A flow birefringence method for the determination of deoxyribonuclease activity

54 °

BIOCHIMIEA ET BIOPHYSCA ,~,¢TA

A FLOW BIREFRINGENCE

VOL. 1 9

(1956)

METHOD F O R T H E D E T E R M I N A T I O N

OF D E O X Y R I B O N U C L E A S E ACTIVITY* by WALTER

J. F R A J O L A , J A C O B G. R A B A T I N AND H A R O L D C. S M I T H

The Herman A. HoMer Research Laboratory o/the Department o/ Medicine and the Department o/ Physiological Chemistry, The Ohio Slate University, Columbus, Ohio (U.S.3.)

Though KUNITZ2 in 1950 reported that deoxyribonuclease (DNase)** caused complete disappearance of the double refraction of flow of deoxyribonucleic acid (DNA), flow birefringence studies have not been utilized for quantitative kinetic measurements of DNase activity. The emphasis, rather, in flow birefringence studies has been directed toward the determination of the size and shape of the DNA molecule a-7 and toward the effect of acid, alkali and various salts on the birefringence of DNA solutions TM. This report describes a new method for the determination of DNase activity based upon the ability of the enzyme to decrease the flow birefringence of DNA or calf thymus nucleoprotein (DNP) solutions. TODESCO in 192817 added a photoelectric cell and a densitometer to the usual birefringence of flow apparatus. Accurate quantitative measurement of the decrease in transmission of elliptically polarized light resulting from enzyme action upon DNA solutions located in the annular gap of the birefringence of flow apparatus is consequently possible. For detailed information on the theory, apparatus and applications of flow birefringence the excellent reviews by EDSALLTM a n d CERF AND SCHERAGA 19 are recommended. EXPERIMENTAL

Materials D N P was p r e p a r e d from calf t h y m u s a c c o r d i n g to the p r o c e d u r e of MIRSKY AND POLLISTER 20. The final p r e c i p i t a t e was d i s s o l v e d in i M NaC1 p H 7.0. D N a s e (from beef pa nc re a s , once c r y s t a l lized) a n d D N A were p u r c h a s e d from W o r t h i n g t o n B i o c h e m i c a l Sales C o m p a n y , Freehold, N e w J e r s e y . T h e D N a s e was d i s s o l v e d in i M NaC1 for D N P e x p e r i m e n t s a n d in H 2 0 for D N A exp e r i m e n t s . D N A was d i s s o l v e d in water.

Methods A s c h e m a t i c d i a g r a m of t h e flow b i r e f r i n g e n c e a p p a r a t u s is s h o w n in Fig. i. The c o n s t r u c t i o n of t h e a p p a r a t u s was b a s e d on t h e design of EDSALL et al. zl. The o u t e r r o t o r h a d a d i a m e t e r of 2.54 cm. T h e i n n e r c y l i n d e r d i a m e t e r was 2.34 cm. The a n n u l a r g a p w a s o . i o cm. T h e l e n g t h of t h e a n n u l a r sp ace t h r o u g h w h i c h t h e polarized l i g h t passed w a s 7.0 cm. To fill t h e a n n u l a r space r e q u i r e d 7.5 ml of solution. W h e n the p o l a r o i d s were crossed a n d t h e a n n u l a r space filled w i t h * This i n v e s t i g a t i o n was s u p p o r t e d in p a r t b y research g r a n t s ~t:C-Io84(C4) a n d ~ C - 2 3 3 o Bio from t h e N a t i o n a l Cancer I n s t i t u t e of t h e N a t i o n a l I n s t i t u t e s of H e a l t h , P u b l i c H e a l t h S e rvi c e a n d f r om t h e D o r o t h y H. a n d Lewis R o s e n s t i e l F o u n d a t i o n . P a r t of t h e d a t a w a s p r e s e n t e d to t h e D i v i s i o n of Biological C h e m i s t r y of t h e A m e r i c a n C h e m i c a l S oc i e t y m e e t i n g a t C i n c i n n a t i , Ohio, M a r c h 29 - A p r i l i, 19551. ** Th e fo llowin g a b b r e v i a t i o n s will be used in this p a p e r : D N a s e = d e o x y r i b o n u c l e a s e ; D N A = d e o x y r i b o n u c l e i c acid ; D N P = calf t h y m u s n u c l e o p r o t e i n .

~e/erences p. ,544.

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a D N P or D N A solution, the area between the concentric cylinders brightened as the outer cylinder was rotated. The a m o u n t of elliptically polarized light reaching the photocell was measured as % t r a n s m i s s i o n by the deflection on the scale of the p h o t o v o l t a p p a r a t u s ( P h o t o m e t e r model

~ Electeonik Recorder

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Fig. i. Flow birefringence a p p a r a t u s for m e a s u r e m e n t of DNase activity. 5oI-A, P h o t o v o l t Corporation, N.Y.). W h e n the a n n u l a r space contained only I M sodium chloride or w a t e r or when the outer cylinder was stationary, practically no light reached the photocell and t h u s % t r a n s m i s s i o n was a p p r o x i m a t e I y zero depending on the light source d i a p h r a g m setting. During enzymic action the large initial % transmission gradually diminished until it approached zero w h e n the reaction was complete. F o r some e x p e r i m e n t s a u t o m a t i c recording of the changes in the transmission of elliptically polarized light as a function of time was o b t a i n e d by connecting a Brown Electronik recorder to the p h o t o v o l t a p p a r a t u s . For greatest accuracy, however, readings were not made while the rotor was in continuous operation b u t at s t a t e d time intervals between which the rotor was stopped. This reduced the heating of the solutions due to friction. All enzymic reactions and flow birefringence m e a s u r e m e n t s were conducted in a 2o°C c o n s t a n t t e m p e r a t u r e room. D N P solutions were made by dilution of a o.85 % D N P stock solution with i M NaC1. F o r comparison of flow birefringence changes with viscosity changes, 5 ml aliquots of the reaction m i x t u r e s were added to i ml of I M sodium citrate to inhibit the enzyme. All viscosity measurem e n t s were made at 25 ° C with an Ostwald viscosimeter.

RESULTS

Because the transmission of light at zero velocity gradient varied from o-5% depending upon the solutions used and upon the extent of the opening of the light source diaphragm some results are expressed as A % transmission. This was obtained by subtracting the % transmission at 100t" .,0.04 % DNA zero velocity gradient from ,ot 1: ° that observed at the velocity gradient under study. Fig. 2 presents the results obtained 5 ~°t / j" 515o for the study of the effect of /" / the rate of flow upon the birefringence of DNA solutions of four different concentrations. 400 800 1200 1600 It is apparent that an increasVelocity gradient (sec -I) ed velocity gradient produced Fig. 2. Effect of concentration of D N A and its rate of flow an increased birefringence of on the percent t r a n s m i s s i o n of elliptically polarized light (flow birefringence) b y the DNA. DNA. The relationship was

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Re/erences p. 544.

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542

w.j.

F R A J O L 4 . , J. G. R A B A T I N ,

H. C. S M I T H

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(1956)

linear for DNA solutions from o.oi to o.o4% for velocity gradients from about 3oo sec -1 to 16oo sec-L Graphs of A % transmission per unit velocity gradient versus concentration of DNA also indicated the direct relationship between flow birefringence, velocity gradient and concentration of the DNA. Similar results were obtained for solutions of DNP in 1 3I NaCI except that at higher concentrations (above o.19 %) the A % transmission reached a maximum at about " 1 "~. ~"~ 4 IlOO sec -1 and then decreased when the flow rate l , a -'~'~-. i ~I 0 10 20 30 was increased. Maximum birefringence was obhfioufes of ONase acfion served at lower flow rates when the concentra- F i g . 3. T h e c h a n g e in f l o w b i r e f r i n g e n c e of D N A ( o p t i c a l d e n s i t y u n i t s tion of the DNP solutions was increased. p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n of It may be assumed on the basis of flow t h e b i r e f r i n g e n t D N A ) a s a f u n c t i o n of birefringence studies that DNA or DNP solu- t h e t i m e of D N a s e a c t i o n . D N A - 0.044 M; DNase -tions may contain elongated rigid particles in a 0 . 0 4 % , ; M g +~ o . o 3 9 7 / m l ; G = 5 2 4 sec -1 distribution of sizes or semi-rigid, moderately flexible, kinked rods in various stages of contraction or elongation s. The A O/:otransmission as determined above is thus directly proportional only to the concentration of those DNA or DNP particles which are birefringent for the given conditions. Then, since A % transmission is directly proportional to the concentration of the birefringent DNA the results of DNase action upon DNA at a given velocity gradient can be expressed as changes in the concentration of the birefringent DNA. In kinetic studies of enzyme action it is customary to plot the logarithm of the substrate concentration versus time to determine the order of the reaction. In these birefringence experiments this is simply accomplished by recording the optical densities directly from the densitometer rather than the % transmission. The term, A OD, as hereafter used, therefore, refers to the logarithm of the concentration of birefringent DNA. Fig. 3 illustrates the effect of DNase and also of NaC1 and MgSO 4 upon the bi-

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ONase (7lint) 1;ig. 4. T h e r a t e of c h a n g e of t h e c o n c e n t r a t i o n of b i r e f r i n g e n t D N A a s a f u n c t i o n of t h e enz y m e c o n c e n t r a t i o n . D N A = o . o 4 % ; M g ++ = o . o 4 4 M ; G = 5 2 4 sec i

Re/erences p. 544.

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F i g . 5. T h e r a t e of c h a n g e of t h e c o n c e n t r a t i o n of b i r e f r i n g e n t D N P a s a f u n c t i o n of t h e c o n c e n t r a t i o n of t h e D N P . D N a s e = o . o o o 8 m g / m l ; M g ++ = o . o 3 1 M ; G = 5 2 4 sec. 1.

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ACTIVITY

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refringence properties of DNA. The addition of MgSO 4 to a o.o4% water solution of DNA resulted in an immediate 37.5% decrease in the A OD (part ..~ A B of curve i, Fig. 3). No further change occurred (BC curve I, Fig. 3) until DNase was added. ~ o S s St°t~Ts;°sity Birefringence of flow then decreased as a first order reaction. When a mixture of NaCI and MgSO 4 was added to the DNA solution (final concentrations /Z 0.33 M NaC1 and 0.044 M MgSO4) a 62.5°; de26 ~ f r l crease in AOD was observed. The rate of the reaction for the water system (curve I) was o . i i 5 1o ; 2; M/nutes of ONase action A OD/min as compared to 0.03 A OD/min for the F i g . 6. A c o m p a r i s o n of D N a s e a c t i o n NaC1 system (curve 2). a s m e a s u r e d b y c h a n g e s in t h e v i s The initial rate of the reaction between DNase c o s i t y of D N P w i t h c h a n g e s in t h e c o n c e n t r a t i o n of b i r e f r i n g e n t D N P . and DNA was directly proportional to the concenD N P - - o. i 7 ° : o ; D N a s e - - 0 . 0 0 0 9 5 tration of DNase for the concentrations measured m g / m l ; M g ++ - - 0 . 0 3 5 M ; G -- 524 sec 1. (O.OI to 0.0 5 ~,/ml). (Fig. 4). Similar results were obtained for the DNase-DNP system for DNase concentrations between o.ooi and o.oo3 mg/ml. Fig. 5 is a typical Michaelis-Menten curve obtained by the flow birefringence method for the action of DNase on DNP in molar salt solution. A comparison of the changes in flow birefringence and viscosity of solutions of DNP was obtained by inhibition of the reaction mixtures with sodium citrate at various time intervals. Inspection of Fig. 6 reveals that the flow birefringence changes occurred somewhat more rapidly than the viscosity changes. I

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DISCUSSION

The variation of the streaming birefringence of DNA solutions as a direct function of the concentration of the DNA or of the velocity gradient was not an unexpected finding. LAUFFER AND STANLEY22 reported that the amount of elliptically polarized light transmitted by a solution of tobacco mosaic virus increased as the concentration of the virus increased. EDSALLTM and others 4, 5, 30 observed a direct relationship between the velocity gradient and the flow birefringence of sodium thymonucleate solutions. Likewise, the observed decrease in flow birefringence of DNA solutions is in accord with results of other investigatorsS, 9,11,12 The flow birefringence method for the determination of DNase activity is relatively simple to perform, fast, accurate and reproducible. Kinetic studies are possible with one enzyme-substrate mixture by recording the optical densities at various times during the continuous reaction. Because of the time required to make the measurements, viscosimetric 23, ultracentrifuga124 and other methods for measuring DNase action require inhibition of the reaction at certain time intervals and thus involve the use of several aliquots or reaction mixtures. Theoretically this method for the study of DNase action should be applicable to the study of any enzyme-substrate system in which the substrate initially exhibits flow birefringence but loses this property after enzyme action. Application of this method to the study of the DNase activity of various sera, serum protein fractions, white blood cells and various tissue extracts is presently being investigated.

ReJerencesp. 544.

544

w.j.

FRAJOLA, J. G. RABATIN, H. C. SMITH

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(1956)

ACKNOWLEDGEMENT The authors wish to thank Ohio State University

Mr. EARL SCHOFIELD a n d Mr. FRANK ZIMMERMAN of t h e

Medical Shop for the construction

of t h e f l o w b i r e f r i n g e n c e

apparatus.

SUMMARY A q u a n t i t a t i v e m e t h o d for the s t u d y of deoxyribouuclease activity by means of a photocell-flow birefringence a p p a r a t u s is described. The a m o u n t of elliptically polarized light t r a n s m i t t e d by D N A or D N P solutions varied directly as the velocity gradieut and the concentration of the D N A or D N P solutions. Salts decreased the flow birefringence. The decrease in flow birefringence due to the action of DNase is comparable to the d a t a obtained by viscosimetry. R~:SUM~ Une m6thode q u a n t i t a t i v e p o u r l'6tude de l'activit6 D N a s e au m o y e n d ' u n appareil A cellule photo-61ectrique p o u r la mesure de la bir6fringence est d6crite. La quantit6 de lumi6re polaris6e elliptiquement t r a n s m i s e p a r des solutions de D N A ou D N P varia directement avec le gradient de la vitesse et les concentrations des solutions de D N A ou DNP. Les sels diminu6rent la bir6fringence d'6coulement. Le d6croissement de la bir6fringence d'6coulement dfi A l'action de la D N a s e est comparable aux donn6es obtenues p a r viscosim6trie. ZUSAMMENFASSUNG Line q u a n t i t a t i v e Methode zur U n t e r s u c h u n g der DNase-Aktivit~tt mittels eines PhotozelleS t r 6 m u n g s d o p p e l b r e c h u n g s - A p p a r a t e s wurde beschrieben. Die Menge des elliptisch polarisierten Lichtes welche die L6sungen von D N A oder D N P durchlassen, variierte mit der Geschwindigkeitssteigung u n d der K o n z e n t r a t i o n der DNA- und D N P - L 6 s u n g e n . Salze v e r m i n d e r t e n die Str6m u n g s d o p p e l b r e c h u n g . Die A b n a h m e der S t r 6 m u n g s d o p p e l b r e c h u n g die v o n d e r W i r k u n g der D N a s e herrfihrt, ist mit den Angaben vergleichbar, die m a n durch Viskosimetrie erhalten hatte. REFERENCES 1 g~'. J. FRAJOLA,J. G. RABATIN AND H. C. SMITH, ,4bsts. Division o/Biol. Chem., r27th meeting Am. Chem. Soc., Cincinnati, Ohio, March 2 9 - A p r i l i, i9552 M. KDNITZ, J. Gen. Physiology, 33 (195 o) 363 • 3 0 . SNELLMAN AND G. WIDSTROM,Arkiv Kemi. Mineral Geol., I9A (I945) No. 3 I. 4 R. SIGNER AND H. SCHWANDER, Helv. Chim. Acta, 32 (1949) 853. 5 H. SCHWANDER AND R. CERF, Helv. Chim. Acla, 32 (1949) 2356. 6 M. GOLDSTEIN AND M. E. REICHMANN,./. l-J?n. Chem. Soc., 76 (I954) 3377 A. R. MATHIESON AND M. R. PORTER, Nature, 173 (1954) 119o. 8 j. i . CREETH, J. M. GULLAND AND D. O. JORDAN, J. Chem. Soc., (1947) 1141. 9 H. SCHVVANDERAND R. CERF, Helv.Chim. Acta, 34 (1951) 1344. 10 R. JEENER, Compt. rend. Soc. Biol., 14o (1946) ti38. 11 R. L. REY, Compt. rend., 237 (1953) 157o. 12 R. L. REY, J. chim. phys., 51 (1954) 85. 13 H. SCHWANDER AND R. SIGNER, Helv. Chim. Acta, 34 (1951) 1344. 14 j. p. GREENSTEIN AND W. ~T. JENRETTE, J. Nat. Cancer Inst., I (194o) 77. 15 A. R. MATHIESON AND M. R. PORTER, Biochim. Biophys. Acta, 14 (1954) 288. 16 j . vv. ROWEN, Biochim. Biophys. Acta, IO (1953) 913. 17 G. TODESCO, Atti accad. Lincei, 7 (1928) 394. 18 j. EDSALL, Advances in Colloid Science, i (1942) 269. 19 R. CERF AND H. SCHERAGA, Chem. Rev., 51 (1952) 185. 2o A. E. MIRSKY AND A. ~V. POLLISTER, .]. Gen. Physiol., 3 ° (1946) 117. 21 j. T. EDSALL, A. RICH AND M. GOLDSTEIN, Rev. Sci. Instrumenls, 23 (1953) 695. 22 M. A. LAUFFER AND "~V. M. STANLEY, J. Biol. Chem., 123 (I938) 507 . 23 j. p. GREENSTEIN AND XV. V. JENRETTE, J. Nat. Cancer Inst., 1 (1941) 845. 24 j. G. RABATIN, R. FRIEDLAND AND W. J. FRAJOLA, dr. Biol. Chem., 203 (1953) 23. R e c e i v e d J u l y 3 o t h , 1955