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Cation activation of desoxyribonuclease
Cation Activation of Desoxyribonuclease Toru Miyaji’ From
the Federal
and Jesse P. Greenstein
Security Agency, Public Health Service, National Cancer Institute, Bethesda,
National
Institutes
of Health,
Maryland
Received March 20, 1951
INTRODUCTION
The activation of desoxyribonuclease by magnesium ions was first reported by Fischer et al. in 1941 (l), and since then has been amply confirmed by others (2-6). Manganese was found by McCarty to be equally effective, zinc very much less so, while calcium and ferrous ions were ineffective (4). Previous studies from this laboratory indicated that a wide variety of salts was capable of activating desoxyribonuclease (5). This broad range of activators for the enzyme was questioned by Kunitz in a very interesting paper (6). Kunitz reported that his crystalline desoxyribonuclease in the absence of added Mg++ was appreciably activated when quartz vessels were used, and much more activated when Pyrex glass containers were used; in the presence of added magnesium, however, the degree of activation was the same whether quartz or Pyrex glass vessels were employed. Kunitz suggested that both Pyrex glass and quartz might furnish Mg++ or Mn++ or both as impurities during the enzymatic digestion. These observations of Kunitz impelled us to reexamine and critically repeat and extend our previous experiments on the cation activation of desoxyribonuclease. In so doing, we have worked with all-Pyrex and with all-quartz vessels, with quartz-distilled water of measured low conductivity, and with spectroscopically-and chemically-analyzed salts. At no time have we ever noted a measurable activation of desoxyribonuclease in either Pyrex or quartz containers in the absence of added salts. We have, moreover, not only confirmed our earlier observations on the activation by calcium, strontium, barium, and zinc, but have made the new observations that cobalt is as potent an activator 1 Special Research Fellow, National Cancer Institute; of Osaka, Japan. 414
on leave from the University
DESOXYRIBONUCLEASE
415
as magnesium or manganese, ferrous ion only a little less so, and nickel and cadmium relatively weaker but appreciable. The conditions under which the various cations are maximally effective may vary from one to the other, and these differences help to explain some of the divergent results in the literature. EXPERIMENTAL The sodium salt of desoxyribonuleic acid was prepared from calf thymus by a modified Hammersten procedure. It was further purified by the Chloroform-gel method. The N content was 14.50/,, P S.9’%. Quartz vessels of various types and sizes were purchased,from the Hanovia Chemical and Mfg. Co. Spectroscopic analysis of a sample of these containers by the National Bureau of Standards revealed less than 1 part/lO,OOO of alkali earth metals. Pipets were prepared and calibrated from quartz tubing by Mr. F. Highhouse of this Institute. Ordinary distilled water was distilled twice in a quartz apparatus and stored in quartz containers; the conductivity of this water varied from 1.03 to 1.49 X 10mB ohm-i cm.-‘. No attempt was made to keep it COyfree. The nucleate was dissolved at 1.0% concentration in this water and dialyzed for 18 hr. in a cellophane bag against frequent changes of this water. The substrate solution was made up fresh every day and kept in a quartz container. The enzyme was a once-crystallized desoxyribonuclease supplied by the Worthington Biochemical Laboratory. The salts employed were mostly the chlorides of Mg, Mn, Co, Fe, Ni, Ba, Ca, Sr, Zn, Cd, Pb, Hg, and Cu. All of these salts except Mg, had less than 1 part/lO,OOO of Mg as measured spectroscopically and by chemical analysis. The enzyme activity was measured by following the appearance of nonacidprecipitable products of the nucleate [of. (6)]. The reaction mixture generally consisted of 1 ml. of a 0.1% solution of nucleate, 1 ml. of enzyme solution containing 5 pg. protein, 1 ml. of activator, 1 ml. of either water or buffer solution, plus water to make the total volume 6 ml. During the digestion period l-ml. aliquots were withdrawn and mixed with 4 ml. of cold 0.25 N HCl, centrifuged at 10,500 r.p.m. for 10 min. in a refrigerated centrifuge, and the supernatant was analyzed at 260 mp in a Beckman spectrophotometer.
Comparative Studies in Quartz and in Pyrex Vessels
Parallel studies were made entirely in quartz vessels and entirely in Pyrex vessels. Digests were first prepared of 1 ml. nucleate, 1 ml. of enzyme solution, 3 ml. water, a few drops of dilute acetic acid to bring the final pH to 5.2, plus sufficient water to bring the final volume to 6 ml. Aliquots were removed and analyzed spectrophotometrically every 10 min. for 50 min. The pH remained nearly constant during this period of time. The temperature was 37.5”. Some twelve experiments were performed in each type of container. In no case was there any
416
TORU
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AND
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P.
GREENSTEIN
evidence of enzymatic activity. When 1 ml. of 0.02 M MgSOc was substituted for 1 ml. of the water in the digest, the rate of activation of the enzyme in Pyrex and in quartz vessels was identical. A sample of the quartz-distilled water possessed a conductivity when freshly prepared of 1.42 X 10m6 mho./cm. After standing for 1 hr. in either a Pyrex or quartz container this value did not change, nor did it change after standing further for 24 hr. If any material dissolved from the vessels into the water, it made no contribution to the conductivity. Digests of 5 ml. of 0.1% nucleate, 5 ml. of enzyme solution, and 20 ml. water possessed an initial conductivity in either Pyrex or quartz vessels of 2.61 X 10e6 mho/cm.; after 1 hr. of incubation at 37.5”, the conductivity in the former vessel was 3.33 X 1O-6 mho/cm. and in the latter 3.32 X 10m5mho/cm. It is interesting to note that some alight reaction is shown by the increase in conductivity, although it cannot be apparently measured by the acid-precipitability teat employed. Nevertheless, the extent of even this very alight reaction is identical in Pyrex and in quartz vessels. A fine Pyrex powder was prepared by grinding in a ball mill, and carefully washed with dilute HCI, water, alcohol, and ether. Amounts of this powder from 20 mg. to 1 g. were added to digests of the enzyme with nucleate in quartz vessels. The surface area of this powder waa not estimated but must have been considerable. When 1 g. of this powder was suspended in 10 ml. water with conductivity of 1.42 X lo+ mho/cm.., the conductivity of the mixture roae after 1 hr. of standing to 1.04 X 1O-s mho/cm. When 150 mg. of the powder was added to a digest of nucleate and enzyme in either Pyrex or quartz vessels, there was no evidence of any activation of the enzyme (Fig. 1.). When the digest contained 1 ml. of 0.5 M sodium acetate-acetic acid buffer at pH 5.0 in place of 1 ml. water (final pH 5.2), there was a marked activation of the enzyme, which was the same in Pyrex or in quartz vessels and the same whether 150 mg. of Pyrex powder was added or not (Fig. 1). However, in the presence of 1 ml. of 0.02 M MgSOd the added powder depressed the activation of the enzyme (Fig. 1). With more powder, the depression of activation was still greater. With 20 mg. or less of the powder, little or no interference with the activation by Mg++ was evident. Each component of the digest, namely nucleate, enzyme, buffer, and Mg++ solution was separately incubated with 300 mg. of Pyrex powder for 1 hr. at 37.5” before mixing with the other compo-
417
DESOXYRIBONUCLEASE
nents. In every case, the results were nearly identical with each other and with a digest in which all the components plus the powder were incubated together. These experiments with added Pyrex powder are rather heroic, for the exposure of an enzymatic reaction to so much surface area may be unreasonable and extreme. However, there was no interference by a large amount of the powder with the buffer activation of the enzyme, and relatively little with the Mg++ activation. It is possible that the silicate which dissolved from the powder, and which increased the elec-
0' 0
I IO
20
30
40
50
MINUTES FIG. 1. Desoxyribonuclease all runs. l Pyrex, 0 quartz, containing Mg++; A quartz,
activity in, Pyrex and in quartz containers; pH 5.2 in 0 quartz plus 150 mg. Pyrex powder; A quartz, digest digest containing Mg++ plus 150 mg. Pyrex powder.
trical conductivity of the mixture, also combined with some of the Mg+-+ and reduced its effective concentration. With lesspowder present (20 mg. or less), this effect is inappreciable, even though such small amounts of powder still possessconsiderable surface area. No activation effects were noted when badly scratched Pyrex vesselswere used. The activation of the enzyme by the acetate buffer (Fig. 1) confirms our earlier observations on the activation by univalent ions (5). Considerable care was taken in the preparation of several samples of the buffer, and no detectable amounts of Mg++ were present. All samples
418
TORU
MIYAJI
AND
JESSE
P.
GREENSTEIN
.6
FINAL MOLARITY FIG.
x6
2. Effect of concentration of added salts on enzymatic activity. Period of incubation at 37.5” was 30 min.; pH 5.2.
gave identical activating results.2 Identical digests of 5 ml. of 0.1% nucleate solution, 5 ml. buffer, 5 ml. of enzyme solution, and 15 ml. water were incubated for 1 hr. in quartz and in Pyrex vessels. The initial conductivity in both vessels was 4.72 X 10m4 mho/cm. After incubation, the conductivity was 5.77 x 10V3 mho/cm. in quartz, and 5.83 X 10m3 mho/cm. in Pyrex. The acid-soluble fraction in each experiment was identical within the experimental error. Activation by Bivalent Ions
Digests were prepared of nucleate, enzyme, and varying amounts of added salts. The pH was set at 5.2 at each concentration of salt. No buffer was present, but the pH did not drift more than 0.2 in any case over the 30-min. digestion period employed. Figures 2 and 3 show the results obtained. Mg++, CO++, and Mn++ were effective to nearly the same degree in the activation of desoxyribonuclease. Under the condiz An earlier report (5) described the activation of desoxyribonuclease by added arginine monohydrochloride. We could confirm this finding with a commercial sample of arginine. When, however, this sample was purified through the benzilidine procedure (7), it no longer activated the enzyme.
419
DESOXYRIBONUCLEASE
tions used, namely 5 pg. enzyme, and 1 mg. nucleate (3 pmoles of nucleic acid phosphorus), the maximum activation for all three ions is achieved with 10 pmoles. Ferrous and nickelous ions exert optimal activation at a concentration one-tenth that of Mg++, Co++,.or Mn++. Indeed, at the maximum effective concentration for the latter three ions, ferrous ion is practically ineffective (Fig. 2). The maximum activation for Fe++ and Ni++ is thus achieved with about 1 pmole of salt. The maximum activation for Ba++ is obtained with about 5 pmoles, for Ca++ and Sr++ with 10, and with Znf+ and Cd++ with about 0.5-l pmole salt (Fig. 3).. Per micromole of nucleate phosphorus, the optimal effectiye amount of each ion in micromoles is: Mg++, Mn++, Co++, Ca++, and Sr++, 3; Ba++ 1.7; Fe++, Ni++, Zn++, and Cd++, 0.3. The marked influence of the concentration of salt, as illustrated by Co++ and Fe++ in Fig. 2, is further emphasized by the time course of the activation shown in Fig. 4. At final concentrations of Co++ of 1.7 X 10m3M and 1.7 X 1O-4 M, the rate of digestion is slightly greater at the higher concentration. At the same concentrations of Fe++, the rate of digestion is considerably different for each concentration, the rate being lower at the higher concentration. The time course of the activation with Ca++, Sr++, and Ba++ shows a considerable lag during the early part of the digestion, as illustrated
lo-5lo-”io-3lo-210-lI FINAL FIG.
3. Effect
of concentration bation
MOLARITY
x6
of added salts on enzymatic activity. at 37.5” was 30 min.; pH 5.2.
Period
of incu-
420
TORU
MIYAJI
AND
JESSE
P.
GREENSTEIN
by the data for Ca++ in Fig. 5. With longer periods of incubation the curves level off as the nucleate is completely digested. It is possible that the shape of the curves is due to complex formation between these ions and the substrate. This lag phenomenon is remindful of the results obtained by Kunitz (6) with high concentrations of nucleate in the presence of enzyme and Mg++. The fact that the concentration-activation function for Mg++ passes through a maximum was reported by McCarty (4), Carter and Greenstein (5), Kunitz (6), and Wroblewski and Bodansky (8). As shown in
.8
MINUTES FIG.
4. Effect
of two enzymatic
different activity.
concentrations of cobalt and ferrous Final concentrations noted; pH 5.2.
chloride
on
Figs. 2 and 3, this phenomenon holds for all the effective salts studied, with the further proviso that the optimal concentration may be different for different salts. As Kunitz pointed out (6), the concentration of Mg++ required for optimal rate of digestion increased with the concentration of the nucleate, and was practically independent of the enzyme concentration. In the experiments of McCarty and of Wr6blewski and Bodansky, 6-12 and 8 pmoles of nucleic acid phosphorus were employed respectively, for the total volume of reaction mixture. The optimal concentration of Mg++ was noted to be 0.003 M, or,
421
DESOXYRIBONUCLEASE
.6,
1 A Ca, I.7xlCF3M 0 Ca ,I.7 x Id%
MINUTES FIG. 5. Effect of calcium chloride on enzymatic activity. Final concentrations pH 5.2.
noted;
approximately, 15 pmoles in the total volume. The Mg++:phosphorus ratio is thus 1.3-2.5, compared with the value of 3 (Fig. 2) obtained with a much lower concentration of substrate. Recognizing that McCarty and Wr6blewski and Bodansky used a viscometric method and a pH (7.5) considerably different from ours, the agreement in order of magnitude between their results and ours may be considered satisfactory, and confirmatory of Kunitz’s conclusion.
3
4
5
6
7
8
9
PH FIG.
6. Effect of pH on activity of enzyme in presence of added salts. Period of incubation at 37.5” was 30 min.
422
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GREENSTEIN
Finally, those ions found ineffective at any concentration in activating the enzyme were Pb++, Hg++, Cu++, Fe+++, Al+++, and Cr+++. Effect 0.f pH Digests were prepared consisting of 1 ml. of 0.1% nucleate, 1 ml. of enzyme solution containing 0.5 pg. protein, 1 ml. of salt solution to give a final concentration pf 5 X 10m4M, sufficient dilute HCl or NaOH to obtain the desired pH, plus water to bring the final volume to 6 ml. The pH was determined before and after incubation. As shown by Kunitz, the optimal pH for the digestion of sodium desoxyribonucleate by nuclease in the presence of Mg++ is about 6.5
0
1
3456789 P”
FIG.
7. Effect
of pH
on
activity
of enzyme incubation at 37.5’
in presence of added was 30 min.
salts.
Period
of
(6). We have confirmed this finding (Fig. 6). With Fe++, however, the optimal pH for the digestion is at 5.7 (Fig. 6). The optimum pH with Co++ is close to that of Mg++ and to Ca++. With Mn++ two peaks are noted, one at 6.8 and the other at about 8.0 (Fig. 7). These values have been repeatedly obtained. SUMMARY
The activation of to occur with Mg++ with Co++, to nearly with Ca++, Ba++,
desoxyribonuclease on desoxyribonucleate, known and Mn+f, has been shown to occur equally well the same extent with Fe++, and to a lesser extent Sr++, Ni++, Cd++, and Zn++. The conditions
423
DESOXYRIBONUCLEASE
under which the optimal activation is revealed vary among these ions. Thus, Mg++, Mn++, and Co++ may show marked activation under conditions in which Fe++ is nearly ineffective. Since too high a concentration of an ion may be as ineffective as too little, concentrationactivation curves were determined for each ion. Per micromole of nucleic acid phosphorus, the optimal effective amount of each ion in micromoles is as follows: Mg ++ 3, Mn++ 3, Co++ 3, Fe++ 0.3, Ni++ 0.3, Ba++ 1.7, Ca++ 3, Sr++ 3, Zn++ 0.3, and Cd++ 0.3. The optimum pH for the activation with Mg++, Co++, and Ca++ is about 6.5, that with Fe++ is at 5.7, while Mn++ shows two optima at pH 6.8 and 8.0. Experiments conducted in Pyrex and in quartz vessels showed the same results, and indicated that there was no activation of desoxyribonuclease in the absence of added salts. REFERENCES 1. FISCHER, F. G., B~TTGER, I., AND LEHMAN-ECHTERNACH, 271, 246 (1941). 2. LASKOWSKI, M., AND SEIDEL, M. K., Arch. Biochem. 7, 3. LASKOWSKI, M., Arch. Biochem. 11, 41 (1946). 4. MCCARTY, M., J. Gen. Physiol. 29, 123 (1946). 5. CARTER, C. E., AND GREENSTEIN, J. P., J. Natl. Cancer 6. ‘KUNITZ, M., J. Gen. Physiol. 33, 363 (1950). 7. BERGMANN, M., AND ZERVAS, L., Z. physiol. Chem. 172, 8. WR~BLEWSKI, F., AND BODANSKY, O., Proc. Sot. Exptl.
H.,
Z. physiol.
Chem.
465 (1945).
Inst.
7, 29 (1946).
277 (1927). Biol. Med. 74, 443 (1950).
the Federal
and Jesse P. Greenstein
Security Agency, Public Health Service, National Cancer Institute, Bethesda,
National
Institutes
of Health,
Maryland
Received March 20, 1951
INTRODUCTION
The activation of desoxyribonuclease by magnesium ions was first reported by Fischer et al. in 1941 (l), and since then has been amply confirmed by others (2-6). Manganese was found by McCarty to be equally effective, zinc very much less so, while calcium and ferrous ions were ineffective (4). Previous studies from this laboratory indicated that a wide variety of salts was capable of activating desoxyribonuclease (5). This broad range of activators for the enzyme was questioned by Kunitz in a very interesting paper (6). Kunitz reported that his crystalline desoxyribonuclease in the absence of added Mg++ was appreciably activated when quartz vessels were used, and much more activated when Pyrex glass containers were used; in the presence of added magnesium, however, the degree of activation was the same whether quartz or Pyrex glass vessels were employed. Kunitz suggested that both Pyrex glass and quartz might furnish Mg++ or Mn++ or both as impurities during the enzymatic digestion. These observations of Kunitz impelled us to reexamine and critically repeat and extend our previous experiments on the cation activation of desoxyribonuclease. In so doing, we have worked with all-Pyrex and with all-quartz vessels, with quartz-distilled water of measured low conductivity, and with spectroscopically-and chemically-analyzed salts. At no time have we ever noted a measurable activation of desoxyribonuclease in either Pyrex or quartz containers in the absence of added salts. We have, moreover, not only confirmed our earlier observations on the activation by calcium, strontium, barium, and zinc, but have made the new observations that cobalt is as potent an activator 1 Special Research Fellow, National Cancer Institute; of Osaka, Japan. 414
on leave from the University
DESOXYRIBONUCLEASE
415
as magnesium or manganese, ferrous ion only a little less so, and nickel and cadmium relatively weaker but appreciable. The conditions under which the various cations are maximally effective may vary from one to the other, and these differences help to explain some of the divergent results in the literature. EXPERIMENTAL The sodium salt of desoxyribonuleic acid was prepared from calf thymus by a modified Hammersten procedure. It was further purified by the Chloroform-gel method. The N content was 14.50/,, P S.9’%. Quartz vessels of various types and sizes were purchased,from the Hanovia Chemical and Mfg. Co. Spectroscopic analysis of a sample of these containers by the National Bureau of Standards revealed less than 1 part/lO,OOO of alkali earth metals. Pipets were prepared and calibrated from quartz tubing by Mr. F. Highhouse of this Institute. Ordinary distilled water was distilled twice in a quartz apparatus and stored in quartz containers; the conductivity of this water varied from 1.03 to 1.49 X 10mB ohm-i cm.-‘. No attempt was made to keep it COyfree. The nucleate was dissolved at 1.0% concentration in this water and dialyzed for 18 hr. in a cellophane bag against frequent changes of this water. The substrate solution was made up fresh every day and kept in a quartz container. The enzyme was a once-crystallized desoxyribonuclease supplied by the Worthington Biochemical Laboratory. The salts employed were mostly the chlorides of Mg, Mn, Co, Fe, Ni, Ba, Ca, Sr, Zn, Cd, Pb, Hg, and Cu. All of these salts except Mg, had less than 1 part/lO,OOO of Mg as measured spectroscopically and by chemical analysis. The enzyme activity was measured by following the appearance of nonacidprecipitable products of the nucleate [of. (6)]. The reaction mixture generally consisted of 1 ml. of a 0.1% solution of nucleate, 1 ml. of enzyme solution containing 5 pg. protein, 1 ml. of activator, 1 ml. of either water or buffer solution, plus water to make the total volume 6 ml. During the digestion period l-ml. aliquots were withdrawn and mixed with 4 ml. of cold 0.25 N HCl, centrifuged at 10,500 r.p.m. for 10 min. in a refrigerated centrifuge, and the supernatant was analyzed at 260 mp in a Beckman spectrophotometer.
Comparative Studies in Quartz and in Pyrex Vessels
Parallel studies were made entirely in quartz vessels and entirely in Pyrex vessels. Digests were first prepared of 1 ml. nucleate, 1 ml. of enzyme solution, 3 ml. water, a few drops of dilute acetic acid to bring the final pH to 5.2, plus sufficient water to bring the final volume to 6 ml. Aliquots were removed and analyzed spectrophotometrically every 10 min. for 50 min. The pH remained nearly constant during this period of time. The temperature was 37.5”. Some twelve experiments were performed in each type of container. In no case was there any
416
TORU
MIYAJI
AND
JESSE
P.
GREENSTEIN
evidence of enzymatic activity. When 1 ml. of 0.02 M MgSOc was substituted for 1 ml. of the water in the digest, the rate of activation of the enzyme in Pyrex and in quartz vessels was identical. A sample of the quartz-distilled water possessed a conductivity when freshly prepared of 1.42 X 10m6 mho./cm. After standing for 1 hr. in either a Pyrex or quartz container this value did not change, nor did it change after standing further for 24 hr. If any material dissolved from the vessels into the water, it made no contribution to the conductivity. Digests of 5 ml. of 0.1% nucleate, 5 ml. of enzyme solution, and 20 ml. water possessed an initial conductivity in either Pyrex or quartz vessels of 2.61 X 10e6 mho/cm.; after 1 hr. of incubation at 37.5”, the conductivity in the former vessel was 3.33 X 1O-6 mho/cm. and in the latter 3.32 X 10m5mho/cm. It is interesting to note that some alight reaction is shown by the increase in conductivity, although it cannot be apparently measured by the acid-precipitability teat employed. Nevertheless, the extent of even this very alight reaction is identical in Pyrex and in quartz vessels. A fine Pyrex powder was prepared by grinding in a ball mill, and carefully washed with dilute HCI, water, alcohol, and ether. Amounts of this powder from 20 mg. to 1 g. were added to digests of the enzyme with nucleate in quartz vessels. The surface area of this powder waa not estimated but must have been considerable. When 1 g. of this powder was suspended in 10 ml. water with conductivity of 1.42 X lo+ mho/cm.., the conductivity of the mixture roae after 1 hr. of standing to 1.04 X 1O-s mho/cm. When 150 mg. of the powder was added to a digest of nucleate and enzyme in either Pyrex or quartz vessels, there was no evidence of any activation of the enzyme (Fig. 1.). When the digest contained 1 ml. of 0.5 M sodium acetate-acetic acid buffer at pH 5.0 in place of 1 ml. water (final pH 5.2), there was a marked activation of the enzyme, which was the same in Pyrex or in quartz vessels and the same whether 150 mg. of Pyrex powder was added or not (Fig. 1). However, in the presence of 1 ml. of 0.02 M MgSOd the added powder depressed the activation of the enzyme (Fig. 1). With more powder, the depression of activation was still greater. With 20 mg. or less of the powder, little or no interference with the activation by Mg++ was evident. Each component of the digest, namely nucleate, enzyme, buffer, and Mg++ solution was separately incubated with 300 mg. of Pyrex powder for 1 hr. at 37.5” before mixing with the other compo-
417
DESOXYRIBONUCLEASE
nents. In every case, the results were nearly identical with each other and with a digest in which all the components plus the powder were incubated together. These experiments with added Pyrex powder are rather heroic, for the exposure of an enzymatic reaction to so much surface area may be unreasonable and extreme. However, there was no interference by a large amount of the powder with the buffer activation of the enzyme, and relatively little with the Mg++ activation. It is possible that the silicate which dissolved from the powder, and which increased the elec-
0' 0
I IO
20
30
40
50
MINUTES FIG. 1. Desoxyribonuclease all runs. l Pyrex, 0 quartz, containing Mg++; A quartz,
activity in, Pyrex and in quartz containers; pH 5.2 in 0 quartz plus 150 mg. Pyrex powder; A quartz, digest digest containing Mg++ plus 150 mg. Pyrex powder.
trical conductivity of the mixture, also combined with some of the Mg+-+ and reduced its effective concentration. With lesspowder present (20 mg. or less), this effect is inappreciable, even though such small amounts of powder still possessconsiderable surface area. No activation effects were noted when badly scratched Pyrex vesselswere used. The activation of the enzyme by the acetate buffer (Fig. 1) confirms our earlier observations on the activation by univalent ions (5). Considerable care was taken in the preparation of several samples of the buffer, and no detectable amounts of Mg++ were present. All samples
418
TORU
MIYAJI
AND
JESSE
P.
GREENSTEIN
.6
FINAL MOLARITY FIG.
x6
2. Effect of concentration of added salts on enzymatic activity. Period of incubation at 37.5” was 30 min.; pH 5.2.
gave identical activating results.2 Identical digests of 5 ml. of 0.1% nucleate solution, 5 ml. buffer, 5 ml. of enzyme solution, and 15 ml. water were incubated for 1 hr. in quartz and in Pyrex vessels. The initial conductivity in both vessels was 4.72 X 10m4 mho/cm. After incubation, the conductivity was 5.77 x 10V3 mho/cm. in quartz, and 5.83 X 10m3 mho/cm. in Pyrex. The acid-soluble fraction in each experiment was identical within the experimental error. Activation by Bivalent Ions
Digests were prepared of nucleate, enzyme, and varying amounts of added salts. The pH was set at 5.2 at each concentration of salt. No buffer was present, but the pH did not drift more than 0.2 in any case over the 30-min. digestion period employed. Figures 2 and 3 show the results obtained. Mg++, CO++, and Mn++ were effective to nearly the same degree in the activation of desoxyribonuclease. Under the condiz An earlier report (5) described the activation of desoxyribonuclease by added arginine monohydrochloride. We could confirm this finding with a commercial sample of arginine. When, however, this sample was purified through the benzilidine procedure (7), it no longer activated the enzyme.
419
DESOXYRIBONUCLEASE
tions used, namely 5 pg. enzyme, and 1 mg. nucleate (3 pmoles of nucleic acid phosphorus), the maximum activation for all three ions is achieved with 10 pmoles. Ferrous and nickelous ions exert optimal activation at a concentration one-tenth that of Mg++, Co++,.or Mn++. Indeed, at the maximum effective concentration for the latter three ions, ferrous ion is practically ineffective (Fig. 2). The maximum activation for Fe++ and Ni++ is thus achieved with about 1 pmole of salt. The maximum activation for Ba++ is obtained with about 5 pmoles, for Ca++ and Sr++ with 10, and with Znf+ and Cd++ with about 0.5-l pmole salt (Fig. 3).. Per micromole of nucleate phosphorus, the optimal effectiye amount of each ion in micromoles is: Mg++, Mn++, Co++, Ca++, and Sr++, 3; Ba++ 1.7; Fe++, Ni++, Zn++, and Cd++, 0.3. The marked influence of the concentration of salt, as illustrated by Co++ and Fe++ in Fig. 2, is further emphasized by the time course of the activation shown in Fig. 4. At final concentrations of Co++ of 1.7 X 10m3M and 1.7 X 1O-4 M, the rate of digestion is slightly greater at the higher concentration. At the same concentrations of Fe++, the rate of digestion is considerably different for each concentration, the rate being lower at the higher concentration. The time course of the activation with Ca++, Sr++, and Ba++ shows a considerable lag during the early part of the digestion, as illustrated
lo-5lo-”io-3lo-210-lI FINAL FIG.
3. Effect
of concentration bation
MOLARITY
x6
of added salts on enzymatic activity. at 37.5” was 30 min.; pH 5.2.
Period
of incu-
420
TORU
MIYAJI
AND
JESSE
P.
GREENSTEIN
by the data for Ca++ in Fig. 5. With longer periods of incubation the curves level off as the nucleate is completely digested. It is possible that the shape of the curves is due to complex formation between these ions and the substrate. This lag phenomenon is remindful of the results obtained by Kunitz (6) with high concentrations of nucleate in the presence of enzyme and Mg++. The fact that the concentration-activation function for Mg++ passes through a maximum was reported by McCarty (4), Carter and Greenstein (5), Kunitz (6), and Wroblewski and Bodansky (8). As shown in
.8
MINUTES FIG.
4. Effect
of two enzymatic
different activity.
concentrations of cobalt and ferrous Final concentrations noted; pH 5.2.
chloride
on
Figs. 2 and 3, this phenomenon holds for all the effective salts studied, with the further proviso that the optimal concentration may be different for different salts. As Kunitz pointed out (6), the concentration of Mg++ required for optimal rate of digestion increased with the concentration of the nucleate, and was practically independent of the enzyme concentration. In the experiments of McCarty and of Wr6blewski and Bodansky, 6-12 and 8 pmoles of nucleic acid phosphorus were employed respectively, for the total volume of reaction mixture. The optimal concentration of Mg++ was noted to be 0.003 M, or,
421
DESOXYRIBONUCLEASE
.6,
1 A Ca, I.7xlCF3M 0 Ca ,I.7 x Id%
MINUTES FIG. 5. Effect of calcium chloride on enzymatic activity. Final concentrations pH 5.2.
noted;
approximately, 15 pmoles in the total volume. The Mg++:phosphorus ratio is thus 1.3-2.5, compared with the value of 3 (Fig. 2) obtained with a much lower concentration of substrate. Recognizing that McCarty and Wr6blewski and Bodansky used a viscometric method and a pH (7.5) considerably different from ours, the agreement in order of magnitude between their results and ours may be considered satisfactory, and confirmatory of Kunitz’s conclusion.
3
4
5
6
7
8
9
PH FIG.
6. Effect of pH on activity of enzyme in presence of added salts. Period of incubation at 37.5” was 30 min.
422
TORU
MIYAJI
AND
JESSE
P.
GREENSTEIN
Finally, those ions found ineffective at any concentration in activating the enzyme were Pb++, Hg++, Cu++, Fe+++, Al+++, and Cr+++. Effect 0.f pH Digests were prepared consisting of 1 ml. of 0.1% nucleate, 1 ml. of enzyme solution containing 0.5 pg. protein, 1 ml. of salt solution to give a final concentration pf 5 X 10m4M, sufficient dilute HCl or NaOH to obtain the desired pH, plus water to bring the final volume to 6 ml. The pH was determined before and after incubation. As shown by Kunitz, the optimal pH for the digestion of sodium desoxyribonucleate by nuclease in the presence of Mg++ is about 6.5
0
1
3456789 P”
FIG.
7. Effect
of pH
on
activity
of enzyme incubation at 37.5’
in presence of added was 30 min.
salts.
Period
of
(6). We have confirmed this finding (Fig. 6). With Fe++, however, the optimal pH for the digestion is at 5.7 (Fig. 6). The optimum pH with Co++ is close to that of Mg++ and to Ca++. With Mn++ two peaks are noted, one at 6.8 and the other at about 8.0 (Fig. 7). These values have been repeatedly obtained. SUMMARY
The activation of to occur with Mg++ with Co++, to nearly with Ca++, Ba++,
desoxyribonuclease on desoxyribonucleate, known and Mn+f, has been shown to occur equally well the same extent with Fe++, and to a lesser extent Sr++, Ni++, Cd++, and Zn++. The conditions
423
DESOXYRIBONUCLEASE
under which the optimal activation is revealed vary among these ions. Thus, Mg++, Mn++, and Co++ may show marked activation under conditions in which Fe++ is nearly ineffective. Since too high a concentration of an ion may be as ineffective as too little, concentrationactivation curves were determined for each ion. Per micromole of nucleic acid phosphorus, the optimal effective amount of each ion in micromoles is as follows: Mg ++ 3, Mn++ 3, Co++ 3, Fe++ 0.3, Ni++ 0.3, Ba++ 1.7, Ca++ 3, Sr++ 3, Zn++ 0.3, and Cd++ 0.3. The optimum pH for the activation with Mg++, Co++, and Ca++ is about 6.5, that with Fe++ is at 5.7, while Mn++ shows two optima at pH 6.8 and 8.0. Experiments conducted in Pyrex and in quartz vessels showed the same results, and indicated that there was no activation of desoxyribonuclease in the absence of added salts. REFERENCES 1. FISCHER, F. G., B~TTGER, I., AND LEHMAN-ECHTERNACH, 271, 246 (1941). 2. LASKOWSKI, M., AND SEIDEL, M. K., Arch. Biochem. 7, 3. LASKOWSKI, M., Arch. Biochem. 11, 41 (1946). 4. MCCARTY, M., J. Gen. Physiol. 29, 123 (1946). 5. CARTER, C. E., AND GREENSTEIN, J. P., J. Natl. Cancer 6. ‘KUNITZ, M., J. Gen. Physiol. 33, 363 (1950). 7. BERGMANN, M., AND ZERVAS, L., Z. physiol. Chem. 172, 8. WR~BLEWSKI, F., AND BODANSKY, O., Proc. Sot. Exptl.
H.,
Z. physiol.
Chem.
465 (1945).
Inst.
7, 29 (1946).
277 (1927). Biol. Med. 74, 443 (1950).