Flow birefringence in deoxyribonucleic acid solutions—II. Effect of thermal denaturation and the ionic strength of the solution on the structure of DNA macro-molecules

Flow birefringence in deoxyribonucleic acid solutions--II

1301

15. 16. 17. 18. 19. 20.

P. I. FLORY and A. M. BUCItER, J. Polymer Sci. 27: 219, 1958 V. Ye. ESKIN, Kolloidn. zh. 22: 117, 1960 W. I~IGBAUM and D. CARPENTER, J. Phys. Chem. 59: 1166, 1955 O. BIANCItI, V. MAGNASCO and C. ItOSSI, Chimica e industria 40: 263, 1958 W. H. STOCKM_I_YEIt, g. Polymer Sci. 15: 595, 1955 W. KItIGBAUM, D. CARPENTER, M. KANEKO and A. ItOIG, J. Chem. Phys. 33: 921, 1960 21. P. I. FLORY and W. It. KItIGBAUM, J. Chem. Phys. 18: 1086, 1950

FLOW BIREFRINGENCE IN DEOXYRIBONUCLEIC ACID SOLUTIONS--II. EFFECT OF THERMAL DENATURATION AND THE IONIC STRENGTH OF THE SOLUTION ON THE STRUCTURE OF DNA MACRO-MOLECULES* E. V. FRISMAN,

V. I. VOROB'YEV,

L. V. SI-ICHAGINA

and N. K. YANOVSKAYA

Leningrad State University Physics Institute Institute of Cytology, U.S.S.R. Academy of Sciences (Received 6 August 1962)

VARIATION in the s t r u c t u r e of the molecules of deoxyribonucleic acid (DNA) is k n o w n to occur as a result of e x t e r n a l factors, i.e. d e n a t u r a t i o n . The results of a n u m b e r o f works h a v e shown t h a t the d e n a t u r a t i o n is c o n n e c t e d w i t h the destruction of the bispiral s t r u c t u r e o f D N A [1, 2]. T h e r m a l d e n a t u r a t i o n occurs in a c o m p a r a t i v e l y n a r r o w t e m p e r a t u r e range with the t e m p e r a t u r e a t which the spiral is b r o k e n depending on the D N A composition, while the e x t e n t of the t r a n s i t i o n range from spiral to r a n d o m chain depends on the h o m o g e n e i t y of t h e specimen [2-6]. Despite the large n u m b e r of works d e v o t e d to the s t u d y of the d e n a t u r a t i o n processes of D N A t h e r e is still a need for new m e t h o d s o f investigation which are sensitive to v e r y slight changes in molecular structure. I t is p a r t i c u l a r l y i m p o r t a n t to glean new i n f o r m a t i o n regarding the initial stage of d e n a t u r a t i o n . I n a previous w o r k [7] the m e t h o d of flow birefringence was used to s t u d y solutions of n a t i v e a n d t h e r m a l l y d e n a t u r e d DNA. W i t h o u t dwelling on the i m p o r t a n c e of the results, here we will simply state t h a t t h e characteristic birefringence In] differs b y more t h a n 100 orders of m a g n i t u d e for the n a t i v e a n d d e n a t u r e d DNA. T h i s indicates the high sensitivity o f this m e t h o d to disturbances o f the molecular structure. * Vysokomol. soyed. 5: No. 4, 622-627, 1963.

E . V . F~ZSM.AN et al.

1302

I n t h i s w o r k t h e m e t h o d o f flow b i r e f r i n g e n c e w a s u s e d t o s t u d y t h e i n i t i a l s t a g e o f t h e t h e r m a l d e n a t u r a t i o n o f D N A . T h i s m e t h o d w a s also u s e d t o s t u d y t h e influence of the ionic strength on the structure of the D N A molecules.

METHOD AND SPECIMEN The flow birefringence measurements of DNA solutions were made by means of an optical apparatus [8] and a dynamo-optimeter, which were both used in the first part of [7]. ~/r the relative viscosity of the solutions was also studied, and their absorption at ,~= 260 m/z. The object of this study was the sodium salt of DNA obtained from calf thymus b y the preliminary separation of the deoxynucleoprotein using the Mirsky method [9] with subsequent deproteinization according to Kirby [10]. The experimental data given below relate to two DNA specimens. One of them, which we shall call No. 1, had already been studied in the previous work [7], in which its characteristics were given. The second specimen, No. 2, contained 13.3% N and 7.4% P. The nitrogen/phosphorus ratio N / P = 1 . 8 , which shows that there is only a very slight a m o u n t of protein in the preparation. The value of the atomic extinction coefficienb E(p)=so=6500, means that the specimen can be regarded as native. The flow birefringence was studied, together with the viscosity and absorption in the ultraviolet range 2 = 2 6 0 m/L, of DNA solutions which had first been heated in a water thermostat and cooled at 0°. After coling the solutions were brought up to 21°at which temperature all the studies were carried out.

E(P)2Eo ~00

~xlO~

2

-0.8

IO00 IZ.-I

o.

-0.6 7500 -t~4

o

0"3 7000

0"2

0"1

-0"2 0

0"4

J

20

I 40

I 60

Fie. 1

I 6500 80 lOOt,°C

0

I

20

I

40

60

80 100 Loc

FIG. 2

FIG. 1. Temperature dependence of n/g (1) and E(p)=6o (2) for D~TA solution (No. 1) in 0.15 M NaC1. c=0-0155 g/100 cm s. :Fie. 2. Temperature dependence of r/t--1 for D:NA solution (No. 1) in 0.15 M NaCl.e =0.0155 g/100 em 3. I n each series of measurements a solution of known concentration was divided into portions which were heated at the given temperatures. For No. 1 three series of measurements were made for different periods of heating of 10 rain, 20 rain, and 30 min. This experiment showed that heating is only important when the temperatures are above 80 °. Solutions of speccimen No. 2 were also studied in two ionic strengths, heated for 20 minutes.

Flow birefringence in deoxyribonucleic acid solutions--II

1303

Besides this the concentration dependence of the birefringence and viscosity of DNA solutions of different ionic strengths were also studied. The DNA concentration (c) was determined from the phosphorus. For all the solutions which we studied the dependence of the birefringence value n on the flow rate gradient g was given by a straight line passing through the coordinate root. The slope of these lines, An/g we will use as the charactersitic of the birefringence of the solutions.

,Io' /E(z)260 45

2

o~ o

-I'0

35

-0"8

- 10,000

-

BOO0

-0"8

- 8000

-0"4 95 o

- 7000 -0"2

I5 I!

0

I 50

21°,407 60 ° I I IOt? g"sec- t 150

= 0

=

i

20

40

Fie. 3

I

60

>_.

80

6000

lOOi~ °C

Fze. 4

FIG. 3. ~-----f(g) dependence for DNA solution (No. 1) in 0.15 M NaC1 at different heating temperatures, c=0-0155 g/100 cmS. FIG. 4. Temperature dependence of An/g and E (P)ls0 for DNA solution No. 2. c = 0.0165 g/100 cma: 1--0.15 lYI NaOl; 2--0.015 M NaC1. RESULTS AND DISCUSSION

F i g u r e 1 shows t h e t e m p e r a t u r e d e p e n d e n c e o f n/g for D N A solutions (No. 1) in 0.15 M NaC1. T h e g r a p h shows t h a t e v e n a t 40 ° h e a t i n g h a s a m a r k e d effect on t h e birefringence value. A t t h e s a m e t i m e , a t (~l----260 mp) t h e a b s o r p t i o n rem a i n s c o n s t a n t u p to a t e m p e r a t u r e o f 60 ° inclusive. N o r is t h e r e a n y change in (~/T--1) t h e specific v i s c o s i t y o f t h e solutions a t these t e m p e r a t u r e s , n o r the orie n t a t i o n o f t h e birefringence, as s h o w n in F i g u r e 2 a n d 3. Similar studies were carried o u t w i t h the D N A No. 2 p r e p a r a t i o n for t w o ionic s t r e n g t h s . To a v o i d a n y difference in t h e c o n c e n t r a t i o n s , a solution was p r e p a r e d in 0.015 M NaC1, h a l f of which w a s t h e n b r o u g h t u p to ionic s t r e n g t h 0.15 M NaC1 b y adding the salt. F o r t h e solution o f lower ionic s t r e n g t h , 0.015 M NaC1, the n/g v a l u e u n d e r w e n t g r e a t e r changes a t t h e s a m e t e m p e r a t u r e s , a n d t h e curves were displaced to t h e left along axis t (Fig. 4).

1304

E . V . FRISMAN ¢t al.

/24.9

/tnxfOZ

_,.o~ / / / "

I1.6

-0'5

0

6.10 5"40 2.70 I

10

I

I

20

30

g,sec"~

:FIG. 5. An=fig dependence for DNA solutions in 1.5 M NaC1. The figures indicate the concentration in 10a g/100 cma. The ratio An/[gt/o (t/r--l)] (t/o is the viscosity of the solvent) is known to be proportional to the optical anisotropy of the macro-molecule [11]. At low temperatures there is no change in the (t/r--1) values; therefore the reduction in n/g must indicate a reduction in the optical anisotropy. These data may provide an indication of the presence of certain "weak spots" in the molecular structure of DNA. It may be that the low temperature heating of DNA breaks a small number of hydrogen bonds in certain parts of the macromolecule. However, the breaking of a few hydrogen bonds is not likely to have a marked effect on the optical anisotropy level, as the broken bonds are easily restored. Besides this, nitrogen bases would not be able to make any great change in their orientation in respect of the main chain sterie reasons. The more reasonable assumption is that several hydrogen bonds are broken in certain sectors of the macro-molecule, and that this may cause the chain to bend in these zones, creating the conditions for the appearance of a certain degree of freedom for the rotation of the nitrous bases. The reduction in ionic strength, which causes an increase in the repulsive forces between the charged particles of the chain, facilitates the breaking of the hydrogen bonds and leads to a closer dependence between the birefringenee and temperature, Figure 4 shows that the ionic strength also affects the birefringence of the native preparation, without changing E(p)~eo. The relative viscosities of the native DNA are almost the same for the two ionic strengths. It is interesting to find out the influence of ionic strength on the characteristic birefringence In] =(,dn/gct/o)g_,o; c--,o.[t/]g=othe characteristic viscosity is known to be independent of the concentration of the salt. For this reason the birefringence of

Flow birefringence in deoxyribonucleic acid solutions--II

1305

solutions of different D N A a n d NaCl c o n c e n t r a t i o n was measured. Figure 5 shows the An ----f(g) dependence for D N A in 1.5 M NaC1. Similar graphs were o b t a i n e d for the o t h e r ionic strength. .4r/ x , ~

i~

g--5-~oJu

-05 -(}25 I

0

I

0.005

I

0~010

I

0.015

I

0"020c, g/iOOml

FIe,. 6. A~/gcvlo)g-,O~f(c ) dependence for DbTA solutions in different ionic s~rengths: 1--0.015 1K NaC1; 2--0.15 M NaC1; 3--1.5 M NaC1.

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40

o

20[ 0

..o

--

,

,

0~005

0"010

0"015

:2.3 0~020

c, g/lO0 crn.~

Fie. 7. (~.--1)/cdependence for DNA solutions in different ionic strengths: 1--0.015 M NaC1; 2--0.15 M NaCl; 3--1.5 M NaC1.

5

o

4 3

0

O~Ol

L

0.02 c,g/lOOcm~

Fie. 8. (@/g)g-,o=f(c) dependence for DNA solutions in 0.15 M NaCI.

The e x p e r i m e n t a l d a t a in Figs. 6 a n d 7 show t h a t a r e d u c t i o n in the NaCl c o n c e n t r a t i o n will cause a n increase in [n] a n d has p r a c t i c a l l y no effect on the characteristic viscosity of the D N A . The m e a n value is [ ~ / ] = ( 3 1 9 0 ± 9 0 ) cma/g. * • We note that the [n] values obtained for specimens Nos 1 and 2 with the same NaC1 concentration are approximately the same, indicating that the preparations are identical. The reason why the [~] value is different from the [7] figures, is that a viscometer with different parameters was used (lower flow gradient).

E . V . FRIS~N et al.

1306

A study of the dependence of a the angle of orientation on g the flow gradient in these solutions was used to study the characteristic orientation [q/g] for each system from the (q~/g)g..,o----f(c) curve extrapolated to inifinite dilution where ~0----45°--~ (Fig. 8). Using the ratio between the diffusion rotation coefficient D r and the [q~/g] value obtained for stable ellipsoids [13]:

- lffDr

(1)

then, independent of the ionic strength of the solution the value D ~ - 3 sec l is obtained. I t is known that the [n]/[ff] value can be used to determine the optical anisotropy of a macro-molecule [14, 15]:

27n~kT

In]

[7]

(2)

Here n, is the refractive index of the solvent, 0i is tile free anisotropy of the macro-molecule, 0/ is the anisotropy due to the asymmetry of the molecular chain (shape anisotropy). As has been shown in [7], the importance of 0/is not very great for our system. We must therefore ascribe the values calculated by equation (2) to 0i the free anisotropy. The tabulated figures show that ionic strength has a considerable effect on the optical anisotropy of DNA molecules, which appears to be due to the poly-electrolytic properties of the chain. If, as suggested b y Tsvetkov [16], one takes account of the segment anisotropy of the shape, which m a y be quite important for fixed particles, then the dependence of 0i on ionic force will be even closer. OPTICAL PARAMETERS OF D N A NaC1 concentration

in solution M 0.015 0.15 1.5

Oi X

MOLECULES W I T H D I F F E R E N T IONIC STRENGTHS

l 0 "2° c m a

according to (2)

( ~ 1 - - ~ 2 ) × l 0 ~° c m a

according (3)

s

-- 1.41 --1-11 --0.85

940 740 570

-- 0.84 --0.67 --0.51

Probably the high 0 i values with constant hydrodynamic parameters of DNA molecules are due either to the absence of correlation in the dependence of these values on the ionic force, or to the high sensitivity of the double refringence value to a change in the strength of the chain. Assuming that the equation in [17] can be applied to a DNA molecule, 3 0i-- -5- (41--~2) '

(3)

Flow birefringence in deoxyribonucleic acid solutions -- I I

1307

where (al--a,) is the segment anisotropy, and using the calculated anisotropy value of the monomer % - - a L = - - 1 5 ×10 -24 cm a, the number of units in the s e g m e n t S-~--(~1--~2)/(~11--~_L) can be calculated (see Table). I n the previous work [7], for n a t i v e D N A in 0.15 M NaC1 we g o t S-~1000. The use of [t/] in t h a t work m e a s u r e d at low g gave S 740. As we showed in [7], if[t/]g= 0 is used we get the figure S_-_500. H o w e v e r , it m u s t be n o t e d t h a t the use of It/]g= 0 to calculate the optical a n i s o t r o p y did n o t affect the relation between the (al--ae) values (or S) o b t a i n e d for different ionic strengths. This is because neither [t/]g= 0 [12] nor [t/]g~0 are d e p e n d e n t on the NaC1 concentration.

CONCLUSIONS (1) The flow birefringence, viscosity a n d a b s o r p t i o n in the ultraviolet range 4 = 2 6 0 m~ h a v e been studied for D N A solutions p r e v i o u s l y h e a t e d a t different t e m p e r a t u r e s a n d cooled at t = 0 °. (2) Below the melting p o i n t h e a t i n g has a m a r k e d effect on the birefringence v a l u e An, b u t does n o t c h a n g e E(p)2eo, the relative viscosity or the birefringence orientation. I t is suggested t h a t this change in An is due to the b r e a k i n g of a small n u m b e r o f h y d r o g e n b o n d s in the D N A molecule. (3) I t has been f o u n d t h a t the characteristic birefringence o f n a t i v e D N A increases as the ionic s t r e n g t h o f the solution falls. The l a t t e r does n o t change the birefringence orientation, n o r the characteristic viscosity or E(p)2eo.

Translated by V. ALFOI~D REFERENCES 1. M. MESELSON and F. STAHL, Prec. Nat. Acad. Sci. U.S.A. 44: 671, 1958 2. P. DOTY, J. MARMUR, I. EIGNER and C. SCHILDKRAUT, Prec. Nat. Acad. Sei. U.S.A. 46: 461, 1960 3. S. A. RICE and P. DOTY, J. Amer. Chem. See. 79: 3937, 1957 4. H. EHRLICH and P. DOTY, J. Amer. Chem. Soc. 80: 4251, 1958 5. P. DOTY, H. BOEDTKER, I. R. FRESCO, R. HASELKORN and M. LITT, Prec. Nat. Acad. Sci. U.S.A. 45: 482, 1959 6. L. F. CAVALIERI, M. ROSOFF and B. H. ROSEN'BERG, J. Amer. Chem. See. 78: 5239, 1956 7. E. V. FRISMAN, V. I. VOROB'YEV, L. V. SHCHAGINA and L. V. YANOVSKAYA, VysokomoL soyed. 4: 762, 1962 8. E. V. FRISMAN and V. N. TSVETKOV, Zh. eksp. teor. fiz. 23: 690, 1952 9. A. E. MIRSKY and A. W. POLLISTER, J. Gen. Physiol. 30: 117, 1946 1O. H. S. KIRBY, Bioehem. J. 66: 495; 1957 ll. A. PETERLIN, J. Polymer Sci. 12: 45, 1954 12. H. EISENBERG, J. Polymer Sci. 25: 257, 1957 13. A. PETERLIN and H. A. STUART, Zs. fiir Phys. 112: 1, 1939 14. W. and H. KUHN, Helv. chem. aeta 26: 1395, 1943 15. V. N. TSVETKOV and E. V. FRISMAN, Dokl. Akad. Nauk SSSR 97: 647, 1954 16. V. N. TSVETKOV, Tezisy I X nauchn, konf. In-ta vysokomolek, soyed. (Theses Presented at the I X Scientific Conference of the Polymer Institute.) Leningrad, 1962 17. W. KUHN and F. GRI~N, Kolloid. Zh. 101: 248, 1942