The ribonuclease activity of crystallized pancreatic deoxyribonuclease

ANALYTICAL

BIOCHEMISTRY

The

14, 269-277

Ribonuclease

Activity

Pancreatic STEVEN From

(1966)

of

Crystallized

Deoxyribonuclease

B. ZIMMERMAN

the Laboratory of Molecular Metabolic Diseases, National

AND Biology, Institutes

Received

July

GEORGIANNA National of Health,

SANDEEN

Institute of Arthritis Bethesda, Maryland

and

12, 1965

Pancreatic DNase is widely used as specific means of degrading or inactivating DNA. The presence of RNase activity in crystallized pancreatic DNase preparations was recognized by Polatnick and Bachrach (1)) who introduced a DEAE-cellulose fractionation to remove the RNase activity. Re-examination of such chromatographed material with a highly sensitive RNase assay (2) indicated that a tenth of the activity remained which resisted further fractionation. However, means of specifically inhibiting either activity were found, suggesting that the low RNase activity was not an intrinsic property of the DNase. Specific inhibition of RNase yielded DNase with no RNase detectable by the new assay. Although modifications of the original (1) DEAE-cellulose chromatography were not more efficient in removing RNase, they did yield a system which resolves DNase from inactive proteins in commercial crystallized preparations. The active fraction has 20 to 100% higher specific activity than the commercial preparations. MATERISLS

AND

METHODS

Methods Absorbance measurements were done with a Zeiss spectrophotometer (model PM& II). The pH of buffers (diluted to 0.0544) was determined with a Beckman meter (model GS) . Enzyme Assays. DNase was assayed by the hyperchromicity method of Kunitz (3) modified as follows: Cuvets containing 3.0 ml of “substrate solution” (3) were equilibrated to 38” in a constant-temperature cell holder. Enzyme (0.01 to 0.10 ml of dilutions in 0.0025N HCl) was rapidly added and mixed and the optical density increase at 260 rn,u was followed. The linear rate of increase of absorbance following the lag period was proportional to the amount of DNase added, 1,2, or 3 ;Lg of a 269

270

ZIMMERMAN

AND

SANDEEN

DEAE-cellulose fraction giving 0.018, 0.035, or 0.050 nODJmin. DNase activity was 7-fold higher under the modified conditions. A unit of DNase activity is the amount giving a rate of increase of 1.0 OD unitjmin. RNase was assayed by release of acid-soluble products from polycytidylate (2). DNase preparations were partially acid-soluble under the original conditions used to terminate the assay. However, substitution of 0.6 ml of cold 12% w/v trichloroacetic acid for the perchloric acid solution of the original procedure precipitated all of the ultraviolet absorbance of the protein being assayed and made no marked change (<15% at any stage of the reaction) in the solubility of degradation products of polycytidylate. The range of proportionality with respect to enzyme and time with DNase preparations (Fig. 1) was similar to that

PROTEIN.

pg

TIME,

HOURS

FIQ. 1. RNase activity of the DEAE-cellulose fraction of DNase: (a) Proportionality to DNase added; incubation was 1 hr. (b) Kinetics. Amounts of DNase added are indicated on the figure. See text for details.

noted previously for purified pancreatic RNase (2). In most cases, RNase activity was determined from two or more points in the linear region of enzyme activity. A unit of RNase activity is the amount producing an optical density at 280 rnp of 1.0 in 1 hr in the above assay. Specific activity of either enzyme is expressed as unitslmg protein. Protein was determined by ultraviolet absorption, using [E]:z = 10. This extinction coefficient agreed within 10% of the values obtained either by directly dissolving weighed amounts of crystallized DNase or by spectra and nitrogen determination on the DEAE-cellulose fraction. DEAE-Cellulose Chromatography. The following operations were carried out at O-5”: a column of DEAE-cellulose (3.8 cm* X 15 cm) was equilibrated with 0.005 il4 potassium phosphate buffer, pH 8.0. Crystallized

RNASE

IN

PANCREATIC

271

DNASE

DNase (100 mg) dissolved in 20 ml of the equilibrating buffer was adjusted to pH 8.0 to 8.2l by slow addition of 0.05 N NaOH (ca. 1.2 ml required) with constant stirring. The DNase was passed through the column and the column was washed with 400 ml 0.015M potassium phosphate buffer, pH 8.0, to remove the bulk of the RNase activity. A constant gradient (1300 ml total volume) was applied from O.O3M, pH 8.0, to O.O53M, pH 6.0, potassium phosphate buffers. The gradient was obtained by open reservoir vessels of equal cross-sectional area. The flow rate for all operations was 4 ml/min; fractions of 20 ml were collected. In the representative chromatography shown (Fig. 2)) a second constant gradient (800 ml total volume from 0 to l.OM NaCI, in 0.053 M potassium buffer, pH 6.0) was used to elute inactive protein.

FRACTION

FIG.

text

for

2. Chromatography

of crystallized

NUMBER

pancreatic

DBase

on DEAE-cellulose.

See

details.

Of the 77% of DNase activity recovered from the column, about half had constant specific DNase activity and reduced RNase activity. This half was pooled and adjusted to pH 2.g2 by addition of about l/40 vol of 1 N HCl with good stirring. This acidified eluate may be stored overnight without activity loss. The chromatographic procedure described above will handle at least 200 mg of DNase with similar resolution. For purifying smaller amounts (5-30 mg DNase) a column 0.9 cm? X 15 cm was used with a wash of 250 ml, 180 ml of each gradient solution, and a 1 Yellow-gray *Faint orange

to thymol to thymol

blue indicator blue indicator

on spot test. on spot test.

272

ZIMMERMAN

AND

SANDEEN

flow rate of 1 ml/min. Resolution of the smaller column was comparable to that described above. The acidified eluate was concentrated with 95-100% recovery of DNase activity by pervaporation. Alternative concentration procedures such as lyophilization, ammonium sulfate precipitation at acid or neutral pH, stepwise DEAE- or TEAE-cellulose columns, or pervaporation without lowering pH were unsatisfactory with regard either to yield of protein or maintenance of specific activity. Accordingly, the acidified eluate was placed in cellulose dialysis tubing (1.7 in. flat width) and at room temperature the tubing was subjected to a warm air stream. After approximately zh of the solvent was removed, the rate of evaporation was insufficient to maintain the sac at less than room temperature and an unheated air stream was used until the volume was reduced to l/lo that initially present. The sac should feel cool to the touch at all times. About 7 hr was necessary for the lo-fold concentration. The concentrated solution was transferred to a fresh smaller casing and dialyzed twice for 24-hr periods at 5’ against several hundred volumes of 0.0025 N HCI. The dialyzed material may be further concentrated by pervaporation with no activity loss, or kept at 5” at least 3 months without change in activity. MATERIALS

DEAE-cellulose (4) (Cellex D, 0.95 meq/gm, Bio-Rad Laboratories) was suspended in 0.25N NaOH for 15 min at room temperature and then exhaustively washed with water and buffer, both before initial use and to regenerate used material. Sodium iodoacetate and N-bromosuccinimide (California Corporation for Biochemical Research) were dissolved in water immediately before use. Calf thymus DNA was prepared by the method of Kay et al. (5). Polycytidylate (Miles Laboratories, Lot 2536) was treated as previously described for use in the RNase assay (2). Various lots of crystallized pancreatic DNase were purchased from Worthington Biochemical Corporation. Cellulose dialysis casing (Visking Company) was soaked in distilled water for several days and thoroughly flushed before use. RESULTS

Purification

of DNase

specific activities found in five lots of crystallized DNase was 10-100. This was reduced approximately lo-fold by DEAE-cellulose chromatography. DEAE-Cellulose Chromatography. Gradient elution of crystallized RNase Levels in Crystallized

DNase. The range of RNase

RNBSE

IN

PANCREATIC

DNASE

273

DNase from DEAE-cellulose (Fig. 2) separated a peak with constant DNase specific activity from the bulk of the RNase activity, and from inactive protein. The gradient employed both changing pH and salt concentrations; phosphate or sodium chloride gradients at constant pH (pH 6.0, 7.0, or 8.0) were similarly effect,ive in reducing RNase content but did not significantly separate inactive protein. Rechromatography on DEAE-cellulose. DNase with RNase activity already reduced ca. lo-fold by the chromatography described above was rechromatographed (Fig. 3). In the second chromatography no significant

t

Wosh-

FRACTION

NUMBER

FIG. 3. Rechromatography of DNase on DEAE-cellulose. DNase (9 mg) concentrated from the last s of the enzyme peak of a chromatograph as in Fig. 2 was run on the small column described in text. Fraction volume = 5 ml. Less than 10% of the RNase was in the wash fractions. For assays of RNase in the DNase peak, the indicated fractions were pooled, concentrated by lyophilization, and dialyzed vs. 0.001 M phosphate buffer, pH 7.0. The DNase recovery in these steps was 5660%.

RNase was found in the wash fraction, RNase activity was found throughout the DNase peak, and as in the first chromatography the RNase specific activity was not constant throughout the peak. The DNase specific activity was constant and not significantly improved by rechromatography. No further separation of DNase and RNase over that obtained with DEAE-cellulose was found in gradient chromatography on TEAE- or CMC-cellulose.

274

ZIMMERMAN

Differential

AND

Inactivation

SANDEEN

of RNase

and DNase

The lack of success in physical separation of the small but readily detectable amount of RNase activity remaining after DEAE-cellulose chromatography led to attempts to inactivate the two activities differentially. Inactivation of DNase by N-Bromosuccinimide. Pancreatic DNase was shown by Okada and Fletcher (6) to be rapidly inactivated by N-bromosuccinimide at molar concentrations only a few times higher than those of the enzyme. The RNase activity was found to be relatively resistant (Fig. 4), allowing destruction of 99% of the initial DNase activity before significant decrease in RNase activity.

0

0

2

4

6

a

N-BROMOSUCCINIMIDE,

IO

I2

14

16

pg

FIG. 4. N-Bromosuccinimide inactivation of DNase and RNase activities of pancreatic DNase. The concentrated DEAE-celhrlose fraction of DNase (0.20 mg) -as incubated for 10 min at 20” in 0.05M sodium acetate buffer, pH 4.0, with the indicated amounts of N-bromosuccinimide (total volume, 0.40 ml). The samples were chilled and immediately assayed for DNase and RNase activities. Different symbols indicate separate experiments. The extent of inactivation was unchanged after 20 min incubation for samples giving ca. 50% inactivation of either activity. Mixtures of untreated and >95% inactivated material (either assay) gave additive results. Activities are expressed relative to untreated enzyme.

Inactivation of RNase by Iodoacetate. Pancreatic RNase is inactivated by iodoacetate (7) ; pancreatic DNase is relatively resistant (6). Kinetics of inactivation of the two activities in either crystallized DNase or its DEAE-cellulose fraction are similar as shown in Figs. 5 and 6, respectively. The pH and temperature conditions are a compromise between those needed for destruction of RNase and those necessary to retain the DNase activity. With either crystallized DNase or the DEAE-cellulose

RNASE

-

IN

20-

PANCREATIC

dNose

275

DNASE

-RNOZ

0

0

I 20

I 40

I 60 TIME

0 AT 55:

20 MINUTES

I/; 43

I 60

FIG. 5. Iodoacetate inactivation of crystallized DNase. Crystallized DNase (5 mg/ ml) was dialyzed for two days VS. two changes of 200 vol of 0.0025 N HCl at 5”; undialyzed enzyme gave erratic and lower recoveries of DNase activity in the following experiment. Reaction mixtures (0.80 ml) contained 0.1 M sodium acetate buffer, pH 5.3, 0.8 mg dialyzed enzyme, and either (a) without iodoacetate, or (b) with 0.15&f sodium iodoacetate. After heating at 55” for the indicated periods, reaction mixtures were chilled and dialyzed, those from (a) and from (b) being dialyzed in separate containers vs. 250 vol of 0.0025 N HCl for 16 hr at 5”. Each sample was then assayed for DNase and RNase as described in text. Activities are expressed relative to the unheated sample of (a).

20 -

0

0

I 20

I 40

RNose

~.

I 60 TIME

0 AT 55:

20 MINUTES

40

60

FIG. 6. Iodoacetate inactivation of the DEAE-cellulose fraction of crystallized DNase. Protocol as in legend of Fig. 5, except the concentrated DEAE-cellulose fraction described in text replaced the crystallized DNase. Activities are relative to the unheated sample of (a).

fraction, treatment with iodoacetate for 40 min under these conditions reduced RNase activity to the limits of detection with the poIycytidyIate assay. In these assays, 0.2 mg of treated DNase was incubated for 2 hr with polycytidylate and produced <0.05 acid-soluble optical density.

276

ZIMMERMAN

AND

SANDEEN

The RNaae specific activity may be c.onservatively estimated to be <0.5, making allowance for the lag in the enzyme proportionality response (Fig. 1). Commercial

“RNase-Free”

DNase

Recently a preparation of pancreatic DNase became commercially available, which is described as “free of RNase” (Worthington Biochemical Corporation). In lots3 assayed by the polycytidylate assay, the RNase specific activity had been reduced below that of the crystallized preparation used as starting material and was generally comparable in level to the DEAE-cellulose fraction described above. DISCUSSION

If the RNase activity in crystallized pancreatic DNase is assumed to be a contamination with the pancreatic RNase crystallized by Kunitz (8), the level of contamination in terms of weight of protein is minute. The activity corresponds to ca. 1 part RNase per million parts DNase. In terms of activity, however, the RNase level can be significant when DNase is used as a specific reagent to degrade or inactivate DNA. The reduction of RNase activity may be approached at present in two ways, by chromatography or by iodoacetate inhibition. DEAE-cellulose chromatography as described by Polatnick and Bachrach (1)) or by the procedure described here, results in about a lo-fold reduction in RNase specific activity. The latter protocol has the advantage of yielding chromatographically homogeneous DNase. Iodoacetate inhibition directly applied to dialyzed crystallized DNase, however, seems the method of choice in gaining a specific enzymic reagent. The RNase level is at least lo-fold further reduced over that obtained by chromatography. Although the present sensitive RNase assay fails to detect activity in iodoacetatetreated DNase, obviously contamination at still lower levels is possible. Chromatography indicates two types of RNase in DNase preparations. One type is not retained by DEAE-cellulose at pH 8.0 and is likely to be a contamination with the Kunitz-type pancreatic RNase (8). The other type is retained. While eluting from DEAE-cellulose under conditions similar to those used to elute the DNase, the profile of RNase activity does not coincide with that of the DNase, even on rechromatography. This lack of cochromatography indicates the two activities reside on physically distinct molecules. The differential inhibition studies indicate that the same enzymic site(s) are not common to both activities. ’ The

authors

thank

Mr.

Charles

Worthington

for samples

of this

material.

RNASE

IN

PANCREATIC

DNASE

277

SUMMARY

Chromatography of crystallized pancreatic DNase on DEAE-cellulose separates DNase of high specific activity from an inactive protein fraction and the bulk of RNase activity. The RNase fraction that resisted physical separation from DNase could be preferent,ially inactivated by iodoacetate. REFERENCES 1. POLATNICK, J., AND BACHRACH, H. L., Anal. Biochem. 2, 161 (1961). 2. ZIMMERMAN, S. B., AND SANDEEN, G., Anal. Biochem. 10,444 (1965). 3. KUNITZ, M., J. Gen. Physiol. 33, 349 (1950). 4. PETERSON, E. A., AND SOBER, H. A., J. Am. Chem. Sot. 78, 751 (1956). 5. KAY, E. R. M., SIMMONS, N. S., AND DOUNCE, A. L., J. Am. Chem. Sot. 74, 1724 (1952). 6. OKADA, S., AND FLETCHER, G. L., Radiation Res. 15, 452 (1961). 7. GUNDLACH, H. G., STEIN, W. H.. AND MOORE, S., J. Biol. Chem. 234, 1754 (1959). 8. KUNITZ, M., J. Gen. Physiol. 24, 15 (1940).