetics. The var ious changes in the physica l and chemical proper t ies

1~ C. Tamm, H. S. Shapiro, and E. Chargaff, J. Biol. Chem. 199, 313 (1952). 18 C. A. Zittle, J. Franklin Inst. 243, 334 (1947). 14 This has been confirmed cytochemically by A. Howard and S. R. Pelc, working in

London, and by H. Gay in the author's laboratory. 1~ L. M. Gilbert, W. G. Overend, and M. Webb, Exptl. Cell Research 2, 138 (1951). 16 S. Zamenhof and E. Chargaff, J. Biol. Chem. 178, 531 (1949); 187, 1 (1950). 17 M. G. Overend and M. Webb, J. Chem. Soc. 1950, 2746.


of D N A catalyzed by D N a s e occur a t unequal rates. 17-19 The change in viscosity and ul t raviolet absorpt ion generally precedes any noticeable change in the precipi tabi l i ty of the nucleate with strong acids or the lib- erat ion of acid groups.

When the concentrat ion of subs t ra te is low, the hydrolysis approxi- mates closely a reaction of the first order, 18,2° the unimolecular constant being independent of the concentrat ion of enzyme. At relat ively higher concentrat ions of substrate, the initial ra te of reaction decreases rapidly with increase in subs t ra te concentrat ion. 18,2°,2~ The products of the reaction are also inhibitory. 21

Activators and Inhibitors. Mg ++ 4.8.22 (or other divalent ions ~3) are ob- l igatory for the enzymic action of pancreat ic DNase . The concentrat ion of Mg ++ required increases with increasing concentrat ion of subs t ra te and is pract ical ly independent of the concentrat ion of enzyme. ~s,2° The relative concentrat ions of Mg ++ and of D N A for the opt imal rate of ac- t iva t ion are such tha t there is always a considerable excess of Mg ++ over the amoun t necessary to change N a - D N A into M g - D N A stoichiometrically. The concentra t ion-act iva t ion function for Mg ++ on D N a s e passes through a max imum; a t concentrat ions of Mg ++ above 0.02 M there is a decrease in the rate of digestion. ~s This inhibi tory effect is also shown by NaC1. 4,~8

Arginine, lysine, and histidine 24,25 have been reported as ac t ivators ~-6 of D N a s e when used in concentrat ions ranging f rom 0.001 to 0.01 M; a t concentrat ions greater than 0.01 M the effect decreases and lysine becomes inhibitory. 24

Fluoride, citrate, s arsenate, 2~ borate, and selenite ions 27 inhibit the action of DNase , owing p robab ly to their abil i ty to remove the ac t iva t - ing Mg ++. Thioglycolic acid, Na2S, Cu ++, Zn ++, Fe ++, Fe +++, Cr ++, and Ni ++ are inhibi tory 27,28 in the presence of Mg ++. Na-usna te 28 inhibits

18 M. Kunitz, J. Gen. Physiol. 33, 363 (1950). 19 G. Jungner, I. Jungner, and L. G. Allg4n, Nature 164, 1009 (1949); R. Vercauteren,

Nature 165, 603 (1950). 20 j. Gr4goire, Compt. rend. 231, 384 (1950). 2, L. F. Cavalieri and B. Hatch, J. Am. Chem. Soc. 75, 1110 (1953). 22 F. G. Fischer, I. BSttger, and H. Lehmann-Echternacht, Z. physiol. Chem. 271, 246

(1941). 23 C. E. Carter and J. P. Greenstein, J. Natl. Cancer Inst. 7, 29 (1946); T. Miyaji and

J. P. Greenstein, Arch. Biochem. and Biophys. 32, 414 (1951). 24 W. Frisch-Niggemeyer and O. Hoffmann-Ostenhof, Monatsh. 81, 607 (1950)

[Chem. Abstr. 44, 9497 (1950)]. 2~ V. L. Nemchinskaya and V. S. Shapot, Biokhimiya 18, 210 (1953) [Chem. Abstr. 47,

8132 (1953)]. 26 This activation may be due to impurities, since a commercial sample of arginine

which activated DNase did not do so after purification. ~ 27 L. M. Gilbert, W. G. Overend, and M. Webb, Exptl. Cell Research 2, 349 (1951). 2s A. Marshak and J. Fager, J. Cellular Comp. Physiol. 85, 317 (1950).


DNase in the presence, but not the absence, of Co ++. Some tissues con- tain a protein(s) which markedly inhibits pancreatic DNase. 29

Physicochemical Properties. Crystalline DNase is a protein of the al- bumin type with the following elementary composition in per cent dry weight: C, 50.16; H, 6.91; N, 14.88; S, 1.09; P, 0; ash, 0.47. 3 No indica- tion of a special prosthetic group is evident from its ultraviolet absorption spectrum which shows a maximum molecular extinction of 70,000 at 280 m~ and a minimum of 26,000 at 250 m~. 3 I t contains about 8% ty- rosine and 2% t ryptophan. 3 Its molecular weight calculated from diffu- sion measurer~.ents (assumed specific gravity, 1.33) is 63,000;3 from inac- t ivat ion by deuteron and electron bombardment , 62,000. 30 The isoelectric point of DNase is in the region of pH 4.7 to 5.0. 3

The stability of solutions of DNase depends markedly on their concentration. Solutions containing > 0.1 mg. of protein per milliliter in dilute buffer in the pH range of 4.0 to 9.0 are stable for at least a week at 50. 3 Solutions containing <0.01 mg. per milliliter require the presence of gelatin or peptone as stabilizers, s The pH of opt imum stability is from 5 to 6. Dilute solutions of the enzyme at pH 2.8 are reversibly denatured when heated for 5 minutes at 900. 3 When inactivated in the dry state by heat, the ent ropy of activation is - 3 7 cal./mole °K, the heat of activation 16,000 cal./mole. 8°

Effect of pH. The optimal pH range for the hydrolysis of D N A by DNase is 6.0 to 7.0. 8

Purity. The specific act ivi ty of DNase becomes constant after one recrystallization. Crystalline preparations are free of measurable traces of trypsin, chymotrypsin, and ribonuclease. All a t tempts to determine the puri ty of the preparations by solubility tests failed becaus'e of the con- t inuous formation of denatured protein at the salt concentrations required for the test.3

II. Partially Puxified Deoxyribonucleases

Purification of Thymus Deoxyribonuclease 31

Step 1. Extraction and Activation. Four hundred grams of calf thymus is freed from fat and connective tissue, cooled in ice, and homogenized in a high-speed mixer for 2 minutes with 1.5 1. of 0.14 M NaC1. The suspension is strained through cloth and centrifuged. The turbid superna- tan t solution (total viscosimetric 8 units, 800) is covered with 5 ml. of

29 W. Dabrowska, E. J. Cooper, and M. Laskowski, J. Biol. Chem. 177, 991 (1949); E. J. Cooper, M. L. Trautmann, and M. Laskowski, Proc. Soc. Exptl. Biol. Med. 73, 219 (1950); H. H. Henstell and R. I. Freedman, Cancer Research 12, 341 (1952).

30 C. L. Smith, Arch. Biochem. and Biophys. 45, 83 (1953). 31 M. Webb, Exptl. Cell Research 5, 27 (1953).


toluene and left at 4 ° for 24 to 48 hours. I t is then adjusted to pH 5.0 to 5.2 with 0.1 N HC1. A heavy white precipitate forms. This is removed by centrifugation, and the supernatant is filtered through cotton wool to remove lipid material.

Step 2. Concentration and Purification. The filtrate (total units, 25,000) 32 is adjusted to pH 6.4 with 0.1 N NaOH and dialyzed for 24 hours against 10 1. of H20 at 4 °. The formed precipitate is removed by centrifugation, and the clear supernatant (total units, 24,800) is con- centrated tenfold by pervaporation in 3/~-in. diameter cellophane dialysis tubes. The solution (total units, 27,000) is centrifuged, and the supernatant (pH 5.2) is left at 35 ° for 10 minutes. A heavy white precipitate forms. The suspension is centrifuged, and the supernatant (total units, 13,200; units/rag. N, 10) is dialyzed for 48 hours at 4 ° against three changes of H20 (total volume of H20, 20 1.). The formed precipitate is removed by centrifugation.

Step 3. Ethanol Fractionation. The supernatant (total units, 9000; units/mg. N, 50) is cooled in a freezing mixture, and cold ( - 10 °) ethanol is added to a final concentration of 29% (44 ml. per 100 ml. of solution), keeping the temperature of the mixture at - 8 °. The resulting suspension is centrifuged, and the precipitate is discarded. The supernatant is again cooled in a freezing mixture, and cold ( - 1 0 °) ethanol is added as above to a final concentration of 37.5% (15 ml. per 100 ml. of solution). The resulting suspension is centrifuged at 0 °, and the supernatant is discarded. The precipitate is dissolved in 40 ml. of H~O (total units, 3400; units/mg. N, 500) and dialyzed for 6 hours against 1 1. of H20 at 4 °. The solution is diluted to 200 ml. with H20 and refractionated with alcohol as before. The precipitate is suspended in H20 and dialyzed to remove the ethanol. The solution is then dried in vacuo from the frozen state, yielding 20 rag. of a pale cream-colored powder containing 15.8% N (total units, 2300; units/mg. N, 750; yield, 9%).

III. Purification of Spleen Deoxyribonuclease ~3

Unless otherwise specified, all operations are performed at 1 to 5 ° and all filtrations are done with suction.

Step 1. Extraction. Twenty pounds of frozen calf spleen is thawed for 24 to 48 hours, then minced in a meat grinder. The mince is suspended in 20 1. of 0.075 N H2SO4; the suspension is stirred for 1 hour and then left for 18 to 24 hours. It is then filtered by gravity through fluted paper

32 This marked increase in act ivi ty by the acid t reatment (or by prolonged incubation at 37 °, M. Webb, Exptl. Cell Research 5, 16 (1953)) has not been observed by the author using frozen thymus or spleen.

83 M. R. McDonald, unpublished data.


(Whatman No. 12) with the aid of 10 g. of Standard Super-Cel and 10 g. of Filter-Cel per liter of suspension. The filtrate is saved. The residue is Suspended in 10 1. of 0.03 N H2S04 and refiltered. The residue is discarded.

Step 2. Fractional Precipitation with (NH4)~S04. The filtrates are combined (total acid-soluble P units, 2 9 million; 34 units/mg, protein, 55), brought to pH 7.5 with 5 N NH40H (about 5 ml./1.), and then to 0.4 saturation of (NH4)2SO4 by the addition of 243 g. of salt per liter of filtrate. The resulting suspension is filtered with the aid of 5 g. of Stand- ard Super-Cel per liter through soft paper (Eaton-Dikeman No. 616). The residue is discarded. The filtrate is brought to 0.75 saturation of (NH4)~SQ by the addition of 241 g. of salt per liter. The resulting suspension is filtered through hardened filter paper (Schleicher and Schuell No. 576), and the filtrate is discarded. The filter cake (about 140 g.) is dissolved in three times its weight of cold H20 (total units, 8.3 million; units/mg, protein, 140), and 66.7 ml. of saturated (NH4)2S04 is added per 100 ml. of H~O (final concentration of (NH4)2SO4, 0.4 saturation). The suspension is filtered through soft paper with the aid of 2 g. of Stand- ard Super-Cel per 100 ml., and the residue is discarded. The filtrate is brought to 0.75 saturation by the addition of 140 ml. of saturated (NH4)2S04 per 100 ml. of filtrate, and the resulting suspension is filtered through hardened paper. The filtrate is discarded.

Step 3. Heat Fractionation. The filter cake (about 55 g.) is dissolved in twenty-five times its weight of H20 (total units, 7.7 million; units/rag. protein, 215), and the solution is brought to pH 4.3 with 1 N HC1. I t is then heated rapidly to 62 ° and left at 62 ° for 10 minutes, after which it is cooled to 25 ° and left at room temperature for 1 hour. The suspension is filtered through soft paper with the aid of 10 g. of Standard Super-Cel per liter; the residue is discarded. The filtrate is brought to pH 7 with 1 N NaOH and then to 0.75 saturation of (NH4)~S04 by the addition of 516 g. of salt per liter. The suspension is filtered, and the filtrate is discarded. The filter cake (about 20 g.) has 75% of the activity originally extracted (total units, 6.7 million; units/mg, protein, 700).

IV. Purification of Yeast Deoxyribonuclease 35

Step 1. Extraction and Activation. 350 g. of fresh baker's yeast is washed with H20, centrifuged, suspended in 60 ml. of H20 and crushed in a bacterial mill. The mixture is centrifuged for 2 hours at 1900 >< g.

3~ Over 90% of the activity present in the minced (or homogenized) spleen is found in the extract.

s5 S. Zamenhof and E. Chargaff, J. Biol. Chem. 180, 727 (1949).


The residue 86 is suspended in 300 ml. of 1 M NaC1, and the viscous mixture is left at 4 ° for four months, during which period its viscosity dis- appears completely and its DNase activity increases markedly (0.6, 0.6, 17, 25, 30, and 30 viscosimetric unitsS/ml, after 0, 14, 19, 52, 90, and 120 days, respectively).

Step 2. Concentration and Purification. The mixture (total units, 15,200) is then centrifuged at 1900 X g for 1 hour, dialyzed with rocking against ice-cold H20 for 4 hours, and dried in vacuo from the frozen state. The residue (total units, 15,000; units/rag, protein, 14) is suspended in 30 ml. of H20 and centrifuged at 31,000 × g for 2 hours. The residue is discarded, and (NH4)2SO4 is added to the yellow supernatant to 0.6 saturation. The resulting precipitate is collected by centrifugation at 31,000 X g, drained with suction on Whatman paper No. 50, suspended in 45 ml. of H20, and dialyzed with rocking against ice-cold H20 for 7 hours. The dialyzed mixture (total units, 10,200) is centrifuged at 31,000 × g for 1 hour, and the supernatant is discarded. The sediment is washed with H20, then extracted, first with 30 ml. and then again with 12 ml. of 1 M NaC1. The combined extracts (total units, 6200) are centrifuged at 31,000 X g for 1 hour, and the supernatant is dialyzed for 6 hours against ice-cold H20. The dialyzate is then dried in vacuo from the frozen state, yielding 27 rag. of pale yellow fluff, insoluble in H20 and soluble in salt solutions (total units, 4300; units/rag, protein, 1250; yield, 35%).

V. Purification of Streptococcal Deoxyribonuclease 37

Group A hemolytic streptococci produce, during growth, appreciable amounts of extracellular DNase. I t is readily precipitated from the cul- ture medium by (NH~)2SO4.

Step 1. Elaboration. Fifteen liters of neopeptone broth 38 is inoculated with a strain of group A hemolytic streptococcus (H105) and incubated at 37 ° for 20 hours. The cells are then removed by centrifugation in a Sharples centrifuge.

Step 2. Concentration and Purification. The slightly turbid superua- tant is brought to 0.4 saturation of (NH4)2SO4 by the addition of 243 g. of salt per liter of supernatant. The suspension is filtered with suction with the aid of 1 g. of Filter-Cel and 1 g. of Hyflo Super-Cel per liter. The residue is discarded, and the filtrate is brought to 0.8 saturation by the addition of 281 g. of (NH4)2SO4 per liter. The resulting precipitate is recovered by filtration and dissolved in 100 ml. of H20. (This solution

3s The supernatant can be used for the preparation of yeast DNase inhibitor. 35 ~7 M. McCarty, J. Exptl. Med. 90, 543 (1949). as V. P. Dole, Proc. Soc. Exptl. Biol. Med. 63, 122 (1946).

[63] DI~OXYRIBONUCLEASES 447

conta ins a lmos t all the original D N a s e act iv i ty . ) The solut ion is b r o u g h t to 0.4 sa tu ra t ion of (NH4)~S04, filtered, and the residue discarded. The fil trate is b r o u g h t to 0.5 sa tu ra t ion of (NH4)~S04, and the prec ip i ta te is recovered b y filtration. I t is dissolved in a small vo lume of H~O, dia lyzed agains t H20, and dried in vacuo f rom the frozen state. The dried ma te - rial (170 mg.) conta ins the bulk of the original D N a s e ac t iv i ty and has 25,000 viscosi ty s u n i t s / m g .

P r o p e r t i e s

& compar i son of some of the proper t ies of several D N a s e s f rom di- verse sources is p resen ted in :Fable I I . The proper t ies of par t ia l ly puri - fied D N a s e f rom spleen and mouse leukemic tissues, and of crude D N a s c ext rac ts of m a n y o ther m a m m a l i a n tissues, appear to resemble those of t h y m u s DNase . 2,~'I.s9,4°

TABLE II SUMMARY OF PROPERTIES OF VARIOUS DEOXYRIBONUCLEASES

Deoxyribonuclease from

Strepto- Pancreas ~ Thymus b Serum c Yeast ~ coccus *

1. pit optimum 7.0 5.2 2. Mg ++ requirement -F -- 3. Inhibition by:

F1 or citrate ions -{- -- Bacterial RNAI Yeast protein s -- -- Pancreatic DNase antisera" + Streptococcal DNase antisera'

7.5 6.0 7.5 + + +

+ + +

4-

+

M. McCarty, J. Gen. Physiol. 29, 123 (1946). b M. Webb, Exptl. Cell Research 5, 27 (1953). c F. Wroblewski and 0. Bodansky, Proc. Soc. Exptl. Biol. Med. 74, 443 (1950).

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[Methods in Enzymology] Volume 2 || [63] Deoxyribonucleases