Studies on the interactions of nogalamycin with duplex DNA

199

Biochimica et Biophysica A cta, 474 (1977) 199--209 © Elsevier/North-Holland Biomedical Press

BBA 98791

STUDIES ON THE INTERACTIONS OF NOGALAMYCIN WITH DUPLEX DNA R.K. SINHA, PAPIYA TALAPATRA, ARUNA MITRA and SANDIP MAZUMDER Biophysics Department, Chittaranjan National Cancer Research Centre, Calcutta-700026 (India) (Received June 8th, 1976)

Summary (1) Electron micrographs of T7 DNA molecules complexed with nogalamycin at r = 0.15, when prepared for electron microscopy by using a m m o n i u m acetate as a hypophase, were remarkably free from the "microkinks" which were present in abundance in free-DNA micrographs prepared under similar conditions. Microkinks could be removed by the stretching resulting from the intercalation of nogalamycin between the adjacent base pairs of T7 DNA. The average length extension of the complexed T7 DNA molecules at r = 0.12--0.17 was 23% of the length of the native T7 DNA. This gives an upper limit of intercalative binding sites of nogalamycin in T7 DNA as 11 nogalamycin molecules intercalating per 100 nucleotides. Calf t h y m u s DNA molecules complexed with nogalamycin at P / D = 0.05--0.10 were thickened structures, with intra- and inter-molecular aggregation, probably indicating the stacking of polymeric nogalamycin molecules on the external surface of native DNA. (2) The relative viscosity of the nogalamycin • DNA complexes in phosphate/ EDTA/saline buffer (0.2 M Na ÷) rose sharply with increasing r, up to r = 0.12 or up to D / P = 0.22 and up to r = 0.12 in 0.001 M Tris • HC1 plus 1 M NaC1. This proves that nogalamycin intercalates in native DNA and indicates that the upper limit of intercalating sites is nearly 12 per 100 nucleotides in high moJar solvents. (3) The binding Df nogalamycin in native calf t h y m u s DNA, as shown by Scatchard plots, seems to be dependent on the ionic strength of the environment. There was a 27% reduction of the total binding sites as the environment was changed from 0.001 M Tris • HC1, pH 7.4 to that plus 1 M NaC1. However, the strong binding sites, as obtained from the binding isotherms, were 0.10 per nucleotide and did not show variation in the above range of ionic strengths. (4) Thermal absorbance profiles of the complexes of nogalamycin, daunomycin and ethidium bromide with native DNA at P / D = 14 showed the release of the bound drug molecules in the helix-coil transition zone of the complexes. However, the profiles of those at P / D = 4 showed that a fraction of the bound

200 drug molecules were released at earlier temperatures, suggesting that this fraction was weakly bound. The results, as a whole, indicated (i) a close similarity between the strong binding sites and the intercalative sites of nogalamycin in native DNA and (ii) the involvement of nogalamycin in weak interactions with native DNA.

Introduction

Nogalamycin, an antitumoral antibiotic of the anthracycline group (the structure of this drug has been described in earlier papers [ 3 - 6 ] ) preferentially inhibits DNA-directed RNA synthesis and is known, from the results of various physicochemical studies [1--6] other than electron microscopy, to bind intercalatively to duplex DNA. In general, the strong binding sites of a drug or dye are also its intercalative binding sites in DNA. The work of Waring [4] on the unwinding and rewinding of super helical ~0x174 RF DNA after interaction with intercalating drugs, through sedimentation analysis, has however left scope for doubt on that generalized hypothesis. From this work, it is claimed that a considerable fraction of the strong binding sites of nogalamycin in the above super-helical DNA are not intercalative. We wanted to verify this in the case of a linear, duplex DNA employing electron microscopy, spectrophotometry and viscometry. Inclusion of electron microscopy in this type of study was encouraged by its successful exploitation that revealed the intercalative binding of ethidium bromide [7,8], acridine orange [8] and proflavine [9] qualitatively as well as quantitatively. There are also contradictory results [2,6] on the ionic .strength-dependent binding of nogalamycin to DNA. In this paper, we have tried to elucidate the secondary binding of nogalamycin with duplex DNA through thermal absorbance profiles, usual-binding isotherms and electron microscopy. The results indicate that the strong binding of nogalamycin is almost the same as the intercalative binding and the drug is also involved in weak interactions with duplex DNA. Materials and Methods Highly polymerised calf thymus DNA (Type I) was purchased from Sigma Chemical Co., U.S.A. Purified, concentrated bacteriophage T7 was supplied by Dr. W.C. Summers, U.S.A. Dr. P.F. Wiley of Upjohn Company, U.S.A., has kindly made a gift of nogalamycin; ethidium bromide and daunomycin were purchased from Sigma Chemical Co., U.S.A. and Farmitalia Co., Italy, respectively. DNA from bacteriophage T7 was isolated by a modified phenol method [10]. For the preparation of solution of nogalamycin in an aqueous buffer, earlier methods [3,6,11] were followed. The binding of this drug with DNA was studied spectrophotometrically following the methods of Peacocke and Skerrett [12] and of Waxing [13] in a Carl Zeiss (G.D.R.) spectrophotometer fitted with a thermostatically controlled water-circulating device. Suitable glassstoppered quartz cuvettes were also supplied with the instrument to be used in thermal absorbance studies. However, with the arrangement available, the tem-

201 perature of the cuvettes in situ could n o t be raised above 88°C. For a single type of binding site the mass action formula should be r/c = (n -- r)Kn, where r is the number of binding sites of the drug per nucleotidic phosphorus of DNA, n is the maximal value o'f r, Kn, the association constant, and c, the concentration of the free drug. Kn is obtained as the intercept on the r/c axis b y extrapolating the plot of r/c versus r to r = 0. The values of n were obtained in t w o ways: (i) from Scatchard plots b y extrapolating the curves to r/c = 0 and taking t h e intercept on the r axis, and (ii) from binding isotherms. The specific viscosities of the free DNA and of the nogalamycin • DNA complexes were measured with a Cannon-Manning semimicroviscometer at 35°C in a thermostatically controlled water bath following the methods and calculations described in earlier papers [6,15]. All measurements were made at a DNA concentration of 25 ~g/ml. Electron microscope preparations of the free T7 DNA and of nogalamycin • T7 DNA complexes were made according to the protein-monolayer technique [16] taking nogalamycin in the hypophase to the same concentration as that in the hyperphase, to minimise the possibility of dissociation of the b o u n d drug molecules due to extreme dilution while spreading drops of the hyperphase on the hypophase surface. Necessary contrast was obtained either b y staining according to Davis et al. [17] or by shadow casting as described elsewhere [ 10]. Electron micrographs were recorded by a Siemens Elmiskop I, operating at 40 kV. Magnification was routinely checked by a cros~grating replica (Polaron, 54 800 lines/inch). The length of the DNA molecules was measured by a map~listance measuring device. The various errors in the linear measurement of DNA were taken into account by using an earlier described method [181. Results

Electron microscopic evidence o f intercalation o f nogalamycin in T7 D N A T7 DNA molecules, prepared for electron microscopy by using an ammonium acetate hypophase and made electron opaque by shadow casting, were very kinky, filamentous structure (Fig. la). This "kinkiness" is the universal feature of the DNA micrographs prepared under similar hypophase conditions [10,18--20] and is thought to reflect the increased flexibility of the DNA polymers [18,20]. The kinkiness disappeared completely from the micrographs (Fig. l b ) of t h e nogalamycin • T7 DNA complexes with r = 0.125. This effect was less conspicuous in the stained, complexed T7 DNA (Fig. 2b', 2b"). In the case of salt-free water as a hypophase, free as well as nogalamycin-complexed T7 DNA presented the same smooth appearance [9,18,20]. The disappearance of the microkinks is a qualitative manifestation of the stretching of the complexed native DNA due to intercalation of the drug molecules between the adjacent base pairs. This diagnostic feature was first demonstrated by Freifelder [7] for the intercalative binding of ethidium bromide as well as acridine orange with k DNA. Our results lend further support to this observation. In the stained samples, however, the kinkiness was only partially removed. The reason is n o t apparent. The remarkable difference in our staining technique was the use of a still higher molar concentration (0.25 M in contrast

202

Fig. 1. (a) E l e c t r o n m i c r o g r a p h of a free T7 D N A m o l e c u l e , p r e p a r e d b y using 0 . 1 2 M a m m o n i u m a c e t a t e as a h y p o p h a s e , c i r c u l a r l y s h a d o w e d . A k i n k y c o n t o u r is t h e special f e a t u r e of the D N A m i c r o g r a p h s u n d e r this p r e p a r a t i v e c o n d i t i o n . 3 5 0 0 0 X. (b) One T 7 D N A m o l e c u l e c o m p l e x e d w i t h n o g a l a m y c i n at P/D = 2; t h e h y p o p h a s e of 0 . 1 2 M a m m o n i u m a c e t a t e plus n o g a l a m y c i n in t h e c o n c e n t r a t i o n t h a t e x i s t e d in t h e h y p o p h a s e . A r e m a r k a b l e f e a t u r e was t h e c o m p l e t e d i s a p p e a r a n c e of kinkiness. 43 0 0 0 ×.

to 0.12 M a m m o n i u m acetate used by Friefelder [7]) of the hypophase. Linear measurements of the DNA micrographs have been summarized in Table I. Assuming, in accordance with Lerman's model [21] of intercalative binding, that the interbase distance in native DNA is doubled per one intercalated nogalamycin, the m a x i m u m average length extension of T7 DNA by 23% {Table I) after nogalamycin binding would mean that the maximum number of the intercalating sites of nogalamycin per 1000 nucleotides of T7 DNA is 115. This agreed well with the strong binding sites of nogalamycin in 0.001 M Tris • HC1 plus 1 M NaC1 (Table II). Since the ionic strength of the hyperphase was equal to 0.8 M (0.5 M a m m o n i u m acetate plus 0.3 M Na*), we hold the above agreement as good proof of a close similarity of the intercalative sites to the strong binding sites.

Viscometry The ratio of the specific viscosity of the nogalamycin • DNA complexes to that of the free DNA when plotted against r, showed a sharp rise up to r = 0.12 or up to a D/P value of 0.225 in phosphate/EDTA/saline buffer (0.002 M NaH2PO4/0.006 M Na2HPO4/0.001 M Na2EDTA/0.179 M NaCl, pH 6.8), {Fig. 3), or up to r = 0.12 in 0.001 M Tris - tIC1 plus 1 M NaC1. It appears that at an r value of 0.12 the intercalative binding of nogalamycin with duplex DNA is complete in high molar solvents. This provides additional support for the electron microscopically determined saturation limit.

203

Fig. 2. ( a r a n d a ' ) T w o free T7 D N A m o l e c u l e s prepared by the s t a i n i n g t e c h n i q u e , s h o w i n g k i n k y app e a r a n c e , a t, 3 8 5 9 9 × ; a ' , 4 0 0 0 0 × . (b t a n d b ' ) T w o n o g a l a m y c i n c o m p l e x e s T7 D N A (P/D = 1) prep a r e d by the s t a i n i n g t e c h n i q u e , w i t h the difference that the h y p o p h a s e contained n o g a l a m y c i n to t h e s a m e c o n c e n t r a t i o n as the h y p o p h a s e . D N A m i c r o g r a p h s s h o w e d the r e m o v a l of t h e k i n k i n e s s t o s o m e extent, b', 41 6 0 0 × ; b " , 41 8 0 0 X.

Interactions of nogalamycin with native DNA in different ionic environments The plots of r/c versus r (Fig. 4) show that the expected linearity is deviated from at high r values, an indication of a second type of binding that is different from the first type (strong binding) and represented by the straight-line portions of the curves. A marked deviation is observed in the case of a salt-free water environment. As there is a possibility of the coexistence of the t w o types of binding above r = 0.1, the theoretical basis of calculating the strong binding sites as well as the association or dissociation constant from the Scatchard plots does n o t seem to be sound. A more rational approach would be to get Ka, the association constant from the intercept on the r/c axis by extrapolating the plots to r = 0. At the level of saturation of drug binding, or just before that, the drug molecules binding to nucleotides should follow a single mode and the b o u n d drugs would also be non-interacting to each other because of the pres-

204 TABLE I LINEAR MEASUREMENTS

ON FREE AND NOGALAMYCIN-COMPLEXED

T7 DNA

T h e figures w i t h i n the b r a c k e t s d e n o t e t h e n u m b e r o f T 7 D N A m o l e c u l e s m e a s u r e d . S a m p l e s 1, 2, 4 a n d 5 were circularly shadowed with metal, samples 3 and 6 were stained with uranyl acetate. Samples

Hypophase

L e n g t h ± S.D.

1. F r e e T7 D N A

Water

1 2 . 7 0 -+ 0 . 6 0 (50)

--

--

2. F r e e T 7 D N A

0.12 M ammonium acetate

11.55 ± 0.56 (47)

--

--

3. F r e e T 7 D N A

0.25 M ammonium acetate

10.8

--

--

4. T7 D N A + n o g a l a m y c i n at D / P = 0 . 2 4 , r = 0 . 1 2

Water + nogalamycin

15.75 ± 0.90

24.0

12.0

5. T 7 D N A + n o g a l a m y c i n a t D / P = 0.5, r = 0 . 1 5

0.12 M ammonium acetate + nogalamycin

14.10 ± 0.62 (42)

22.0

11.0

6. T 7 D N A + n o g a l a m y c i n at D / P = 1 , r = 0 . 1 7

0.25 M ammonium acetate

13.30 ± 0.55 (40)

23.0

11.5

Percentage length extension

± 0.46 (35)

N u m b e r of interc a l a t e d sites p e r hundred nucleotides o f T7 D N A

ence of a large number of binding sites. Therefore, the n values of binding may be calculated from the binding isotherms of that stage. Table II summarizes the results. The n values determined from the Scatchard plots have also been included in Table II, since these n values, as we have discussed, are ionic-strength dependent and so help the assessment of the electrostatic-binding process, if any, of a ligand. The association constants, Kn, were of the order of 106, reflecting a strong mode of binding, with the binding energy of 8--9 kcal/mol ( A G = - - R T In Kn, (25)). To ascertain whether the complexes of higher r values contain weakly bound nogalamycin molecules, the following studies were made.

T A B L E II BINDING PARAMETERS OF THE COMPLEXES FERENT IONIC CONDITIONS

Ionic condition

OF NOGALAMYCIN

AND CT DNA UNDER DIF-

Kn (M - ! )

n ( f r o m binding isotherms)

n ( f r o m Scatchard plots)

Triple-distilled w a t e r

0.21

0.20

3 . 0 0 × 106

0 . 0 0 1 M T r i s ' HCI, pH 7.4

0.11

0.15

3 . 9 3 × 106

0.01 M T r i s • HC1, pH 7.4

0.10

0.14

2 . 5 0 × 106

Phosphate/EDTA/saline buffer ( 0 . 2 M N a +)

0.14

0.13

1 . 9 5 × 106

0 . 0 0 1 M T r i s • H C I plus 1 M NaCl, p H 7.4

0.10

0.11

5.21 × 106

205 //

1

1"6

1"2

3"0

'~ x 0'8 r..i~

2.0

.l

I'7

1"6

2

4"0

I"5

i

0 0"~I0

1.4

I

"20

~ 0'0 "30 "I0

t

.20

,2

,,

1'2

l'2

1.2

x O.O

0'8

1"I

0'4

0"4

I'0

'

'15

//

0:1

;2

0"3

0"0, o

t o|

,o

Fig. 3. P l o t o f the specific visco~ty versus r of nogalamycin • calf thymus D N A complexes in phosphate/ EDTA/saline butter at 35°C. Shear gradient ~ 1 . 0 X 103 s -1 .

Fig. 4. T h e P l o t s b e t w e e n r/c versus r d r a w n f r o m t h e a b s o r b a n e e data o f n o g a l a m y c i n • calf t h y m u s D N A c o m p l e x e s , S o l v e n t c o n d i t i o n s w e r e as f o l l o w s : ( 1 ) salt-free w a t e r , ( 2 ) p h o s p h a t e / E D T A / s a l l n e b u f f e r (0.2 M Ha+), (3) 0.001 M Trls • HCI, pH 7.4, (4) 0.001 M Tris. HCI plus 1 M N a t l .

Thermal dissociation o f the bound nogalamycin molecules The enhanced thermal stability of the nogalamycin-bound native D N A has been exhibited in the thermal absorbance profiles drawn from the A260nm values [3] or in the thermal viscosity profiles [6]. Here we were interested in the thermal absorbance profiles of nogalamycin • D N A complexes at visible wavelengths (460 and 470 nm) to get an idea of the thermal dissociation of the bound nogalamycin molecules. It has been shown elsewhere [22] that the weakly bound acridines are released well before the onset of the helix-coil transition of the complexes, whereas the intercalated acridines and daunomycin are released strictly in the helix~coil transition zone [22,23]. Our studies (Fig. 5) indicated that for a nogalamycin • D N A complex of P/D = 14 (at this complexing ratio all the nogalamycin molecules were expected to b e i n the intercalated state) the drug release occurred sharply (co-operatively) between 73 and 87 ° C. This temperature zone was found, however, from the profile of the absorbances at 260 nm, to be the helix~oil transition zone of the same complex. At P/D = 4, the release of nogalamycin molecules occurred at lower temperatures. Similar profiles were obtained for ethidium bromide • D N A as well as daunomycin • D N A complexes. As both ethidium bromide and daunomycin are involved in

206

2"0

1",9

/'8

1"7

t t.6

g Q ~

1"5

l'5

1"2

1"1

~91

30"

t

4o °

~'¢

,

60"

7'0"

J

~o °

90"

Temperature "C Fig. 5. Thermal absorbance profiles of the d r u g . D N A complexes in 0 . 0 0 1 M T r i s . HC], p H 7.4. have been drawn from the average value of the relative absorbanee at 460 and 4 7 0 rim. Curves 1, 2 ethidium bromide • DNA, d a u n o m y e i n • D N A and noga/amycln • D N A respectively, all eomplexed = 4; curves 3 and 4, ethidium bromide • DNA and nogalaraycin • DNA respectively, eomplexed at

Curves a n d 5,

at P/D P/D

=

14.

weak, electrostatic interactions with native DNA in low molar solvents [13, 24], it appears, as also observed elsewhere for the aminoacridines [23], that the hyperchromic profiles at sub-melting temperatures represented the release of the weakly bound ligands. Thus, nogalamycin also seems to be involved in weak interactions with native DNA. In 0.001 M Tris • HC1 plus 1 M NaC1, the complex of nogalamycin • DNA at P / D = 1 did not however show any significant hyperchromicity up to 75 ° C, indicating the absence of the weakly bound fraction and suggesting a probable electrostatic nature of the weak interactions. We attempted to demonstrate the weakly bound nogalamycin molecules electron microscopically through some structural changes. The complexes of P / D = 4--1 did not however present any remarkable structural differences. But

207

Fig. 6. E l e c t r o n m i c r o g r a p h s o f n o g a l a m y c i n • C T D N A c o m p l e x e s at P / D = 0 . 0 5 . ( C T D N A 0 . 0 2 • 10 - s M), n o g a l a m y c i n ( 0 . 4 • 10 - s M) i n p h o s p h a t e / s a l i n e b u f f e r . A t t h i s c o n c e n t r a t i o n free n o g a l a m y c i n m o l e c u l e s w e r e in the m o n o m e r i c f o r m .

as the proportion of the drug was increased further (complexes of P / D = 0.01--0.05), the DNA micrographs were much thickened as well as intra- and inter-molecularly aggregated (Fig. 6). It appears that at very high r values nogalamycin molecules are probably stacked on the external surface of DNA in the polymeric form. Discussion

The upper limit of strong binding sites of nogalamycin per nucleotide of native DNA, obtained from binding isotherms as well as from the Scatchard plots at high ionic strength, was found to be 0.10, which agreed very closely with that of the intercalative binding as reflected in the length extension of T7 DNA as well as in the saturation limit of the viscosity effect of nogalamycin • DNA complexes. Such a striking agreement is possible when the strong binding is s y n o n y m o u s with intercalation [21]. From this, we can imagine an intercalative binding model in which four sites are excluded per one intercalated nogalamycin molecule. Similar observations were also made in t h e binding of proflavine with native calf thymus and T7 DNAs [9]. The findings are, however, n o t in agreement with the calculations of Waring [4], which showed that for nogalamycin as well as proflavine binding with a duplex DNA only 70% of the strong binding sites were intercalative. The calculations were based on the uncoiling of a super-helical DNA. The uncoiling was thought to be brought a b o u t by intercalation of the ligand molecules. Since even non-intercalative mechanisms can also uncoil a super-helical DNA [4], such calculations would n o t perhaps give a correct estimation of the intercalative sites. This has been discussed elsewhere [9]. The deviation of the r / c versus r plots from linearity, the heat-induced dissociation of the b o u n d nogalamycin molecules at submelting temperatures of the

208 complex, and the thickened electron micrographs at very low P/D values were clearly indicative of a weak mode of binding of the drug with native DNA. Some earlier workers [2] also recorded a similar indication by noting that the binding was appreciably reduced after addition of a sufficient a m o u n t of NaCl to the complex of nogalamycin. DNA. Other workers [6] using phosphate/ EDTA]saline buffer almost missed the effect. This is because of the ability of EDTA to bind to free nogalamycin, which would obviously lessen the stacking of nogalamycin on the external surface of DNA. And that is w h y in our results too, the n values for this buffer, obtained in either way, were the same. However, the strong binding, as reflected in the n values determined from binding isotherms, does not seem to be d e p e n d e n t on the ionic strength from 0.001 M Tris • HC1 to that plus 1 M NaC1. In this molarity range the native nature of DNA is found, from the h y d r o d y n a m i c studies, to be retained [25]. Whereas in the case of a water environment, where partial denaturation of DNA should occur [26,27], the n values were appreciably higher. That the strong binding sites are considerably higher in denatured DNA than in native DNA has also been shown elsewhere [28]. So, it appears that the availability of the strong binding sites is dependent on the secondary structure of DNA. The n values of the cationic drugs, obtained from the Scatchard plots, show a strong dependence on the ionic strength of the environment. This is evident from the results: n changes from 0.25 to 0.15 for proflavine, 0.30 to 0.17 for 9-aminoacridine, 0.125 to 0.07 for 9-amino-l,2,3,4-tetrahydroacridine, 0.005 M Na ÷ to 0.1 M Na ÷ [28] and 0.18 to 0.10 for ethidium bromide [13]. The considerable reduction of binding in high molar solvent is obviously due to the existence of an electrostatic, weak binding (process II binding) of the cationic dyes in low molar solvents. In the case of nogalamycin, n changes from 0.15 to 0.11 with the change of the environment from 0.001 M Tris • HC1 to that plus 1 M NaC1, thus suggesting the presence of the process II (using the terminology of ref. 12) type of binding. However, in this case, the reduction of the binding sites due to removal of the process II type of binding is 27% only, whereas in the case of ethidium bromide and the other cationic dyes, this is around 40%. The thermal absorbance profiles also support this, where the hyperchromic effect of nogalamycin is appreciably smaller than that of ethidium bromide or of daunomycin. The forces involved in the weak mode of binding appear to be electrostatic, and for that the ligand should be cationic. The available structure of nogalamycin does n o t contain any cationic site, b u t one o f its t w o sugar moieties is still unsolved. This sugar residue may contain a cationic site, probably in its nitrogen atom. However, a clearer physical picture will emerge only when the complete structure of nogalamycin is available. Acknowledgements The authors are thankful to the Director, CNCRC, Calcutta, for his kind permission to carry o u t this work. Mr. K.L. Bhattacharya advised and encouraged us throughout. We are thankful to Dr. P. Sadhukhan of S.I.N.P., Calcutta, for allowing us to use the shadow-casting instrument. The technical assistance of Mr. Kalyan Ghosh and Ranjit Das Gupta is gratefully acknowledged. This work received financial support from I.C.M.R., New Delhi.

209

References 1 2 3 4 5 6 7 6 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 26

Ward, D.C., Reich, E. and Goldberg, I.H. (1965) Science 149, 1259--1263 Bhuyan, B.K. and Smith, C.G. (1965) Proc. Natl. Acad. Sci. U.S, 54, 566--572 Kersten, W., Kersten, H. and Szybalski, W. (1966) Biochemistry 5, 236--244 Waring, M.J. (1970) J. Mol. Biol. 54, 247~279 Saunders, G.F., Reese, W.N. and Saunders, P.P. (1969) Biochim. Binphys. Acta 190, 406--417 Das, G.C., Dasgupta, S. and Das Gupta, N.N. (1974) Biochim. Biophys, Acta 353, 274--282 Freifelder, D. (1971) J. Mol, Biol. 60,401--403 Lang, D. (1971) Phil. Trans. R. Soc. Lond, Ser. B 261,151 Dasgupta, S., Misra, D.N. and Das Gupta, N.N. (1973) Biochim. Binphys. Acta 294, 241--244 Misra, D.N., Sinha, R.K. and Das Gupta, N.N. (1969) Virology 39,183--193 Neogy, R.K., Choudhury, K. and Guha-Thakurata, G. (1973) Biochim. Biophys. Acta 299,241--244 Peacoeke, A.R. and Skerrett, J.N.H. (1956) Trans. Farad. Soc. 52, 261--279 Warlng, M.J. (1965) J. Mol. Biol. 13, 269--282 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 5 1 , 6 6 0 Das, G,C. and Das Gupta, N,N. (1974) Z. Naturforsch. 29C, 133--135 Kleinschmidt, A.K. and Zahn, R.K. (1959) Z. Naturforsch. 14b, 770--779 Davis, R.W., Simon, M. and Davidson, W. (1971) Methods in Enzymology, (Grossman, L, and Moldave, K., eds.), Vol. 21D, pp. 413--428, Academic Press, New York Lang, D., BuJard, H., Wolff, B. and Russell, D. (1967) J. Mol. Biol. 23, 163--161 Inman, R.B. (1967) J. Mol. Biol. 23, 18--30 Sinha, R.K., Misra, D.N. and Das Gupta, N.N. (1974) Ind. J. Biochim. Biophys. 11, 162---169 Lerman, L.S. (1961) J. Mol. Biol. 3, 18--30 Iehlmura, S., Zama, M., Fujlta, H. and Ito, T, (1969) Biochim. Biophys. Acta 190, 406--417 Kleinwachter, V., Bakarava, Z. and Bohacek, J. (1969) Biochim. Biophys. Acta 174, 168--201 Zunino, F., Gambetta, R., Di-Marco, A. and Zaccara, A. (1972) Biochim. Biophys. Acta 277, 489--496 Rosenberg, A.H. and Studier, F.W. (1969) Biopolymers 7,765--774 Inman, R.B. and Jordan, D.C. (1960) Biochim. Biophys. Acta 42, 421--426 Cavalieri, L.F., Roshoff, M. and Rosenberg, B.H. (1956) J. Am. Chem. Soe. 78, 5239 D r u m m o n d , D.S., Simpson-Gildemeister, V.F.W. and Peacocke, A.R. (1965) Biopolymers 3, 135--153