Interaction of daunomycin and its derivatives with DNA

489

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 97366

INTERACTION OF DAUNOMYCIN AND ITS DERIVATIVES W I T H DNA F. Z U N I N O , R. G A M B E T T A , A. D I M A R C O AND fl-. Z A C C A R A

Experimental Oncology Division, National Cancer Institute, Via Venezian i, 2oi33-Milano (Italy) (Received M a r c h 29th, 1972)

SUMMARY

The interaction of daunomycin and its derivatives with calf thymus DNA has been studied by visible absorption spectrophotometry, equilibrium dialysis, low-shear viscosimetry and thermal denaturation of the complex. It has been found that the binding of daunomycin to DNA involves more than one class of sites. A comparison of association constants confirms quantitatively the previously reported suggestion that the amino sugar residue of daunomycin is involved in the stabilization of the complex. The reduced binding constant for the derivative containing D-glucosamine suggests the importance of the structure and the stereochemistry of the amino sugar moiety in the binding process. In addition, interesting influences of substitutions in the acetyl side chain were observed. The results are consistent with the proposed intercalative model. Measurements of the effect of disorganization of the double-helical structure of DNA upon dannomycin binding indicate that the double-helical structure is a necessary condition of the strong binding process. Complex formation with DNA accounts adequately for the biological properties of the antibiotics.

INTRODUCTION Daunomycin, a glycosidic anthracycline antibiotic, has been isolated from Streptomyces peucetius I and shown to have cytotoxic and antimitotic activities ~. Chemical studies have established 3-5 the total absolute configuration of this antibiotic. Daunomycin has been reported to inhibit both enzymatic RNA and DNA synthesis 6'7, probably by binding to DNA s, thus interfering with its template function. When daunomycin binds to native DNA, there is a decrease in the buoyant density and the sedimentation and an increase in the viscosity of the DNA 8'9. Since the latter two effects are similar to those produced by acridine dyes x°, daunomycin is also believed to intercalate between adjacent base pairs of double-helical DNA n. In addition, Waling TMreported that daunomycin affects the supercoils of closed circular DNA in the same qualitative fashion as other drugs that are believed to intercalate. Recently, viscosity measurements 13 and X-ray diffraction studies 14 of the daunomycin-DNA complex have been found to be consistent with the intercalation model. Biochim. Biophys. dcta, 277 (1972) 489-498

49 °

l;. ZUNINO e[ c/].

Previous studies JS.~ of the physical properties and biological activity of related compounds suggested an important iole of the amino sugar residue in complex formation and in the mechanism of action. However, until now there has been no systematic investigation for establishing the critical groupings on the molecule for binding. In an attempt to inlprove our understanding of the binding mechanism we have approached the problem using a variety of daunomycin derivatives a'~v'18 and employing a combination of different types ot physical measurements, including spectrophotometric analysis, equilibrium dialysis, low-shear viscosimetry and elevation of the DNA melting temperature.

MATERIALS AND METHODS

Daunomycin and its derivatives (Table I) were supplied by Farmitalia, Milan. All antibiotics were stored in the dark in a dessicator at 4 °C. Solutions in buffer were freshly prepared immediately before use. TABLE [ DAUNOMYCIN

A N D ITS D E R I V A T I V E S

0

OH

a e n s r s-I s t r u c t u r e

H OH

daunosamine

I H

H NH2

_4 nlibiotic

31ol. wt R

D a u n o m y c i n - HCI Adriamycin • I4CI I 3 - D i h y d r o d a u n o r n y c i n • HCI 2-Amino-z-deoxyglucosyl d a u n o m y c i n o n e • HCI N - G u a n i d i n e acetamide d a u n o m y c i n • HCI N - A c e t y l d a u n o m y c i n • ffCl

563. 5 CO • CH 3 58o.o -CO - C H , O H 566.o -CHOI-[ • CH 3 596. 5 "~ ) 662.5 CO • CHu 606.0

IU ] j Daunosamine 2-Amino-2 ~x-I)-deoxyglucose N - G u a n i d i n e acetamide daunosamine N-Acetyldaunosamine

H

Origin Natural Natural Natural Synthetic Synthetic Synthetic

Calf thymus DNA was prepared according the procedure of ZamenhoP 9. Some samples were further purified by phenol extraction. The DNA sample was stored in solid state. When needed, a stock solution of DNA was prepared in o.I M Tris-HC1 (pH 7.0). The primary stock solution was stored flozen until just prior to use. The same DNA sample was used in all experiments. Concentrations of DNA solutions were determined spectrophotometrically at 260 nm. Unless otherwise indicated, all experiments were performed in o.I M Tris-HC1 (pH 7.0). H e a t - d e n a t m e d DNA was prepared by heating a solution of native DNA in o.I M Tris-HC1 (pH 7.0) in a boiling water bath for IO rain and then inunediately cooling the solution in a water bath at o °C. To study the effects of daunomycin on Biochim. B i o p h y s . . q c l a , 277 (1972) 489-49 S

BINDING OF DAUNOMYCINDERIVATIVESTO DNA

491

the viscosity of denatured DNA, denaturation was accomplished by heating at 90 °C for IO min in 0.0oi M NaC1, followed by fast cooling. Sonicated DNA was prepared as reported by Doty et al. ~°. The binding parameters of antibiotics to DNA were determined spectrophotometrically with a Zeiss PMQ II spectrophotometer, using 2o-cm light path cells. The cuvettes were filled with a DNA solution (2.5 • 10-5-3 • lO -5 M). Aliquots of a concentrated antibiotic solution (5 • lO-*-6 • i0-4 M) were added with the aid of a microliter syringe. The absorbance was measured at 480 nm, about 20 min after antibiotic addition. These measurements were done at 20 °C, in o.I M Tris-HC1 (pH 7.0). The absorbance data were used to calculate the Scatchard plot, according a method employed by Miiller and Crothers 2~. The expression used:

rim = Kapp( B a p p

--

r)

is appropriate to the plot of binding isotherms, where r is the ratio of bound antibiotic to total DNA nucleotides, m is the concentration of free antibiotic, Kapp is the apparent binding constant, and Bapp is the apparent number of binding sites. Equilibrium dialysis experiments were carried out according a method similar to that employed by Chambron et al. ~, except that tritiated antibiotics were used. The DNA concentration was held at 1.3 • io-* M throughout, and the concentration of tritiated antibiotic (labelled antibiotics were obtained from Farmitalia, spec. act. I • io13-io • lO is dpm/mole) was varied from 16. IO-~ to 12 • lO -5 M. Experiments were performed in the dark at constant temperature (20 °C). Control experiments showed that equilibrium was achieved in about 72 h; no degradation of the antibiotics was detected at this time by thin-layer chromatography. Samples (IOO#1) from both sides of the membrane were dried, dissolved in Soluene (Packard) and added to 15 ml liquid scintillator composed of PPO and dimethyl-POPOP in toluene. Radioactivity was determined in a Tlicarb liquid scintillation counter with an internal standard. Viscosities were measured at 20 °C in a Zimm-Crother low-shear viscosimeter adjusted to give a shear stress of less than 0.002 dyne/cruZ. Dilutions of D N A antibiotic mixtures were carried out at constant r by adding solvent which contains a concentration m of free drug according to the method of Miiller and Crother 2'. Thermal denaturation studies were carried out with a Zeiss PMQ I I spectrophotometer. The temperature of thermal transition (Tin) was obtained from absorbance changes at 260 nm of drug-DNA complexes in Tris-HC1 buffer (pH 7.0) in thermostated cuvettes in which the temperature was measured with a YSI rood. 42 SC tele-thermometer.

RESULTS

Equilibrium measurements Some equilibrium aspects of the interaction of daunomycin with DNA have already been investigated le,23. The present measurements attempt to examine the above finding further and extend the observations to daunomycin derivatives. Because of the limited quantities of some daunomycin derivatives available, the binding Biochim. Biophys. Acta, 277 (1972) 489-498

492

F. ZUNINO el al.

of daunomycin and its derivatives to calf thymus DNA was measured spectrophotometrically. The spectrophotometric analysis is based on the fact that at 48o nm tile absorbance of bound antibiotic is 2o-4o % less than the absoibance of free antibiotic. A typical binding isotherm calculated from the experimental data is shown in Fig. I. Extrapolation of the curve for small values of r yields the apparent association constant, Kapp, and the apparent number of binding sites per nucleotide, Bapp, for stronger binding process. The binding parameters, Kap,, and Bam~ derived from r / m versus r plots are presented in Table II. This table summarizes the results of several measurements.

3.~

0

\

,c -~. \

31

2~

\

\

\ $'20

5 E

El5

\ L.

\ L.

\.

1.0

O~

°ol 0

I

0

O 05

t

0.1

r

0

0.05

olo

o ls

•

o'2o 7

Fig. i. I s o t h e r m for t h e b i n d i n g of d i h y d r o d a u n o m y c i n to calf t h y m u s D N A . D a t a were o b t a i n e d f r o m s p e c t r o p h o t o m e t r i c analysis. D e t a i l s are described in Materials a n d M e t h o d s . Fig. 2. S c a t c h a r d plot of t h e b i n d i n g of d a u n o m y c i n to calf t h y m u s D N A . D a t a were o b t a i n e d f r o m e q u i l i b r i u m dialysis s t u d i e s u s i n g [ 3 H q d a u n o m y c i n . Details are given u n d e r Materials a n d Methods.

As a check on the method, the binding of daunomycin and adriamycin to DNA was measured by equilibrium dialysis, using 3H-labelled antibiotics. An analysis of the results obtained foi the DNA-daunomycin interaction is shown in Fig. 2. The numerical values of this and other binding measurements are listed in Table II. I t m a y be seen that the spectrophotometric analysis have results for the daunomycin and adriamycin-DNA interaction in reasonable agreement with those obtained from the equilibrium dialysis method under similar conditions. As expected trom previous data 8' 15, acetylation of the amino group of daunosamine markedly reduces the affinity. In addition, other modifications on the amino Biochim. Biophys. ,4cta, 277 (I972) 489-498

BINDING OF DAUNOMYCIN DERIVATIVES TO D N A

493

TABLE II BINDING PARAMETERSFOR THE INTERACTIONOF DAUNOMYCINAND ITS DERIVATIVESWITH DNA All experiments were carried out at 2o°C in o.i M Tris-I-~C1 (pH 7.o). Kapp and Bappwere determined from binding isotherms as described in the test and indicated in I~ig. I and Fig. 2. Each value represents an average of at least three values determined by the indicated method. Unless otherwise indicated, native calf thymus DNA was used. A ntibiotic

Ka~~(M -1)

B a~

Method

Daunomycin

2.9 • lOs 3.3 'lOS 1.5 • lOs

o.16 o.18 o.17

Adriamycin

2.8 • lOs 2. 3 - lOs i.I • lOs

0.20 o.19 o.io

Equilibrium dialysis Optical method Optical method (using denatured DNA) Equilibrium dialysis Optical method Optical method

7.2 " lOs

0.09

Optical method

7.1 • lO4 1.8 • lO4

0.o9 o.12

Optical method* Optical method*

Dihydrodaunomycin N-Guanidine acetamide daunomycin 2-Amino-2-deoxyglucosyl daunomycinone N-Acetyldaunomycin

* In this case, i-cm cells were used.

function (N-guanidine acetamide d a u n o m y c i n ) or exchange of d a u n o s a m i n e for D-glucosamine (2-amino-2-deoxyglucosyl d a u n o m y c i n o n e ) considerably decrease the affini t y for DNA. H e a t - d e n a t u r e d D N A caused a hypochromic shift of the s p e c t r u m of daunom y c i n similar to t h a t observed with double-stranded DNA, b u t is r e m a r k a b l y different from n a t i v e D N A in its b i n d i n g characteristics. The straight line in the r i m versus r plot i n d i c a t e d t h a t there is only one type of site, whose b i n d i n g c o n s t a n t is 1.5 " lO5 i -I. I t is worth n o t i n g t h a t the total n u m b e r of b i n d i n g sites (Bapp c~ o.16) is not affected b y d e n a t u r a t i o n . Viscosimetry

Sevelal investigatorss,9,13,15 have n o t e d t h a t the viscosity of D N A is increased b y d a u n o m y c i n . The increase in intrinsic viscosity of D N A is accepted as a diagnostic feature of an i n t e r c a l a t i o n process 1°. We t h o u g h t it, therefore, of i n t e i e s t to compare the specific effects of d a u n o m y c i n derivatives on the h y d r o d y n a m i c b e h a v i o u r of D N A with those reported for d a u n o m y c i n ~3. I n t r i n s i c viscosities of the a n t i b i o t i c - D N A complexes were o b t a i n e d b y linear e x t r a p o l a t i o n of the reduced viscosity measured at different D N A concentrations. D i l u t i o n was carried out at c o n s t a n t r as already described a3. A typical set of results is shown in Fig. 3. The effects of d a u n o m y c i n a n d its derivatives on the intrinsic viscosity of n a t i v e D N A are compared in Fig. 4. I n this figure the intrinsic viscosity of each D N A complex has been divided b y the intrinsic viscosity of D N A alone a n d the ratio has been plotted against the degrees of b i n d i n g r. U n d e r identical conditions of r a n d ionic strength, the intrinsic viscosity is m a r k e d l y less e n h a n c e d b y all the derivatives with altered a m i n o sugar t h a n b y other derivatives c o n t a i n i n g unsubstituted daunosamine. Bioehim. Biophys. Acta, 277 (x972) 489-498

494

C

~:. zuNi~xo et al.

i

t - - * - -

~ * -

r=O.O00

i 05

7£

2C

!~ Lx

25

3-C

t~ ~

Fig. 3- A representative set of reduced viscosity curves used to determine the intrinsic viscosity of a n t i b i o t i c - D N A complexes for different values of r with native calf t h y m u s DNA. D a t a apply to N - a c e t y l d a u n o m y c i n - D N A complexes at 2o°C, in o.I M Tris-HC1, p H 7.o.

2'

/ ° x

/ /

005

010

015

OL20

r

Fig. 4. Ratio of intrinsic viscosity of antibiotic D N A complexes to t h a t of D N A alone plotted against v. Conditions of the viscosity m e a s u r e m e n t s : 2o°C, o.i M Tris-HC1, pH 7.0. O , d a u n o m y cin; ×, adriamycin; 0 , I3-dihydrodaunomycin; + , 2-amino-2-deoxyglucosyldaunomycinone; A, N-guanidine acetamide daunomycin; A, N - a c e t y l d a u n o m y c i n .

In contrast to its effects on the native DNA, daunomycin caused a marked decrease in intrinsic viscosity of d e n a t m e d DNA. The intrinsic viscosity of denatured D N A - d a u n o m y c i n complex at r -: o.178 and at ionic strength o.ooi is about 80 ~)"o below that of denatured DNA alone. The effect of binding of daunomycin to denatured DNA parallels the results reported for acridine dyes 24. In the case of acridine dyes, the effect of binding on the intrinsic viscosity of denatured DNA is quantitatively the same as that achieved by adding neutral electrolyte and has usually been attributed to decrease in mutual repulsion between the charged groups of a flexible polyelectrolyte chain (phosphate groups of DNA). Biochim. Biophys. Acla, 277 (1972) 489-498

BINDING OF DAUNOMYCIN DERIVATIVES TO

DNA

495

Our results on the effect of daunomycin binding on denatured DNA could be satisfactory explained by the effects of the positively charged amino group of daunosamine on the mutual repulsion of the phosphate groups of the polynucleotide chain, suggesting that daunomycin, like the acridines, binds in the non-helical regions of DNA in the denatured state.

Thermal denaturation The interaction of daunomycin with native DNA produces not only the configurational changes mentioned above, but it also stabilizes the double helical structure of the macromolecule towards thermal denaturation. This effects depends on antibiotic/DNA ratio. At a daunomycin to DNA-P ratio of o.I, the Tm of calf thymus DNA was raised from 70.5 to 83.5 °C in o.oi M Tris-HC1 (pH 7) (Fig. 5, Curve 2). In this figure is also shown (Curve 3) the increase in absorbance at 480 nm, arising from dissociation of the daunomycin-DNA complex.

i

x x-x (~

f.?

/

16

i.p o

io/ t3

/

V

2

/

t2

/2

t!

•!

,J

o i x/

g~

10 I ' 0

I x x~--x---'~..x,.x 50 60 70 rEMPERA?URE

t 80

9xO

tO0

--

(°C)

Fig. 5. T h e r m a l d e n a t u r a t i o n c u r v e s for t h e D N A - d a u n o m y c i n c o m p l e x a t a t o t a l a n t i b i o t i c to D N A - P r a t i o of o.i. i, D N A alone a t 26o n m ; 2, D N A - d a n n o m y c i n a t 26o n m ; 3, D l q A d a u n o m y c i n a t 48o nm. S o l v e n t is o.oi M Tris-HC1 (pH 7.o). D N A c o n c e n t r a t i o n is a b o u t I • lO -4 M in all e x p e r i m e n t s . I n C u r v e 2 all a b s o r b a n c e r e a d i n g s w e re c o r r e c t e d b y s u b t r a c t i o n of t h e A2s 0 nm in t h e b l a n k c o n t a i n i n g no DI~A, c o n t a i n i n g d a u n o m y c i n a t t h e s a m e c o n c e n t r a t i o n as t h e s a m p l e a n d h a v i n g t h e s a m e t e m p e r a t u r e as t h e sample.

Biochim. Biophys. Acta, 277 (1972) 489-498

496

I;. ZUNINO et al.

The effects of daunomycin and its derivatives on the thermal denaturation of DNA at an antibiotic to DNA-P molar ratio of o.I are presented in Table I I I , In the same experimental conditions, we observed that adriamycin was the most effective in stabilizing the secondary structure of DNA. Scruting of the data in Table I I I reveals that in general a small value of z] Tm is found with the derivatives containing alterated aminosugar. TABLE II[ tgFFI~CT

OF

DAUNOMYCIN

AND

ITS

DERIVATIVES

ON" T H E

THERMAL

TRANSITION

TEMPERATURE

(Tin) OF CALF THYMUS D N A All e x p e r i m e n t s were carried o u t in o.oi M Tris-FIC! (pH 7) at an antibiotic to D N A - P ratio of o.I. D N A c o n c e n t r a t i o n was a b o u t I • IO -4 M in all experiments.

Antibiotic

Tm (26o n m )

ATm(26o n m )

None Daunomycin Adriamycin 13-Dihydrodaunomycin N - G u a n i d i n e acetamide daunomycin 2-Amino-2-deoxyglucosyl daunomycinone N-Acetyldaunomycin

7o.5 83.9 85.3 8o.3

-t 3.4 14.8 9-

78.8

8. 3

78.5 71.5

8.o i .o

DISCUSSION

Our results of binding equilibrium in this system confirm the genelal picture provided b y previous workers 8'1a'16'2~. The shape of the isotherms for the binding of daunomycin to DNA suggests the existence of more than one class of binding sites for daunomycin on DNA. This conclusion is clearly supported by the chromatographic behaviour of daunomycin on DNA-cellulose column 13. The picture which has emerged so far m a y be summarized as follows: at least two modes of interaction between antibiotic molecules and DNA are distinguished; the "strongly" bound antibiotic molecules are understood to be intercalated between successive base pairs of the double helix; the "weakly" bound antibiotic molecules are thought to be attached to DNA by means of electrostatic interaction involving the DNA phosphate groups and the daunomycin amino group. Association constant for the strong binding type is of the same order of magnitude as those found for acridine dyes 2a and actinomycin D ~. The apparent number of binding sites per nucleotide (Baop) obtained for the strong binding mode of daunomycin is in agreement with the value of r (approx. O . 1 6 - O . 2 0 ) at which the limiting viscosity is attained JS. This value is understood to correspond to the end of the process of strong binding of daunomycin 1~. The availability of natural and synthetic daunomycin derivatives has led to some information on the requirements of the antibiotic for complex formation. Nacetylation of the sugar residue of daunomycin markedly reduces the affinity for DNA. This finding confirms quantitatively the qualitative conclusions previously reported 8'15. The importance of amino sugar residue for the binding reaction was emphasized by experiments in which substitution of daunosamine for N-guanidine Biochirn. Biophys. Acta, 277 (I972) 489--498

BINDING OF DAUNOMYCIN DERIVATIVES TO D N A

497

acetamide daunosamine or for o-glucosamine considerably reduced the apparent binding constants. In addition, the equilibrium binding data of the derivative containing n-glucosamine suggest the importance of the structure and stereochemistry of the amino sugar moiety in the binding process. It has been shown ~e that daunosamine, the amino sugar moiety of daunomycin, is an L-sugar, characterized by Llyxo conformation. Substitutions or modifications in the acetyl side chain may cause only minor changes in the binding properties. In particular, adfiamycin binds to DNA as strongly as daunomycin itself, in contrast to the behaviour of IB-dihydrodaunomycin which binds appreciably less tightly. The binding properties of these compounds parallel other physical properties of the complex reported in this paper. The in vivo activity 15 and the inhibitory properties in the DNA-dependent RNA polymerase reaction (P. Chandra, F. Zunino, A. Gbtz, S. A. Hausmann and A. di Marco, unpublished) of daunomycin and its derivatives correlate well with their ability to bind to DNA. Then complex formation appears to account for the biological properties of the antibiotics. The double helix structure is not necessary for the formation of a complex. A complex may also be formed with denatured DNA. However, our measurements of binding equilibrium indicate that the double-helical structure is a necessary condition of a strong binding process. The absence of a specific increase in the viscosity of denatured DNA suggests that this effect is a specific feature of a strong binding with native DNA. The much stronger binding to double-helical DNA probably accounts for the insensitivity of Ec 9, a single-stranded DNA phage, to daunomycin2L ACKNOWLEDGEMENTS

The authors wish to thank Dr L. Lenaz for reading the manuscript and for helpful comments. REFERENCES I A. Grein, C. Spalla, A. Di Marco and G. Canevazzi, Giorn. Microbiol., II (1963) lO9. 2 A. Di Marco, M. Gaetani, P. Orezzi, B. M. Scarpinato, R. Silvestrini, M. Soldati, T. Dasdia and L. Valentini, Nature, 2Ol (1964) 706. 3 F. Arcamone, G. Franceschi, P. Orezzi and S. Penco, Tetrahedron Lett., 3 ° (1968) 3349. 4 F. Arcamone, G. Cassinelli, G. Franceschi, P. Orezzi and R. Mondelli, Tetrahedron Lett., 30 (1968) 3353. 5 R. Angiuli, E. Foresti, L. R i v a di Sanseverino, N. W. Isaacs, O. Kennard, ~Ar. D. S. Motherwell, D. L. W a m p l e r a n d F. Arcamone, Nature New Biol., 234 (1971) 78 6 D. C. Ward, E. Reich, and I. H. Goldberg, Science, 149 (1965) 1259. 7 G. H a r t m a n n , H. Goller, K. Koschel, W. Kersten and H. Kersten, Biochem. Z., 341 (1964) 126. 8 E. Calendi, A. di Marco, R. Reggiani, B. Scarpinato and L. Valentini, Biochim. Biophys. Acta, lO3 (1965) 25. 9 W. Kersten, H. Kersten and W. Szybalski, Biochemistry, 5 (1966) 236. IO L. S. Lerman, J. Cell. Comp. Physiol., 64 (1964) I. II A. di Marco, in D. Gottlieb and P. D. Shaw, Antibiotics, Vol. I, Springer Verlag, Berlin, 1967, p. 19o. 12 M. Waring, J. Mol. Biol., 54 (197 o) 247. 13 1~. Zunino, F E B S Lett., 18 (1971) 249. 14 W. J. Pigram, W. Fuller and L. D. Hamilton, Nature New Biol., 235 (1972) 17.

Biochim. Biophys. Mcta, 277 (1972) 489-498

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15 A. di Marco, IT. Zunino, R. Silvestrini, C. G a m b a r u c c i and A. R. G a m b e t t a , Biochem. Pharma. col., 20 (1971 ) 1323 . 16 H. Berg a n d t~. Eckardt, Z. Naturforsch., 25b (197o) 362. 17 F. Arcamone, G. Cassinelli, G. Fantini, A. Grein, P. Orezzi, C. Pol and (L Spalla, BiotechJ~. Bioeng. I I (1969) i i o i . 18 S. Penco, Chimica e Industvia, 5 ° (1968) 9o8. 19 S. Zamenhof, in C. \¥estling, Biochemical Preparalio**s, Vol. 6, J. Wiley and Sons, New York, 1958, p. 8. 20 P. Doty, B. B. McGill and S. A. Rice, Proc. Natl. Acad. 5"ci. U.S., 44 (t958) 432. 21 ~V. Miiller and D. M. Crothers, J. ~VIol. Biol., 35 (1968) 251. 22 J. C h a m b r o n , M. D a u n e and Ch. Sadron, Bioehim. Biophys. Acta, 123 (1966) 306. 23 I. Niculescu-Duvaz, R. A. G a m b e t t a and A. di Marco, Oncol. Rctdiol., 9 (197 o) 42I24 D. S. D r u m m o n d , N. J. Pritchard, V. t v. \~r Simpson-Gildenleister and A. 1¢. I'eacoeke Biopolymers, 4 (1966) 971. 25 A. Blake and A. R. Peacocke, Biopolymers, 6 (1968) 1225. 26 F. Arcamone, C. Cassinelli, P. Orezzi, (;. Franceschi, and R. Mondelli, . ] . . 4 ~ . Chem. Noe. 86 (1964) 533527 E. Calendi, R. D e t t o r i and M. G. Neri, Gior~. Microbiol., 14 (1966) 227.

Biochim. Biophys. Acla, 277 (1972) 489-498