The viscometric assay of streptococcal deoxyribonuclease

AN.4LYTIC.4L

BIOCHEMISTRY

The Viscometric J. H. MOWAT, From

11,

327-334 (1965)

Assay

of Streptococcal

Deoxyribonuclease

G. C. KRUPKA, C. F. WOLF, AND B. L. HUTCHUSGS

F. WILCOXON,

the Biochemical Research Section, Led(Jrle Laboratories DiLlision, American Cyanamid Company, Pearl Riiw. Neu York

Received October

12, 1964

A viscometric procedure for the assay of streptococcal deoxyribonuclease has been described by McCarty (1). This method, as modified by Christensen (2)) and by Hazlehurst (3)) was used in our biological control laboratory, but experience indicated that, under our operating conditions, further investigation of the met,hod was necessary if reliable results were to be obtained. The numerous assays to be made required the use of many Ostwaldtype viscometers and these were difficult to obtain with reasonably uniform flow times. In addition, it was thought that accuracy would be improved if flow times could be measured automatically, thus eliminating the personal factor to a considerable degree. Therefore, a modified viscometer (Fig. 1) was designed which has proved very satisfactory. Even more important than the purely mechanical aspects of the determination was the kinetics of the enzyme-substrate reaction, since it was apparent that substrates of different initial viscosity or concentration gave widely different assay values for the same sample of enzyme. Comparison of unknowns with an arbitrary standard enzyme preparation did not entirely eliminate discrepancies, and it appeared that further investigation of the assay procedure was desirable. The procedure described herein has been tested in thousands of assays over a period of several years by various operators and appears to be consistent,ly accurate and reproducible. F:SPERIME?;T.ZI>

PROCEDVRE

Materials

Sodium Deoxyribonucleate. Various procedures for the preparation of sodium deoxyribonucleate have been described in the chemical literature. As typical examples, the methods of Sevag, Lackman, and Smolens (4), Marko and Butler (5), Kay, Simmons, and Dounce (6), Hammarsten 327

328

MOWAT,

KRUPKA,

WOLF,

WILCOXON,

T O TIMER

AND

HUTCHINGS

MECHANISM

MERCURY

WELLS

ELECTRODE CARRIER rn.““fedrlr No4 RUBBER STOPPER SIDE ARM TUBE NICHRDME

(

;::F$&

ELECTRODES

30° WATER BATH LEVEL UPPER CHAMBER (P~EC/.voN 8Ofl.T ) I NICHROME BREATHER

ELECTRON -

TUBE

GROUND

JOINT

QROUND

JOINT

SPRINGS OR RUBBER BANDS

I

FIG.

1. The

LOWER

viscometer

CHAMBER

assembly.

(7), and Frick (8), may be cited. In our work we have used either the Sevag et al. method (4), or the procedure of Kay, Simmons, and Dounce (6). We have also purchased satisfactory material from Nutritional Biochemicals Corp., Cleveland 28, Ohio. The dry product (150 mg) was dissolved with frequent vigorous shaking in about 75 ml of warm (SO’C) 1M/40 Verona1 buffer containing 1.0 gm of dissolved Neopeptone@ (Difco Laboratories, Inc., Detroit, Michigan) at pH 7.5. After 1 hr, the mixture was cooled to room temperature, 0.3 ml of 1 M magnesium sulfate1 solution was added, and the volume ‘This concentration of magnesium sulfate (0.003 M) was used by our biological control laboratories following the procedures of Christensen (2) and Hazlehurst (3). Laskowski and Seidel (9) showed that the sensitivity of the assay can be increased somewhat by raising the magnesium content, and we have found that some batches of substrate require more magnesium sulfate in order to obtain a satisfactory K, from the standard enzyme.

THE

VISCOMETRIC

ASS.AT

OF

329

STREPTODORNASE

was made up to 100 ml by the addit,ion of the required amount of Verona1 buffer. The solution was then filtered through a coarse sinteredglass funnel and stored at 5°C in stoppered test tubes. When properly prepared, this solution had a relative viscosity in the range of 5 to 8, was completely clear, and was stable for weeks. cleaned It is essential that all glassmarc, stoppers, etc. be thoroughly to prevent contamination of the substrate with even the slightest trace of stSreptococcal deoxyribonuclease. Neopeptone’B D&e&. A 1% solution of Difco Neopcptone@ in 1lP/40 Verona1 buffer at pH 7.5 was freshly prepared each day and was used for making up the necessary serial dilutions of the deoxyribonuclease prior to assay. visco,neter. The digaram (Fig. 1) is self-explanatory. These viscometers may be obtained from Metro Industries, Inc., 11-38 31st Avenue, Long Island City 6, New York. Si

% 12AU7 o ClOCk -

- DPST

Switch

s2 - SPST

Switch

Rl,RJ

- 3.3 Meqohmr

R2,R4

- 200

RS - 30,000

Ohms Ohms

l/z W

R7 - 1,000

Ohms

1W

Cl, C2 - 20-20 C3,C4

- .05

T - %ncore

Ohms

l/z W

MM Hfd

250V

ELECTROLYTIC

CONDENSER

200~

PA 0421

RLl - RL2 - Potter

FIG.

I W

R6 - 5,000

R8 - 100,000 ‘Z2A”7

‘/z W

Ohms ‘/a W

125V-50mo

L Brumfitld

‘AWove LH-5

6-JV

2Amp

5OOD Ohms SpD

2. Wiring diagram of timing mechanism

Timing Mechanism. An electric stopclock (or constant-speed counter) was attached through an electronic relay device (see Fig. 2 for circuit diagram) to the electrodes of the viscometer to measure flow times. The timer should be sensitive to at least 0.2 sec. A similar stopclock should be used to measure the total elapsed time during the assay. Reset electric stopclocks were obtained from Arthur H. Thomas Company, Vine Street at Third, Philadelphia 5, Pennsylvania.

330

MOWAT,

KRUPKA,

WOLF,

WILCOXON,

AND

HCTCHINGS

Constant-Temperature Water Bath. A glass water bath with a thermostat set at 30°C is required. Temperature variation should not. exceed 0.05”. METHODS

Assay Procedure. The sample of enzyme, if solid, is weighed and dissolved at a concentration of 1.0 mg/ml in water at about, pH 11.0-11.2. The pH of liquid samples should also be raised to this level if any doubt exists as t,o complete solubility of the enzyme. If necessary, the alkaline solution may be clarified by centrifugation; then the required serial dilutions in Neopeptone-Verona1 buffer should be made without delay, since prolonged exposure to high pH will cause loss of enzyme activity. The final dilution should contain from 5 to 20 units ‘of streptococcal deoxyribonuclease per milliliter, preferably about 10 units/ml. A clean, dry viscometcr is mounted in the constant-temperat,ure wat,er bath, and 3.5 ml of substrate is pipetted into the upper chamber. The longest electrode is inserted in the breather tube, with care being taken that the end of the electrode reaches the bottom of the lower chamber. The stopper carrying the double electrode assembly is inserted in the top of the upper chamber with the two electrodes centered. The timer is connected to the three electrodes and the flow time of the substrate is determined until a constant value is obtained. The substrate is then partially drawn into the upper chamber, precisely 0.1 ml of the diluted enzyme solut,ion is added just above the surface of the substrate, and the continuous timer is started. The stopper carrying the double electrode assembly is put in place again with the electrodes centered. The substrate-enzyme mixture is then drawn up and blown down vigorously and repeatedly during the next 4 min to ensure thorough mixing. Care must be exercised that no bubbles form in the upper chamber. With the flow timer connected and set at zero, the substrate-enzyme mixture finally is drawn up until the ends of the double electrodes are covered, and held there by gentle suction with about 2 mm of the short electrode showing below the meniscus. At about 15 set before the 5-min time (as shown on the continuous time clock), the substrate-enzyme mixture is allowed to flow freely, and if the operation has been correctly judged (easily done after a little practice), the liquid meniscus will break contact with the short electrode exactly at 5 min and start the clock (or counter) which measures flow time. When the meniscus breaks contact with the longer electrode the clock stops, registering the flow time. After determining flow time, the reaction mixture is drawn up and allowed to flow down to maintain continuous mixing, but without timing.

THE

VISCOMETRIC

ASSAY

OF

STREPTODORNASE

331

The flow time is determined again at 5-nun intervals until six readings have been taken. The flow time of Verona1 buffer, divided by the flow time of the substrat.e-enzyme mixture, is plotted against time on a large-scale chart. The six points should lie on a straight line. The slope K of this line is a measure of the amount of enzyme, and is expressed as the change in reciprocal relative viscosity during a period of 50 min, or more briefly as K,,. In practice, twice the change in reciprocal relative viscosity during 25 min is taken as the value of K,,. The K,, of the unknown divided by the KY,,, of one unit of the primary standard enzyme and multiplied by 10 times the dilution factor gives the potency of the unknown in units/ml. For the most precise work, the assay should, of course, be run in duplicate or triplicate and the results averaged. After a little experience the assay should be accurate to within 3 to 5%, based on singIe determinations. The difference in length of the double electrodes (10 mm) should be checked frequently with an appropriate gauge. When assaying unknowns, if the approximate K,, (10 times the difference of the first two reciprocal relative viscosity readings) is outside the acceptable range, the necessary change in dilution can be readily calculated so that, on reassay, a K,, value near that of one unit of the standard will be obtained. DISCUSSIOK

To clarify the course of the reaction between streptococcal deoxyribonuclease and sodium deoxyribonucleate, a number of reaction rate curves were determined using various batches of substrate and various levels of the same batch of enzyme. Relative viscosity readings were taken at about 2- or 3-min intervals over a period of 60 to 90 min, but no attempt was made to take these readings at exactly equal intervals. For the purposesof this presentation, however, it was felt that viscosity values taken at uniform (5-min) intervals would be more convenient. The 5-min readings, shown in Table 1, were therefore obtained from the actual data by interpolation. Large-scale plots were made of relative viscosity vs. time. A study of these reaction rate curves showed that, within the range of the observations, all of the curves could be represented by the general equation: n = nl - Bnt

or

nl

n=I

(1)

where n = relative viscosity at any time t, n, = initial relative viscosity of the substrate (corrected for the volume of enzyme sample added (see Eq. 4)) and B = a constant dependent upon the amount of enzyme and also upon n,.

n

1.0

a The first reading low levels of enzyme.

xas ignored

K

ON RATE

in calculating

0.00358 0.00356 0.00361 0.00367 0.00367 0.00367 0.00367 0.00367 0.00369 0.00367 0.00363 0.00363 0.00365

unit S.D.

A

CONCENTRATION

n1 = 6.20 5.58 5.08 4.64 4.26 3.95 3.68 3.45 3.24 3.05 2.90 2.77 2.64

OF ENZYME

15 20 25 30 35 40 45 50 55 60 Meana

10

5

0

Time, min

EFFECT

the mean

6.06 5.74 5.44 5.20 4.97 4.76 4.57 4.39 4.23 4.05 3.94 3.81

nl = 6.32

n

TABLE 1

R.D.

of K, since

0.00136 0.00160 0.00169 0.00171 0.00172 0.00172 0.00172 0.00174 0.00174 0.00174 0.00174 0.00174 0.00171

K

n

0.25

there

appears

c

0.00072 0.00088 0.00091 0.00093 0.00094 0.00094 0.00094 0.00094 0.00096 0.00096 0.00096 0.00096 0.00094

K

to be a slight

unit S.D.

~E~xYRIB~N~cLE.~~E-S~DIUM

nl = 6.25 6.11 5.92 5.76 5.60 5.45 5.31 5.18 5.05 4.93 4.81 4.71 4.60

OF THE STREPTOCOCCAL ACID REACTION

value

0.5 .I%

CONSTANT

1.0 .t%

induction

period,

n1 = 5.00 4.60 4.32 3.91 3.65 3.40 3.20 3.02 2.8s 2.74 2.61 2.50 2.38

n

K

-

with

0.00348 0.00370 0.00372 0.00370 0.00376 0.00374 0.00374 0.00368 0.00366 0.00366 0.00364 0.00366 0.00369 especially

S.D.

DEOXYRIBONUCLEIC

THE

The reciprocal

VISCOMETRIC

ASSAY

OF

STREPTODORNASE

333

of this equation: 1 -= IL

(2)

is the equation of a st,raight line with a slope of B/n, or K, where K represents a reaction rate constant which, under specified conditions, is dependent only on the amount of enzyme. Differentiating Eq. (1) gives: r!,1 -= ut

-K,l”

from which it is seen that the slope of the reaction rate curve is proportional to the square of the viscosity. In the assay procedure described by McCarty (l), Christensen (2), and Hazlehurst (3), the measure of enzyme activity is defined in terms of the average slope of the reaction rate curve during the first 10 min, but it is now clear from Eq. (3) that assay values obtained by such a procedure will be proportional to the square of the initial viscosity of t,he substrate, other factors being equal. Similar conclusions may be reached from the experimental evidence, since a comparison of the react,ion rate curve data in Table 1A and 1D indicates that the two curves may be converted from one to the other by a transposition of the ?J (or n) axis. In other words, the two curves differ only by a constant time interval, in this case about 11 min, or the time required for the viscosity of the substrate used in Table 1A (nl = 6.20) to drop to the viscosity of the substrate used in Table 1D (n, = 5.00) under identical experimental conditions. On the other hand, if the reciprocal relative viscosity (or fluidity) is plotted against time (see Eq. 2), the slope K of t,he resulting straight line will be independent of the initial viscosity of the substrate. In order that K shall be dependent only upon the amount of enzyme, it is necessary that the amount of substrate used in a given assay, the concentration of the substrate solution, and the temperature shall be kept constant. Since different preparations of the substrate are seldom of equal purity, each batch of substrate solution must be calibrated against a highly uniform batch of standard enzyme whose potency has been arbitrarily defined in terms of streptococcal deoxyribonuclease units per milligram. In the assay, the K value of the unknown is compared with t.he K value given by one unit of the standard enzyme acting on the same batch of substrate solution. This use of K as a measure of enzyme concentration is therefore analogous to the procedure described by Laskowski and Seidel (9). Earlier in this discussion it was pointed out that the effective value of n, was the initiaI viscosity of the substrate solution n?, corrected for

334

.VO\1’.4T,

KRIiPh.4,

WOLE’,

WILCOXON.

AND

HUTCHIN(;S

the dilution effect caused by the addition of the enzyme solution. The decrease in viscosity caused by dilution with 0.1 ml of enzyme solution, alt.hough appreciable, was too small to measure with the desired accuracy. Measurements made under more appropriate conditions indicated that the viscosity-concentration relationship could be expressed with reasonable accuracy by the following empirical equation: 5

(nz - 1) = (a - 1)

where Vj? = original volume, VI = volume relative viscosity, and n1 = relative viscosity

after dilution, after dilution.

n, = original

SUMMARY

The reaction rate curves representing the reaction between streptococcal deoxyribonuclease and sodium deoxyribonucleate are discussed with respect to the viscometric assay of the enzyme. An apparatus is described which permits automatic measurement of flow time in the viscometer, and a reliable and reproducible assay procedure for streptococcal deoxyribonuclease is given. REFERENCES 1. MCCARTY, 2. CHRISTENSEN,

3. HAZLEHURST, 4. SEVAG, 5. MARKO,

M., J. Gen. Physiol. 29, L. R., J. Clin. Invest. G. N., J. Zmmunol.

123

(1946).

28, 163 (1949). 65, 85 (1950).

M. G., LACKMAN, D. B., AND SMOLENS, J., J. Biol. Chem. A. M., AKD BUTLER, G. C., J. Biol. Chem. 190, 165 (1951).

6. KAY, E. R. M., SIMONS, N. S., AND DOUNCE, A. (1952). 7. HAMMARSTEN, E., Biochem. Z. 144,383 (1924). 8. FRICK, G., Biochem. Biophys. Acta 13, 374 (1954). 9. LASKOWSKI, M., AND SEIDEL, M. K., Arch. Biochem.

L., J. Am.

7, 465 (1945).

124, 425 (1938). Chem.

Sot.

74, 1724