- Email: [email protected]
The reaction of anthramycin with DNA
288
Biochimica et Biophysica Acta, 361 ( 1 9 7 4 ) 2 8 8 - - 3 0 2 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 98081
THE REACTION OF ANTHRAMYCIN WITH DNA II. STUDIES OF KINETICS AND MECHANISM*
K U R T W. K O H N , D A N I E L G L A U B I G E R a n d C A R L O S L. S P E A R S
Laboratory o f Molecular Pharmacology, Division of Cancer Treatment, National Cancer Institute, Bethesda, Md. 20014 (U.S.A.) (Received January 4th, 1974) (Revised m a n u s c r i p t received May 1 4 t h , 1 9 7 4 )
Summary Anthramycin is k n o w n to react firmly with DNA, but no chemical basis for this interaction has been elucidated. Since anthramycin readily undergoes hydrolytic changes at C-11, the possible relation of this reaction to the interaction with DNA was considered. Both the hydrolysis of an l l - m e t h o x y group and the reaction with DNA were f o u n d to be H * catalysed, thus supporting a relation between the two processes. The titration of H ÷ from the phenolic group at Position 9 of anthramycin was f o u n d to be absent in the DNA complex, suggesting that Position 9 also is involved in the complex. The rate of reaction of a n t h r a m y c i n - l l - m e t h y l ether with DNA was found to be increased by prior hydrolysis. The rate of reaction with DNA, however, does not involve a rate-limiting conversion of anthramycin to a reactive component, since the kinetics of the reaction were found to be basically bimolecular. This excluded the possibility that a slow conformational change of the DNA was rate-limiting. The findings support the proposal that the binding of anthramycin to DNA is covalent, and that Positions 11 and 9 are involved in the binding. The bimolecular rate constant was found to decrease exponentially with the extent of DNA reaction, probably due to neighboring-site exclusion effects. The site of reaction involves guanine, since only guanine-containing deoxypolynucleotides were found to be reactive, and since reaction of DNA with anthramycin caused stochiometric elimination of binding sites of actinomycin.
* Paper I of this series is ref. 4.
289 Introduction Anthramycin, an antibiotic derived from Streptomyces refuineus, has antit u m o r as well as antibacterial activity [1,2]. It inhibits both R N A and DNA synthesis in mammalian cells, while having relatively little effect on protein synthesis [3]. The antibiotic reacts with DNA [3,4] and inactivates DNA as a template for DNA and R N A synthesis and as a substance for nuclease action [5,6]. The structure of anthramycin (Fig. 12, I) has been confirmed by total synthesis [7--9]. The interaction with DNA is of special interest because, although the anthramycin molecule lacks structural features currently k n o w n to favor tight binding to DNA, it nevertheless reacts specifically with DNA to form a nearly irreversible complex [4]. The specificity of the reaction is shown by the absence of reaction with R N A or m o n o d e o x y r i b o n u c l e o t i d e s and by a marked preference for reaction with double-stranded relative to single-stranded DNA. Anthramycin stabilizes the D N A helix, as shown by an increase in melting temperature, b u t does n o t prevent strand separation in alkali [3,4]. Strand separation by alkali does not dissociate b o u n d anthramycin. Further indications of irreversibility are that the complex resists dissociation b y dialysis, gel filtration, detergent, or silver ions, under conditions that readily dissociate the D N A complexes of actinomycin, daunomycin and c h r o m o m y c i n [4]. These antibiotics have side chain structures that help to explain their tight binding to DNA. Anthramycin is a relatively small molecule and lacks such side-chains, b u t nevertheless binds to DNA more firmly than the above antibiotics. These findings suggested that the reaction of anthramycin with DNA may be covalent [4]. One of the peculiar features of the binding is its time-dependence. Unlike DNA-binding agents such as actinomycin [10] and acridines [11] which react over a time scale of seconds or milliseconds, anthramycin reacts over a period of minutes to hours [4]. The structure of the anthramycin molecule (Fig. 12, I) gives no obvious clue w h y the reaction should be slow. The slow reaction may be due to covalent bond formation or to the necessity for a slow conformational change. However, another possibility would be that anthramycin undergoes a slow conversion to a reactive species that then binds rapidly. This was of special concern since the form of anthramycin used in our early experiments [3] was the l l - m e t h y l ether (ll-MeO-anthramycin) which is k n o w n to be susceptible to hydrolytic changes under mild conditions [7,12]. The hydrolytic changes must be taken into account in kinetic studies of the reaction with DNA. The anthramycin molecule {Fig. 12, I) is susceptible to chemical changes at C~ ~ under mild conditions [7,12]. The --OH group is readily displaced by an --OCH3 group from methanol to form the l l - m e t h y l ether (ll-MeO-anthramycin). In aqueous solution, ll-MeO-anthramycin readily hydrolysis to form anthramycin and other species that are in equilibrium with anthramycin. These include ll-epi-anthramycin and 10,11-anhydroanthramycin. Spontaneous hydrolysis under physiological conditions is a feature c o m m o n to a variety of biological alkylating agents, and is a manifestation of the same reactivity involved in the reaction of these agents with DNA. Although ll-MeO-anthramy-
290 cin does not have chemical grouping that would make it a member of a known class of alkylating agents, its hydrolysis property may be a clue to the manner of its reaction with DNA. Materials and Methods Anthramycin-11-methyl ether (ll-MeO-anthramycin) was obtained from H o f f m a n - - L a R o c h e (Lot F-60). Stock solutions in methanol, isopropanol or d i m e t h y l f o r m a m i d e were stored at --15°C. Concentrations were based on a molar extinction coefficient of 35 000 at 333 nm in aqueous solution at pH 7. Calf-thymus DNA, obtained from Worthinton Biochemical Corp., was reprecipitated with isopropanol, dissolved at 2--3 mg/ml in 1 mM Na3 EDTA, and centrifuged 10 min at 18 000 × g (in order to remove any undissolved gel particles). DNA concentrations were calculated from absorbance measurements, assuming e (260 n m ) = 6700. Sodium diethyl phosphate was prepared by slowly neutralizing diethyl phoshoric acid (Estman 5764) with NaOH in the cold. Poly(dG), poly(dC), p o l y [ d ( A - - T ) ] , p o l y ( d A ) - p o l y ( d T ) , poly(dG) • poly(dC), and poly(dI)" poly(dC) were obtained from Biopolymers, Chagrin Falls, Ohio. Absorption spectra were recorded using a Cary Model 115 spectrophotometer. Kinetic experiments were performed with a Gilford automatic absorbance recorder. The temperature of the sample c o m p a r t m e n t was controlled + 0.2 ° C. DNA solutions in the desired buffers were equilibrated for at least 10 min in the spectrophotometer chamber. A small volume of drug solution was then added to start the reaction. Because of the possibility that the presence of methanol may cause some back reaction to the methyl ether and thereby affect the reaction kinetics, experiments were also performed in the absence of methanol. For these experiments, aliquots of a fresh solution of ll-MeOanthramycin in methanol were evaporated under a stream of N2. The fine residue of solid l l - M e O - a n t h r a m y c i n could then be rapidly dissolved in neutral aqueous buffers: solution was found to be complete within 1 min. It was found that methanol concentrations up to 5% in the reaction mixtures did not significantly affect the results. H ÷ consumption during the reaction of anthramycin with DNA was tested in a Metrohm Model 30 pH-stat. Solutions were prepared in boiled water and maintained under Ar. 11-MeO-anthramycin ( 2 - 1 0 - 4 M ) in 0.03 M NaC1 was incubated in the pH-stat until fully hydrolysed. During this process, the pH rose from 5.6 to 6.4, presumably due to a slight differences between the pKa of 11-MeO-anthramycin and anthramycin. The solution was then adjusted to pH 7.0 and kept under Ar until used. 5 ml of 4 mM calf-thymus DNA in 0.03 M NaC1, 5 • 10-SM Na3 EDTA was adjusted to pH 7.0 in the pH-stat. 5 ml of the 2 . 1 0 - 4 M anthramycin solution was then added. The reaction was monitored for completeness by means of absorption spectra. After completion of the reaction, the sensitivity of the system to detection of H ÷ consumption was checked by the addition of small amounts of NaOH; it was demonstrated that 1% of a stochiometric uptake of H ÷ by anthramycin could have been detected. The anthramycin--DNA complex used for pH titation was prepared by
291 reacting 3.4 mg/ml calf-thymus DNA (Worthington) with 2 . 1 0 - 3 M 11-MeOanthramycin for 3 h at 23 ° C. The complex was precipitated and spooled twice by addition of isopropanol and NaC1 to final concentrations of 50% and 0.15 M, respectively. The spooled fibers were rinsed in 80% methanol and dissolved in 0.01 M NaCI, 2 . 1 0 - S M Na3EDTA. The resulting anthramycin/ DNA nucleotide ratio was 0.10. pH titrations were conducted under At. The extent of reaction of anthramycin with DNA was determined either spectrophotometrically or by solvent extraction. The spectrophotometric m e t h o d employed the absorbance ratio, A 321 n m /A ~ 49 n m, taking the ratios for free and b o u n d anthramycin to be 1.134 and 0.697, respectively [4]. The solvent extraction m e t h o d permitted extention of the measurements to high anthramycin concentrations. In this procedure, unreacted anthramycin was removed by extracting rapidly with 5 volumes of isoamyl alcohol. The extraction was repeated 3 times, which removed all detectable free anthramycin. (The isoamyl alcohol was pre-saturated with water to avoid depletion of the aqueous phase). The remaining aqueous phase was mixed with a small volume of methanol to avoid phase separation due to slight temperature changes, and the concentration of b o u n d anthramycin remaining in the aqueous phase was then determined spectrophotometrically. Results
Hydrolysis of 11-MeO-anthramycin The hydrolysis of ll-MeO-anthramycin was studied with the view that the reactivity of the l l - p o s i t i o n may play a role in the reaction with DNA. When ll-MeO-anthramycin was diluted into neutral aqueous solution, a small but highly reproducible change in absorption spectrum was observed {Fig. 1). The m a x i m u m change was at 375 nm, where the absorbance dropped 22%. There was also a detectable shift in the 334-nm absorption peak to a b o u t
0.8 ~
1
~ 0.4 .~f/ Q2
0.0
,
,
,
300 nm 400 Fig. 1. Spectral change o f 1 1 - M e O - a n t h ~ m y c i n ( A M E ) i n n e u t r ~ aqueous solution. 11-MeO-anthramycin d i s s o l v e d in i s o p r o p a n o l w a s d i l u t e d a p p r o x . 20-fold in 0.01 M s o d i u m p h o s p h a t e buffer, 0.5 m M EDTA, (pH 7.14) to a final concentration of 4.5 • 10-5M. The initial s p e c t r u m ( . . . . . . ) w a s c o m p l e t e d a b o u t 1 m i n after mixing. R e p e a t e d s p e c t r a s h o w e d a g r a d u a l c h a n g e up to a final curve ( ) obtained at 80 min, after w h i c h t h e r e w a s n o f u r t h e r c h a n g e , o . . . . . . o, calculated d i f f e r e n c e b e t w e e n t h e two spectra.
292 L0
'
,
'
J
'
i
,
r
'
,
,
o
,
pH
,
7.89
g _~
•
pH 7.23
z O.I <
5 < a-
0.0
'
20
'
410
'
60
'
MIN.
Fig. 2. K i n e t i c s of h y d r o l y s i s o f l l - M e O - a n t h r a m y e i n ( A M E ) , as m e a s u r e d b y c h a n g e in a b s o r b a n c e at 3 7 5 rim. 1.6 • 1 0 --4 M A M E , 0 . 1 5 M NaC1, 0 . 0 2 M s o d i u m p h o s p h a t e b u f f e z , 0.5 m M E D T A , 8% m e t h a n o l , 22.7 + 0.3 °C. ©, p H 7.89 (tl/2 = 3 1 . 2 rain); A p H 7.23 (tl/2 = 15.1 r a i n ) ; e, p H 6.41 (tl/2 = 4.2 min).
333 nm. This spectral change was presumed to be due to the conversion of ll-MeO-anthramycin to anthramycin and its equilibration products. In order to test this further, a sample of anthramycin (kindly provided by W. Leimgruber) was compared with l l-MeO-anthramycin. Both substances were dissolved in dimethylformamide in order to avoid the displacement reactions that occur in hydroxylic solvents. When the l l-MeO-anthramycin solution was diluted into 0.01 M sodium phosphate buffer, (pH 6.9) 0.5 mM EDTA, a spectral change similar to that shown in Fig. 1 was observed. In the case of anthramycin, however, there was no spectral change upon dilution into the aqueous buffer, and the spectrum was indistinguishable from the final aqueous spectrum produced by 11-MeO-anthramycin. This supports the view that the product after completion of the spectral change in Fig. 1 is anthramycin (in equilibrium with its epimer and anhydro derivative). The spectral change due to reaction with DNA was the same with 11-MeO-anthramycin and anthramycin [ 3,4]. The kinetics of the absorbance change at 375 nm (Fig. 2) indicate that the hydrolysis of 11-MeO-anthramycin is a first-order acid-catalysed reaction. The hydrolysis rate constant is plotted as a function of pH in Fig. 3a. The reaction of anthramycin (fully hydrolysed ll-MeO-anthramycin) with DNA also was found to be acid-catalysed (Fig. 3b), thus supporting the possibility that the t w o processes may be related.
Catalysis of 11-MeO-anthramycin hydrolysis by phosphate It was noted that at a given pH the hydrolysis rates were slightly faster in 0.15 M NaC1, 0.02 M sodium phosphate buffer (Fig. 3a, curve A) than in 0.04 M triethanolamine buffer (Curve B). Upon further exploration of this difference, it was found that the hydrolysis of l l-MeO-anthramycin is specifically catalysed by phosphate ions. This is demonstrated in Fig. 4 and Table I, which compare the hydrolysis rates in various aqueous solutions at a fixed pH and temperature. The reaction in 0.1 M triethanolamine-HC1 buffer(pH 7.2,
293 -0,5
o: ~
'
I
'
~
'
~
I
'
~
~
;
-I.0
A
1.0 m
I
i
l
i
I
i
I
i
I
-2.0
b A
2.5
_z bJ Ic
~
2o
z
1.5
1.0
\o
0.1
I I L L I I I I l l
6
~
0
7 pH
0 MIN
Fig. 3 (a) p H - d e p e n d e c e o f f l r s t - o r d e r r a t e c o n s t a n t (k in r a i n - I ) f o r h y d r o l y s i s o f l l - M e O - a n t h r a m y c i n ( A M E ) a t 2 2 . 9 ° C . Buffers: ( A ) 0 . 1 5 M NaCI, 0 . 0 2 M s o d i u m p h o s p h a t e , 5 • 1 0 -4 M E D T A ; (B) 0 . 0 4 M t r l e t h a n o l a m i n e - - H C l , 2 • 10-4 E D T A . ( T h e s o l u t i o n s also c o n t a i n e d 5% m e t h a n o l . ) (b) p H - d e p e n d e n c e of t h e r e a c t i o n o f a n t h r a m y c i n w i t h c a l f - t h y m u s D N A . l l - M e O - a n t h z a m y c i n was fully h y d r o l y s e d p r i o r t o a d d i t i o n o f D N A . T h e r e a c t i o n w a s f o U o w e d a t 321 n m , t h e m a x i m u m o f t h e d i f f e r e n c e s p e c t r u m [ 3 ] . T h e a p p a z e n t initial r a t e c o n s t a n t f o r t h e d i s a p p e a r a n c e o f free a n t h r a m y c i n w a s d i v i d e d b y t h e D N A n u c l e o tide c o n c e n t r a t i o n t o give t h e s e c o n d - o ~ l e r ~ate c o n s t a n t , k 2 . T h e line w a s d r a w n h a v i n g t h e slope t h e o r e t i c a l l y e x p e c t e d f o r a r e a c t i o n r a t e p r o p o r t i o n a l t o H + c o n c e n t r a t i o n . R e a c t i o n c o n d i t i o n s : Initial a n t h r a m y c i n c o n c e n t r a t i o n , 2 . 1 0 -5 M; 2 2 . 6 ° C . Buffers: o, 0 . 0 4 M c i t r a t e ; ~, 0 . 0 2 M p h o s p h a t e (6 • 10-4 M E D T A ) . Fig. 4. E f f e c t s o f p h o s p h a t e a n d NaC1 o n r a t e of l l - M e O - a n t h r a m y c i n h y d r o l y s i s . All s o l u t i o n s c o n t a i n e d 1.2 • 10-4M l l - M e O - a n t h r a m y c i n , 5 • 1 0 - 4 M E D T A , a n d w e r e a d j u s t e d t o p H 7 . 1 7 -+ 0 . 0 5 ; 2 2 . 7 ° C . Curve
Sodium phosphate b u f f e r c o n c n (M)
NaC1 c o n c n (M)
Triethanolamine b u f f e r c o n c n (M)
0 1.0 0 0.85 0
0.I 0.I 0 0 0 o r 0.1
A
0
B
0
C D E
0.I 0.I 0.4
t 1/2 (rain)
2.14 18.1
6.1 9.0 2.7
23°C} is shown by Curve A in Fig. 4. Addition of 1 M NaC1 to this buffer (Curve B) caused only a slight increase in rate. Addition of 0.4 M sodium phosphate, however, increased the rate about 7-fold {Curve E), and 0.1 M sodium phosphate increased the rate 3.5-fold {Curve C). These rates were independent of the presence or absence of triethanolamine. The rate increase produced by 0.1 M sodium phophate was partially reversed by adding 0.85 M NaC1 {Curve D), indicating that the catalytic effect of phosphate is reduced by high ionic strength. Interestingly, high ionic strength also retards the reaction
294 TABLE
I
FIRST-ORDER 0.04
HYDROLYSIS
RATES
(k) OF
ll-MeO-ANTHRAMYCIN
o A T 2 2 . 7 -+ 0 . 5 C , p H 7 . 1 6 -+
( k w a s d e t e r m i n e d f r o m the s l o p e s o f s e m i - l o g a r i t h m i c p l o t s such as t h o s e s h o w n in Figs 2 and 4. k is related to the h a l f - t i m e o f the r e a c t i o n by t I/2 = 0 . 6 9 3 / k . ) T E A , t r i e t h a n o l a m i n e - - H C l .
Solvent
k (min -I )
0.1 M TEA buffer 1 M NaC1 + 0 . 1 M T E A b u f f e r 1 M NaBr + 0.1 M TEA buffer 1 M NaN 3 + 0.1 M TEA buffer 0.021 M sodium phosphate buffer 0.11 M sodium phosphate buffer 0.11 M sodium phosphate buffer + 0.85 M NaCl 0.43 M sodium phosphate buffer 0.43 M sodium phosphate buffer + 0.1 M TEA buffer 0.4 M Na2SO 4 + 0.1 M TEA buffer 1 M imidazole + 0.1 M TEA buffer 0 . 0 5 M GMP
0.0324 0.0382 0.0388 0.0523 0.0531 0.118 0.0775 0.256 0.260 0.0493 0.07* 0.074
* R e a c t i o n d e v i a t e d f r o m first-order k i n e t i c s , and w a s a c c o m p a n i e d b y an overall fall in l l - M e O - a n t h r a mycin absorption peak
of anthramycin with native D N A [ 4 ] . This suggests that both reactions may be promoted by the electrostatic effects of phosphate groups. We attempted to detect a complex between phosphate and 11-MeO-anthramycin by looking for an effect o f phosphate on the absorption spectrum of ll-MeO-anthramycin, but no effect was detected. Various anions were compared for their ability to stimulate ll-MeOanthramycin hydrolysis (Table I). Of the ions tested, only phosphate had a large effect. Whereas 0.4 M phosphate increased the hydrolysis rate more than 7-fold, equal or greater concentrations of azide or sulfate caused less than a doubling in rate, and chloride or bromide produced barely detectable increases. Imidazole produced an apparent doubling in hydrolysis rate, but this value may be erroneously high because the reaction was accompanied by a decrease in the absorption band at 333 nm (without noticable change in position or shape of the peak), indicating another process "besides hydrolysis. The spectral changes in the presence o f the other salts were the same as occurred in the absence of salt. Guanylic acid (GMP) produced an effect similar to that which would be expected with an equimolar concentration of phosphate. Phosphate might act in two possible ways to catalyse the hydrolysis: (1) it may act as a nucleophile in displacement of the ---OCH3 group, or (2) it may act as a proton donor facilitating the acid-catalysed reaction (Fig. 12, III). The t w o possibilities are equally capable of explaining the data to this point. In order to distinguish between them, we tested sodium diethyl phosphate which lacks a donatable proton. Like phosphate, this c o m p o u n d produced no spectral change in anthramycin, indicating that no complex affecting the chromophore is produced. Unlike phosphate, diethyl phosphate did not catalyse the hydrolysis of ll-MeO-anthramycin (Fig. 5). This indicates that phosphate acts by donating a proton and facilitating an acid-catalysed reaction (Fig. 12, III).
295 1.0 '
I.O
'
I
'
i
,
I
,
I
'
F
i
0.8
'•0.5
~. 0 . 6
5 z z <
•
•
4;
0.4
0
0.I
0.2
~0.05 L
I
i
IO
I
20
MINUTES
,
I
30
,
I
i
20
I
40
J
60
MINUTES
Fig. 5, C o m p a r i s o n b e t w e e n e f f e c t s o f p h o s p h a t e (D) and d i e t h y l p h o s p h a t e ( A ) o n rate of l l - M e O a n t h r a m y c i n h y d r o l y s i s , relative t o c o n t r o l ( o ) . C o n c e n t r a t i o n s o f p h o s p h a t e and d i e t h y l p h o s p h a t e w e r e 0 . 5 M. 1 1 - M e O - a n t h r a m y e i n w a s f r e s h l y dissolved in d i m e t h y l f o r m a m i d e . Final c o n c e n t r a t i e s : 1 1 - M e O a n t h r a m y c i n , 1 . 2 5 • 1 0 -4 M; N - t r i s - ( h y d r o x y m e t h y l ) - m e t h y l - 2 - a m i n o s u l f o n a t e b u f f e r , 0 . 1 2 M; t o t a l Na+, 1 . 0 M ( m a d e u p w i t h N a C 1 ) : d i m e t h y l f o r m a m i d e , 1%. p H = 7 . 0 0 -+ 0 . 0 2 , 2 3 . 2 ° C . Fig. 6. E f f e c t o f 1 1 - M e O - a n t h r a m y c i n p r e - i n c u b a t i o n o n r e a c t i o n w i t h D N A . ( e ) no p r e - i n c u b a t i o n ; ( A ) p r e - i n c u b a t i o n for 1 8 0 r a i n a t 2 2 . 5 ° C i n 0 . 0 6 M t r i e t h a n o l a m i n e - - H C 1 b u f f e r ( p H 7 . 3 4 ) , 0 . 0 2 M triethan o l a m i n e , 0 . 0 2 M N a C 1 , 0 . 4 m M E D T A . T h e r e a c t i o n w i t h c a l f - t h y m u s D N A w a s carried o u t u n d e r the s a m e c o n d i t i o n s e x c e p t that t h e b u f f e r w a s d i l u t e d to 7 5 % o f t h e a b o v e c o n c e n t r a t i o n s . P r e - i n c u b a t i o n w a s b e g u n b y r a p i d l y dissolving solid 1 1 - M e O - a n t h r a m y c i n in b u f f e r as d e s c r i b e d u n d e r Materials and M e t h o d s . T h e h a l f - t i m e f o r t h e h y d r o l y s i s o f l l - M e O - a n t h r a m y c i n u n d e r t h e s e c o n d i t i o n s (in t h e a b s e n c e o f D N A ) w a s d e t e r m i n e d to b e 2 7 , 3 rain.
Relation between I1-MeO-anthramycin hydrolysis and DNA reaction kinetics In order to determine the relative reactivities of 11-MeO-anthramycin and anthramycin with DNA, 11-MeO-anthramycin was pre-incubated in neutral aqueous solution for various times and then mixed with DNA. Pre-incubation of 11-MeO-anthramycin was found to increase the rate of reaction with DNA. As the pre-incubation time was increased, the initial rate of reaction with DNA increased up to a maximum beyond which longer pre-incubation had no effect. The extreme cases, no pre-incubation and maximal pre-incubation, are shown in Fig. 6. Intermediate times of pre-incubation produced a family of curves between the t w o extremes shown. The initial rate of reaction with DNA was correlated with the fraction of the 11-MeO-anthramycin hydrolysed at the time of the earliest DNA rate measurement (about 2 rain after mixing with DNA) (Fig. 7). The fraction of 11-MeO-anthramycin hydrolysed was determined under similar conditions in the absence of DNA by following the change in absorbance at 375 nm. The linear relation in Fig. 7 indicates that the increase in reaction rate with DNA is due to the same change in 11-MeO-anthramycin that is responsible for the spectral change of 11-MeO-anthramycin in aqueous solution. It is seen that hydrolysed 11-MeO-anthramycin reacts about 5 times as fast with DNA as does
296 I
I
7
8
I
p
9
I0
6
80
0
g E
4 60
o
uJ
I
z w :L
4O
2
20 2'
0.5 FRACTION
OF
AME
1.0 HYDROLYZED
pN
Fig. 7. R e l a t i o n b e t w e e n initial r e a c t i o n rate w i t h D N A a n d e x t e n t o f h y d r o l y s i s o f 1 1 - M e O - a n t h r a m y e i n ( A M E ) . D a t a d e r i v e d f r o m e x p e r i m e n t s like t h o s e d e s c r i b e d in Fig. 6. T h e h a l f - t i m e ( i n rain) c a n be c a l c u l a t e d f r o m t h e o r d i n a t e v a l u e s ( k ) b y , tl/2 = 0 . 6 9 3 / ( k [ D N A ] ) , w h e r e [ D N A ] is in m o l e s o f n u c l e o t i d e u n i t s p e r I. ( S m a l l c o r r e c t i o n s w e r e a p p l i e d t o k f o r c h a n g e s in a b s o r b a n c e at 3 2 1 n m d u e t o l l - M e O a n t h r a m y c i n h y d r o l y s i s . T h e s e c o r r e c t i o n s d i d n o t m a t e r i a l l y a f t e r t h e r e s u l t s a n d n e v e r e x c e e d e d 15%.) Fig. 8. p H t i t r a t i o n o f a n t h r a m y c i n ( ~ ) a n d a n t h r a m y c i n / D N A c o m p l e x ( o ) . A n t h r a m y c i n c o n t e n t in e a c h c a s e w a s 4 . 1 # m o l e s at a c o n c e n t r a t i o n o f a p p r o x . 1 0 - 3 M . A n t h r a m y c i n / D N A n u c l e o t i d e ratio = 0 . 1 0 ; t h e c o m p l e x w a s p r e p a r e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s . ( o ) s o l v e n t ( 0 . 0 1 M N a C 1 , 2 • 1 0 - 5 M N a 3 E D T A ) ; a d d i t i o n o f D N A d i d n o t alter t i t r a t i o n c u r v e o f s o l v e n t . ( A slight d i f f e r e n c e w a s n o t e d between the titration curves of ll-MeO-antbxamycin before and after hydrolysis, indicating a small i n c r e a s e in p Ka u p o n h y d r o l y s i s . T h e c u r v e s h o w n is f o r f u l l y h y d r o l y s e d 1 1 - M e O - a n t h r a m y c i n .
unhydrolysed 11-MeO-anthramycin. Unhydrolysed 11-MeO-anthramycin also, however, reacts at a rate significantly greater than zero, as is seen by the zero-time intercept in Fig. 7. Hence, ll-MeO-anthramycin (or a product to which it is very rapidly converted} is able to react with DNA directly without first undergoing the hydrolysis reaction that produces the spectral change at 375 nm.
Role o f I-£ in the reaction with DNA The proportionality of the DNA reaction rate to H ÷ concentration (Fig. 3b) indicates that H ÷ is kinetically involved in the rate-limiting step of the reaction. In order to determine whether H ÷ is actually consumed, the reaction was carried out in a pH-stat (see Materials and Methods}. It was found that there is neither consumption nor loss of H ÷ in the reaction. Therefore, the kinetic dependance on H ÷ concentration must be in the rate-limiting formation of a transient intermediate. In order to determine whether there is a change in dissociable protons during the reaction, the pH titration curves of anthramycin and anthramycin-DNA complex were compared. Free anthramycin was found to have a pKa at about 8.7. In the complex, however, this pKa was completely absent (Fig. 8). Hence, the dissociable proton of anthramycin either is missing or is stabilized in the complex.
297
Kinetics of the reaction of anthramycin with DNA We have previously noted [4] l l - M e O - a n t h r a m y c i n that the reaction of anthramycin (fully hydrolysed ll-MeO-anthramycin) with DNA slows down with time more than would be expected on the basis of depletion of free anthramycin or DNA. It is as if the reactivity of either the anthramycin or the DNA decreased with time. In order to distinguish between these two possibilities, either fresh anthramycin or fresh DNA was added to a reaction mixture after it had reached the slow phase (Fig. 9). If one of the two reactants has lost reactivity, then addition of fresh reactant should increase the reaction rate more than in proportion to the total concentration of free reactant. At the time of addition of fresh reactant, the extent of binding, r, was approx. 0.03, far from the saturation value of r = 0.1 bound anthramycin per nucleotide [4]. Therefore, about 70% of the DNA sites were still available for reaction. Anthramycin could n o t have been depleted by reaction with DNA because its initial concentration was fax in excess of the available DNA sites. Addition of fresh anthramycin to give a 3-fold increase in total anthramycin concentration (Fig. 9A) produced approx, a 3-fold increase in reaction rate, indicating that fresh anthramycin was no more reactive than t h a t present in the reaction mixture. Addition of fresh DNA to give a 2-fold increase in concentration of total DNA (equivalent to approx, a 3-fold increase in unreacted DNA sites), however, produced far more than a 3-fold increase in reaction rate (Fig. 9B). The kinetics were in fact identical to the original reaction. This confirms that the reactivity of the anthramycin has n o t changed, and demonstrates that the reduced reaction rate is due to reduced reactivity of the DNA sites remaining after a partial reaction.
to
i A
0
i
i
I
i
i
~
i
I
i
L
I
t
I
I
I
I
i
i
~
FRESH ANTHRAMYCIN
~
I
I
I
I
I
i
I
Z
i IO REACTION
I
[
I
20 TIME
(minutes)
Fig. 9. E f f e c t s o f a d d i t i o n o f f r e s h a n t h r a m y c i n ( A ) o r f r e s h D N A (B) d u r i n g t h e slow p h a s e o f a n a n t h r a m y c i n - - D N A r e a c t i o n . (a) I n i t i a l c o n c e n t r a t i o n : a n t h r a m y c i n , 80/~M; D N A , 1 2 0 ~M. A n t h r a m y e i n a d d e d at 5 m i n : 1 7 0 JzM. (B) I n i t i a l c o n c e n t r a t i o n s : a n t h r a m y c i n , 87/~M; D N A , 1 2 5 / ~ M . D N A a d d e d at 5 m i n : 1 2 5 ~zM. R e a c t i o n m e d i u m : 0 . 0 1 M s o d i u m p h o s p h a t e b u f f e r ( p H 7 . 3 3 ) , 2 3 . 0 - - 2 3 . 5 ° C. " A n t h r a mycin" was derived from 1 1 - M e O - a n t h r a m y c i n by complete hydrolysis prior to use in the DNA reactions. Bound
anthramycin
was determined
by isopropanol
extraction
(see Materials and Methods).
298 Partial reaction of DNA with anthramycin therefore reduces the reactivity of the remaining DNA sites to further reaction with anthramycin. When reaction rates were compared at fixed values of r, however, the rates were found to be proportional to both anthramycin and DNA concentration, as expected for a bimolecular reaction. The reaction can therefore be expressed in terms of a bimolecular reaction rate constant (k') that is a function of the extent of reaction, r: dr
- - = k'(r)AC(~---r) dt
where A is the concentration of unreacted anthramycin, C is the total concen. tration of DNA, and v is the fraction of nucleotide residues that are potential binding sites. A plot of k ' ( r ) = ( d r / d t ) / a C ( v - - r ) against r should therefore obey a consistant function of r for various reaction times and reactant concentrations. This is demonstrated in Fig. 10. The apparent reaction rate constant, k ' ( r ) , is seen to decrease exponentially with r. Specificity of the reaction
Previous studies, based on spectral measurements, have shown that anthramycin reacts with p o l y ( d G ) , poly(dC), but not with poly[d(A--T)] (alternating) or p o l y ( r G ) , poly(rC) [4]. It was also found that although anthramycin reacts preferentially with double-stranded DNA, there is a substantial reaction rate with single stranded DNA at moderate ionic strength. We have therefore tested the reactivity with single-stranded poly(dG) and poly(dC) in order to determine which of these bases is required for reaction (Table II). Poly(dG) reacted to an extent similar to denatured calf-thymus DNA (Table II). A significant extent of reaction was, however, observed also with poly(dC) (Sample 1). This sample of poly(dC) was therefore analysed by acid hydrolysis and high-voltage paper electrophoresis, and was found to be contaminated with
&
•
[
r
t
I
r
•,
107
~
10s
~
10 5
AAA~ I
,
l
I
I
1
0.02 0.04 0.06 O.OS 0.I0 MOLES ANTHRAMYCIN BOUND PER MOLE DNA NUCLEOTIOES (r)
Fig, 10. D e p e n d a n c e o f a p p a r e n t r e a c t i o n r a t e c o n s t a n t ( k ' ) o n e x t e n t o f r e a c t i o n w i t h D N A (r). k ' w a s c a l c u l a t e d b y t h e e q u a t i o n g i v e n in t h e t e x t , a s s u m i n g ~ = 0.1 [ 4 ] . R e a c t i o n s w e r e c a r r i e d o u t in 0.01 M s o d i u m p h o s p h a t e b u f f e r . ( p H 6 . 7 - - 7 . 4 ) , A p p a r e n t r a t e c o n s t a n t s w e r e a d j u s t e d t o p H 7.0, b a s e s o n proportionality between reaction rate and H÷ concentration [ 4 ] . Reactant concentrations were anthram y c i n , 50---970 ~zM; D N A n u e l e o t i d s , 60---1800 /~M. R e a c t i o n s w e r e t e r m i n a t e d at v a r i o u s t i m e s . A n t h r a m y c i n b i n d i n g w a s d e t e r m i n e d e i t h e r s p e c t r o s c o p i c a l l y (o) or b y e x t r a c t i o n w i t h i s o a m y l a l c o h o l (A).
299 T A B L E II EXTENTS OF REACTION OF ANTHRAMYCIN
WITH POLYNUCLEOTIDES
1.5 X 1 0 - ~ M p o l y n u c l e o t i d e w a s r e a c t e d w i t h 1 0 _ 3 M a n t h r a m y c i n in 0.1 M s o d i u m p h o s p h a t e b u f f e r , ( p H 6 . 1 ) a t 2 3 ° C f o r 8 h. T h e e x t e n t o f b i n d i n g (r) w a s d e t e r m i n e d b y r e p e a t e d d i a l y s i s f o l l o w e d b y s p e c tral determination of anthramycin nucleotide ratio [4]. Polynucleotide Calf-thymus DNA (native) Calf-thymus DNA (denatured) p o l y ( d G ) : p o l y (dC) poly(dG) poly(dC) (Sample I ) p o l y ( d C ) ( S a m p l e 2) poly (dI)• poly(dC) poly [d(A--T)] (alternating) poly(dA) • poly(dT) poly(rG) poly(rC)
E x t e n t o f b i n d i n g (r) 0.099 0.032 0.099 0.031 0.012 0.006 <:0.002 <:0.002 <:0.002 <:0.002 <:0.002
about 10% poly(dG). A second, more highly purified, sample of poly(dC) exhibited substantially lower reactivity, consistant with the view that poly(dC) itself does not react. This was confirmed by the finding that poly(dI) • poly(dC) exhibited no detectable reaction.
Competition with actinomycin The finding that the reaction of anthramycin with DNA requires guanine suggested that anthramycin should compete with actinomycin for some of the same binding sites, since actinomycin also requires guanine for binding to DNA [14]. In order to test this, a series of anthramycin-DNA complexes of various extents of reaction were prepared, and the actinomycin-binding abilities of these cOmplexes were determined. Since the binding of anthramycin to DNA is firmer than the binding of actinomycin, it is possible to consider the anthramycin--DNA bonds to be essentially irreversible while the actinomycin--DNA reaction is a reversible equilibrium [4]. It is seen from Fig. l l a that the actinomycin binding capacity varied inversely with the extent of anthramycin binding. When the DNA was saturated with anthramycin (r = 0.10), the actinomycin binding capacity was nil. At lower extents of anthramycin binding, the capacity for actinomycin binding increased in proportion to the fraction of the anthramycin binding capacity that was unfilled. The binding constant of actinomycin with DNA, however, was unaffected by the presence of anthramycin, since the slopes in the Scatchard plots were similar for DNA and anthramycin--DNA complex (Fig. l l a ) . Discussion
The hydrolysis of ll-MeO-anthramycin is accompanied by a small change in absorption spectrum (Fig. 1) which was utilized to study the hydrolysis
300 O. I0
I
I
I
•
~
f
•
=o
[
I
Anthromycin: DNA 000
~c
0
~ o.os ~ru
~-~ :,= o
0.05 r (mol. onthromYcin bound per tool. DNA nucleoflde)
0.10
002
0.04 0.06 r ~Actinomycin)
0.08
Fig. 11. E f f e c t o f b o u n d a n t h r a m y c i n o n t h e i n t e r a c t i o n of D N A w i t h a c t i n o m y c i n . ( A ) A c t i n o m y c i n b i n d i n g c a p a c i t y ; (B) S c a t c h a r d p l o t . A n t h r a m y c i n - - D N A c o m p l e x e s w e r e d i a l y s e d to r e m o v e a n y u n b o u n d a n t h r a m y c i n . T h e c o m p l e x e s w e r e t i t r a t e d s p e c t r o p h o t o m e t r i c a l l y b y successive a d d i t i o n s o f a c t i n o m y c i n , a n d t h e d a t a w e r e a n a l y s e d as d e s c r i b e d b y H y m a n a n d D a v i d s o n [ 1 3 ] . T h e t i t r a t i o n s w e r e c a r r i e d o u t in 0 . 0 0 1 M s o d i u m p h o s p h a t e , ( p H 7.0).
' C
OH
H
Jg
N~/
OR
O
3
~/
\CONH 2
I
H
I
O--R OH
H ~)/
~"
O
\CONH 2
"rr O~.~
~O
o/P"~O H
e
O
-"
\CONH 2
"n'r
Fig. 12. S t r u c t u r e of a n t h r a m y c i n (R~----I-I) a n d l l - M e O - a n t h r a m y c i n (R--~CH3). ( I I ) P r o p o s e d p r o t o n a t e d i n t e r m e d i a t e . ( I I l ) Possible m o d e o f c a t a l y s i s b y p h o s p h a t e a c t i n g as a p r o t o n d o n o r .
301 kinetics. The hydrolysis obeys first-order kinetics and is acid-catalysed (Fig. 2). The pH-dependance of the kinetics (Fig. 3a) indicates that a protonated intermediate determines the reaction rate. Protonation of the oxygen atom at Position 11 would favor displacement of the m e t h o x y group and is a likely intermediate in the hydrolysis (Fig. 12, II). The fact that the reaction of anthramycin with DNA also is acid-catalysed (Fig. 3b) suggests that protonation of the oxygen at Position 11 may be an intermediate step in this reaction as well. Position 11 may, in fact, be the site through which anthramycin becomes covalently linked to DNA. This would be analogous to the case of alkylating agents, where reactivity with nucleophiles and ready hydrolysis have a c o m m o n chemical basis. The chemistry differs from alkylation, however, in that hydrolysis of alkylating agents inactivates them, whereas hydrolysis of 11-MeOanthramycin does not inactivate. The relationship of H ÷ to the interaction of anthramycin with DNA may be summarized as follows: (1) H ÷ (above pH 4) stimulates the reaction with DNA; (2) H ÷ is n o t consumed during the reaction; (3) the dissociation of H ÷ from the phenolic group of anthramycin is n o t seen in the titration curve of the complex; (4) below pH 4, H ÷ stimulates the dissociation of the complex [4]. A possible interpretation of these data is that the interaction involves both the C , , group and the d e p r o t o n a t e d phenolic group. This conforms with the finding that the 9-methoxy and l l - k e t o derivatives of anthramycin do not react with DNA and are biologically inactive [9] (Leimgruber, W., personal communication). The evidence that ll-MeO-anthramycin can react directly with DNA b u t at a slower rate than anthramycin (Fig. 7) is compatible with a displacement reaction at Position 11, since either a hydroxyl or a m e t h o x y group could undergo displacement by the same mechanism (Fig. 9, II) although the rates may differ. The slowness of the interaction of ll-MeO-anthramycin with DNA is one of the factors that suggested that the binding involves formation of a covalent bond [4]. An alternative possibility, however, would be that ll-MeO-anthramycin is slowly converted to a reactive species that then reacts rapidly, and perhaps non-covalently, with DNA. The increased rate of reaction of 11-MeOanthramycin after pre-incubation {Figs 6 and 7) tended to support this suggestion. Closer examination, however, reveals that the pre-incubation effect is limited: b e y o n d an optimal pre-incubation time, the reaction rate is not further increased and still remains slow compared to non-covalent DNA interactions with agents such as acridines or actinomycin, which occur on a time scale of seconds or milliseconds [ 1 0 , 1 1 ] . It may still be argued however that even after complete hydrolysis, anthramycin still must undergo a rate-limiting conversion to a reactive species normally present in very low concentration. If this were so, the rate of binding of anthramycin should be governed by the rate of its conversion to the reactive form and should b e c o m e independent of DNA concentration. Since DNA concentration did affect the rate of binding, this argument t o o is ruled out. We therefore conclude that the slowness of the reaction of anthramycin with DNA is n o t due to a rate-limiting conversion of anthramycin to a rapidly reactive species. Similarly, the possibility that the reaction rate is limited by a requirement for a slow conformational change in the DNA is ruled o u t by the finding that the reaction rate does n o t b e c o m e independent
302
of anthramycin concentration. These findings therefore support our previous inference that the slow reaction is due to covalent bond formation [ 4 ] . The site of anthramycin reaction apparently is guanine, since reaction occurs only with guanine-containing deoxypolynucleotides, and since there is competition with actinomycin for bindings sites. The exacting structural requirements for the anthramycin reaction are shown by the loss of reactivity when dG is replaced by dI, the absence of reactivity with helical ribo-polymers, and by the greatly reduced reactivity with single-stranded D N A [4]. A characteristic of the reaction of anthramycin with D N A is the marked slowing of the reaction with time. This apparently is not due to heterogeneity of sites with preferential reaction at more reactive sites, because the slowing was also seen in the reaction with poly(dG) • poly(dC} [4]. The most likely explanation is that reacted sites interfere with further reaction at nearby sites. Acknowledgement We thank Dr V.H. Bono, Jr for assistance with the pH stat and pH titration experiments. References 1 2 3 4 5 6 7 S 9 10 11 12 13 14
Tendler, M.D. and Korman, S. (1963) Nature 199, 501 Adamson, R.H., Hart, L.G. Devita, V.T. and Oliverio, V.T. (1968) Cancer Res. 28, 343--347 Kohn, K.W., Bono, Jr, V.H. and Kann, Jr, H.E. (1968) Biochim. Biophys. Acta 155, 121--129 Kohn, K.W. and Spears, C.L. (1970) J. Mol. Biol. 51, 551--572 Bates, H.M., Kuenzig, W. and Watson, W.B. (1969) Cancer Res. 29, 2 1 9 5 - - 2 2 0 5 Horwitz, S.B. and Grollman, A.P. (1968) Antimicrob. Agents Chemother. 21--24 Leimgruber, W., Stefanovic, V., Schenker, F., Karr, A. and Berger, J. (1965) J. Am. Chem. Soc. 87, 5791--5793 Leimgruber, W., Bateho, A.D. and Schenker, F. (1965) J. Am. Chem. Soc. 87, 5793--5795 Leimgruber, W., Batcho, A.D. and Czajkowski, R.C. (1968) J. Am. Chem. Soc. 90, 5641--5643 Miiller, W. and Crothers, D.M. (1968) J. Mol. Biol. 35, 251--290 Li, H.J. and Crothers, D.M. (1969) J. Mol. Biol. 39, 461--477 Leimgruber, W., Batcho and Schenker, F. 4th Int. Syrup. Chem. Nat. Prod. IUPAC Congr. Stockholm, 1966, Abstr. p. 106 Hyman, R.W. and Davidson~ N. (1971) Biochim. Biophys. Acta 228, 3 8 - 4 8 Jain, S.C. and Soben, H,M~ (1972) J. Mol. Biol. 68, 1--20
Biochimica et Biophysica Acta, 361 ( 1 9 7 4 ) 2 8 8 - - 3 0 2 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 98081
THE REACTION OF ANTHRAMYCIN WITH DNA II. STUDIES OF KINETICS AND MECHANISM*
K U R T W. K O H N , D A N I E L G L A U B I G E R a n d C A R L O S L. S P E A R S
Laboratory o f Molecular Pharmacology, Division of Cancer Treatment, National Cancer Institute, Bethesda, Md. 20014 (U.S.A.) (Received January 4th, 1974) (Revised m a n u s c r i p t received May 1 4 t h , 1 9 7 4 )
Summary Anthramycin is k n o w n to react firmly with DNA, but no chemical basis for this interaction has been elucidated. Since anthramycin readily undergoes hydrolytic changes at C-11, the possible relation of this reaction to the interaction with DNA was considered. Both the hydrolysis of an l l - m e t h o x y group and the reaction with DNA were f o u n d to be H * catalysed, thus supporting a relation between the two processes. The titration of H ÷ from the phenolic group at Position 9 of anthramycin was f o u n d to be absent in the DNA complex, suggesting that Position 9 also is involved in the complex. The rate of reaction of a n t h r a m y c i n - l l - m e t h y l ether with DNA was found to be increased by prior hydrolysis. The rate of reaction with DNA, however, does not involve a rate-limiting conversion of anthramycin to a reactive component, since the kinetics of the reaction were found to be basically bimolecular. This excluded the possibility that a slow conformational change of the DNA was rate-limiting. The findings support the proposal that the binding of anthramycin to DNA is covalent, and that Positions 11 and 9 are involved in the binding. The bimolecular rate constant was found to decrease exponentially with the extent of DNA reaction, probably due to neighboring-site exclusion effects. The site of reaction involves guanine, since only guanine-containing deoxypolynucleotides were found to be reactive, and since reaction of DNA with anthramycin caused stochiometric elimination of binding sites of actinomycin.
* Paper I of this series is ref. 4.
289 Introduction Anthramycin, an antibiotic derived from Streptomyces refuineus, has antit u m o r as well as antibacterial activity [1,2]. It inhibits both R N A and DNA synthesis in mammalian cells, while having relatively little effect on protein synthesis [3]. The antibiotic reacts with DNA [3,4] and inactivates DNA as a template for DNA and R N A synthesis and as a substance for nuclease action [5,6]. The structure of anthramycin (Fig. 12, I) has been confirmed by total synthesis [7--9]. The interaction with DNA is of special interest because, although the anthramycin molecule lacks structural features currently k n o w n to favor tight binding to DNA, it nevertheless reacts specifically with DNA to form a nearly irreversible complex [4]. The specificity of the reaction is shown by the absence of reaction with R N A or m o n o d e o x y r i b o n u c l e o t i d e s and by a marked preference for reaction with double-stranded relative to single-stranded DNA. Anthramycin stabilizes the D N A helix, as shown by an increase in melting temperature, b u t does n o t prevent strand separation in alkali [3,4]. Strand separation by alkali does not dissociate b o u n d anthramycin. Further indications of irreversibility are that the complex resists dissociation b y dialysis, gel filtration, detergent, or silver ions, under conditions that readily dissociate the D N A complexes of actinomycin, daunomycin and c h r o m o m y c i n [4]. These antibiotics have side chain structures that help to explain their tight binding to DNA. Anthramycin is a relatively small molecule and lacks such side-chains, b u t nevertheless binds to DNA more firmly than the above antibiotics. These findings suggested that the reaction of anthramycin with DNA may be covalent [4]. One of the peculiar features of the binding is its time-dependence. Unlike DNA-binding agents such as actinomycin [10] and acridines [11] which react over a time scale of seconds or milliseconds, anthramycin reacts over a period of minutes to hours [4]. The structure of the anthramycin molecule (Fig. 12, I) gives no obvious clue w h y the reaction should be slow. The slow reaction may be due to covalent bond formation or to the necessity for a slow conformational change. However, another possibility would be that anthramycin undergoes a slow conversion to a reactive species that then binds rapidly. This was of special concern since the form of anthramycin used in our early experiments [3] was the l l - m e t h y l ether (ll-MeO-anthramycin) which is k n o w n to be susceptible to hydrolytic changes under mild conditions [7,12]. The hydrolytic changes must be taken into account in kinetic studies of the reaction with DNA. The anthramycin molecule {Fig. 12, I) is susceptible to chemical changes at C~ ~ under mild conditions [7,12]. The --OH group is readily displaced by an --OCH3 group from methanol to form the l l - m e t h y l ether (ll-MeO-anthramycin). In aqueous solution, ll-MeO-anthramycin readily hydrolysis to form anthramycin and other species that are in equilibrium with anthramycin. These include ll-epi-anthramycin and 10,11-anhydroanthramycin. Spontaneous hydrolysis under physiological conditions is a feature c o m m o n to a variety of biological alkylating agents, and is a manifestation of the same reactivity involved in the reaction of these agents with DNA. Although ll-MeO-anthramy-
290 cin does not have chemical grouping that would make it a member of a known class of alkylating agents, its hydrolysis property may be a clue to the manner of its reaction with DNA. Materials and Methods Anthramycin-11-methyl ether (ll-MeO-anthramycin) was obtained from H o f f m a n - - L a R o c h e (Lot F-60). Stock solutions in methanol, isopropanol or d i m e t h y l f o r m a m i d e were stored at --15°C. Concentrations were based on a molar extinction coefficient of 35 000 at 333 nm in aqueous solution at pH 7. Calf-thymus DNA, obtained from Worthinton Biochemical Corp., was reprecipitated with isopropanol, dissolved at 2--3 mg/ml in 1 mM Na3 EDTA, and centrifuged 10 min at 18 000 × g (in order to remove any undissolved gel particles). DNA concentrations were calculated from absorbance measurements, assuming e (260 n m ) = 6700. Sodium diethyl phosphate was prepared by slowly neutralizing diethyl phoshoric acid (Estman 5764) with NaOH in the cold. Poly(dG), poly(dC), p o l y [ d ( A - - T ) ] , p o l y ( d A ) - p o l y ( d T ) , poly(dG) • poly(dC), and poly(dI)" poly(dC) were obtained from Biopolymers, Chagrin Falls, Ohio. Absorption spectra were recorded using a Cary Model 115 spectrophotometer. Kinetic experiments were performed with a Gilford automatic absorbance recorder. The temperature of the sample c o m p a r t m e n t was controlled + 0.2 ° C. DNA solutions in the desired buffers were equilibrated for at least 10 min in the spectrophotometer chamber. A small volume of drug solution was then added to start the reaction. Because of the possibility that the presence of methanol may cause some back reaction to the methyl ether and thereby affect the reaction kinetics, experiments were also performed in the absence of methanol. For these experiments, aliquots of a fresh solution of ll-MeOanthramycin in methanol were evaporated under a stream of N2. The fine residue of solid l l - M e O - a n t h r a m y c i n could then be rapidly dissolved in neutral aqueous buffers: solution was found to be complete within 1 min. It was found that methanol concentrations up to 5% in the reaction mixtures did not significantly affect the results. H ÷ consumption during the reaction of anthramycin with DNA was tested in a Metrohm Model 30 pH-stat. Solutions were prepared in boiled water and maintained under Ar. 11-MeO-anthramycin ( 2 - 1 0 - 4 M ) in 0.03 M NaC1 was incubated in the pH-stat until fully hydrolysed. During this process, the pH rose from 5.6 to 6.4, presumably due to a slight differences between the pKa of 11-MeO-anthramycin and anthramycin. The solution was then adjusted to pH 7.0 and kept under Ar until used. 5 ml of 4 mM calf-thymus DNA in 0.03 M NaC1, 5 • 10-SM Na3 EDTA was adjusted to pH 7.0 in the pH-stat. 5 ml of the 2 . 1 0 - 4 M anthramycin solution was then added. The reaction was monitored for completeness by means of absorption spectra. After completion of the reaction, the sensitivity of the system to detection of H ÷ consumption was checked by the addition of small amounts of NaOH; it was demonstrated that 1% of a stochiometric uptake of H ÷ by anthramycin could have been detected. The anthramycin--DNA complex used for pH titation was prepared by
291 reacting 3.4 mg/ml calf-thymus DNA (Worthington) with 2 . 1 0 - 3 M 11-MeOanthramycin for 3 h at 23 ° C. The complex was precipitated and spooled twice by addition of isopropanol and NaC1 to final concentrations of 50% and 0.15 M, respectively. The spooled fibers were rinsed in 80% methanol and dissolved in 0.01 M NaCI, 2 . 1 0 - S M Na3EDTA. The resulting anthramycin/ DNA nucleotide ratio was 0.10. pH titrations were conducted under At. The extent of reaction of anthramycin with DNA was determined either spectrophotometrically or by solvent extraction. The spectrophotometric m e t h o d employed the absorbance ratio, A 321 n m /A ~ 49 n m, taking the ratios for free and b o u n d anthramycin to be 1.134 and 0.697, respectively [4]. The solvent extraction m e t h o d permitted extention of the measurements to high anthramycin concentrations. In this procedure, unreacted anthramycin was removed by extracting rapidly with 5 volumes of isoamyl alcohol. The extraction was repeated 3 times, which removed all detectable free anthramycin. (The isoamyl alcohol was pre-saturated with water to avoid depletion of the aqueous phase). The remaining aqueous phase was mixed with a small volume of methanol to avoid phase separation due to slight temperature changes, and the concentration of b o u n d anthramycin remaining in the aqueous phase was then determined spectrophotometrically. Results
Hydrolysis of 11-MeO-anthramycin The hydrolysis of ll-MeO-anthramycin was studied with the view that the reactivity of the l l - p o s i t i o n may play a role in the reaction with DNA. When ll-MeO-anthramycin was diluted into neutral aqueous solution, a small but highly reproducible change in absorption spectrum was observed {Fig. 1). The m a x i m u m change was at 375 nm, where the absorbance dropped 22%. There was also a detectable shift in the 334-nm absorption peak to a b o u t
0.8 ~
1
~ 0.4 .~f/ Q2
0.0
,
,
,
300 nm 400 Fig. 1. Spectral change o f 1 1 - M e O - a n t h ~ m y c i n ( A M E ) i n n e u t r ~ aqueous solution. 11-MeO-anthramycin d i s s o l v e d in i s o p r o p a n o l w a s d i l u t e d a p p r o x . 20-fold in 0.01 M s o d i u m p h o s p h a t e buffer, 0.5 m M EDTA, (pH 7.14) to a final concentration of 4.5 • 10-5M. The initial s p e c t r u m ( . . . . . . ) w a s c o m p l e t e d a b o u t 1 m i n after mixing. R e p e a t e d s p e c t r a s h o w e d a g r a d u a l c h a n g e up to a final curve ( ) obtained at 80 min, after w h i c h t h e r e w a s n o f u r t h e r c h a n g e , o . . . . . . o, calculated d i f f e r e n c e b e t w e e n t h e two spectra.
292 L0
'
,
'
J
'
i
,
r
'
,
,
o
,
pH
,
7.89
g _~
•
pH 7.23
z O.I <
5 < a-
0.0
'
20
'
410
'
60
'
MIN.
Fig. 2. K i n e t i c s of h y d r o l y s i s o f l l - M e O - a n t h r a m y e i n ( A M E ) , as m e a s u r e d b y c h a n g e in a b s o r b a n c e at 3 7 5 rim. 1.6 • 1 0 --4 M A M E , 0 . 1 5 M NaC1, 0 . 0 2 M s o d i u m p h o s p h a t e b u f f e z , 0.5 m M E D T A , 8% m e t h a n o l , 22.7 + 0.3 °C. ©, p H 7.89 (tl/2 = 3 1 . 2 rain); A p H 7.23 (tl/2 = 15.1 r a i n ) ; e, p H 6.41 (tl/2 = 4.2 min).
333 nm. This spectral change was presumed to be due to the conversion of ll-MeO-anthramycin to anthramycin and its equilibration products. In order to test this further, a sample of anthramycin (kindly provided by W. Leimgruber) was compared with l l-MeO-anthramycin. Both substances were dissolved in dimethylformamide in order to avoid the displacement reactions that occur in hydroxylic solvents. When the l l-MeO-anthramycin solution was diluted into 0.01 M sodium phosphate buffer, (pH 6.9) 0.5 mM EDTA, a spectral change similar to that shown in Fig. 1 was observed. In the case of anthramycin, however, there was no spectral change upon dilution into the aqueous buffer, and the spectrum was indistinguishable from the final aqueous spectrum produced by 11-MeO-anthramycin. This supports the view that the product after completion of the spectral change in Fig. 1 is anthramycin (in equilibrium with its epimer and anhydro derivative). The spectral change due to reaction with DNA was the same with 11-MeO-anthramycin and anthramycin [ 3,4]. The kinetics of the absorbance change at 375 nm (Fig. 2) indicate that the hydrolysis of 11-MeO-anthramycin is a first-order acid-catalysed reaction. The hydrolysis rate constant is plotted as a function of pH in Fig. 3a. The reaction of anthramycin (fully hydrolysed ll-MeO-anthramycin) with DNA also was found to be acid-catalysed (Fig. 3b), thus supporting the possibility that the t w o processes may be related.
Catalysis of 11-MeO-anthramycin hydrolysis by phosphate It was noted that at a given pH the hydrolysis rates were slightly faster in 0.15 M NaC1, 0.02 M sodium phosphate buffer (Fig. 3a, curve A) than in 0.04 M triethanolamine buffer (Curve B). Upon further exploration of this difference, it was found that the hydrolysis of l l-MeO-anthramycin is specifically catalysed by phosphate ions. This is demonstrated in Fig. 4 and Table I, which compare the hydrolysis rates in various aqueous solutions at a fixed pH and temperature. The reaction in 0.1 M triethanolamine-HC1 buffer(pH 7.2,
293 -0,5
o: ~
'
I
'
~
'
~
I
'
~
~
;
-I.0
A
1.0 m
I
i
l
i
I
i
I
i
I
-2.0
b A
2.5
_z bJ Ic
~
2o
z
1.5
1.0
\o
0.1
I I L L I I I I l l
6
~
0
7 pH
0 MIN
Fig. 3 (a) p H - d e p e n d e c e o f f l r s t - o r d e r r a t e c o n s t a n t (k in r a i n - I ) f o r h y d r o l y s i s o f l l - M e O - a n t h r a m y c i n ( A M E ) a t 2 2 . 9 ° C . Buffers: ( A ) 0 . 1 5 M NaCI, 0 . 0 2 M s o d i u m p h o s p h a t e , 5 • 1 0 -4 M E D T A ; (B) 0 . 0 4 M t r l e t h a n o l a m i n e - - H C l , 2 • 10-4 E D T A . ( T h e s o l u t i o n s also c o n t a i n e d 5% m e t h a n o l . ) (b) p H - d e p e n d e n c e of t h e r e a c t i o n o f a n t h r a m y c i n w i t h c a l f - t h y m u s D N A . l l - M e O - a n t h z a m y c i n was fully h y d r o l y s e d p r i o r t o a d d i t i o n o f D N A . T h e r e a c t i o n w a s f o U o w e d a t 321 n m , t h e m a x i m u m o f t h e d i f f e r e n c e s p e c t r u m [ 3 ] . T h e a p p a z e n t initial r a t e c o n s t a n t f o r t h e d i s a p p e a r a n c e o f free a n t h r a m y c i n w a s d i v i d e d b y t h e D N A n u c l e o tide c o n c e n t r a t i o n t o give t h e s e c o n d - o ~ l e r ~ate c o n s t a n t , k 2 . T h e line w a s d r a w n h a v i n g t h e slope t h e o r e t i c a l l y e x p e c t e d f o r a r e a c t i o n r a t e p r o p o r t i o n a l t o H + c o n c e n t r a t i o n . R e a c t i o n c o n d i t i o n s : Initial a n t h r a m y c i n c o n c e n t r a t i o n , 2 . 1 0 -5 M; 2 2 . 6 ° C . Buffers: o, 0 . 0 4 M c i t r a t e ; ~, 0 . 0 2 M p h o s p h a t e (6 • 10-4 M E D T A ) . Fig. 4. E f f e c t s o f p h o s p h a t e a n d NaC1 o n r a t e of l l - M e O - a n t h r a m y c i n h y d r o l y s i s . All s o l u t i o n s c o n t a i n e d 1.2 • 10-4M l l - M e O - a n t h r a m y c i n , 5 • 1 0 - 4 M E D T A , a n d w e r e a d j u s t e d t o p H 7 . 1 7 -+ 0 . 0 5 ; 2 2 . 7 ° C . Curve
Sodium phosphate b u f f e r c o n c n (M)
NaC1 c o n c n (M)
Triethanolamine b u f f e r c o n c n (M)
0 1.0 0 0.85 0
0.I 0.I 0 0 0 o r 0.1
A
0
B
0
C D E
0.I 0.I 0.4
t 1/2 (rain)
2.14 18.1
6.1 9.0 2.7
23°C} is shown by Curve A in Fig. 4. Addition of 1 M NaC1 to this buffer (Curve B) caused only a slight increase in rate. Addition of 0.4 M sodium phosphate, however, increased the rate about 7-fold {Curve E), and 0.1 M sodium phosphate increased the rate 3.5-fold {Curve C). These rates were independent of the presence or absence of triethanolamine. The rate increase produced by 0.1 M sodium phophate was partially reversed by adding 0.85 M NaC1 {Curve D), indicating that the catalytic effect of phosphate is reduced by high ionic strength. Interestingly, high ionic strength also retards the reaction
294 TABLE
I
FIRST-ORDER 0.04
HYDROLYSIS
RATES
(k) OF
ll-MeO-ANTHRAMYCIN
o A T 2 2 . 7 -+ 0 . 5 C , p H 7 . 1 6 -+
( k w a s d e t e r m i n e d f r o m the s l o p e s o f s e m i - l o g a r i t h m i c p l o t s such as t h o s e s h o w n in Figs 2 and 4. k is related to the h a l f - t i m e o f the r e a c t i o n by t I/2 = 0 . 6 9 3 / k . ) T E A , t r i e t h a n o l a m i n e - - H C l .
Solvent
k (min -I )
0.1 M TEA buffer 1 M NaC1 + 0 . 1 M T E A b u f f e r 1 M NaBr + 0.1 M TEA buffer 1 M NaN 3 + 0.1 M TEA buffer 0.021 M sodium phosphate buffer 0.11 M sodium phosphate buffer 0.11 M sodium phosphate buffer + 0.85 M NaCl 0.43 M sodium phosphate buffer 0.43 M sodium phosphate buffer + 0.1 M TEA buffer 0.4 M Na2SO 4 + 0.1 M TEA buffer 1 M imidazole + 0.1 M TEA buffer 0 . 0 5 M GMP
0.0324 0.0382 0.0388 0.0523 0.0531 0.118 0.0775 0.256 0.260 0.0493 0.07* 0.074
* R e a c t i o n d e v i a t e d f r o m first-order k i n e t i c s , and w a s a c c o m p a n i e d b y an overall fall in l l - M e O - a n t h r a mycin absorption peak
of anthramycin with native D N A [ 4 ] . This suggests that both reactions may be promoted by the electrostatic effects of phosphate groups. We attempted to detect a complex between phosphate and 11-MeO-anthramycin by looking for an effect o f phosphate on the absorption spectrum of ll-MeO-anthramycin, but no effect was detected. Various anions were compared for their ability to stimulate ll-MeOanthramycin hydrolysis (Table I). Of the ions tested, only phosphate had a large effect. Whereas 0.4 M phosphate increased the hydrolysis rate more than 7-fold, equal or greater concentrations of azide or sulfate caused less than a doubling in rate, and chloride or bromide produced barely detectable increases. Imidazole produced an apparent doubling in hydrolysis rate, but this value may be erroneously high because the reaction was accompanied by a decrease in the absorption band at 333 nm (without noticable change in position or shape of the peak), indicating another process "besides hydrolysis. The spectral changes in the presence o f the other salts were the same as occurred in the absence of salt. Guanylic acid (GMP) produced an effect similar to that which would be expected with an equimolar concentration of phosphate. Phosphate might act in two possible ways to catalyse the hydrolysis: (1) it may act as a nucleophile in displacement of the ---OCH3 group, or (2) it may act as a proton donor facilitating the acid-catalysed reaction (Fig. 12, III). The t w o possibilities are equally capable of explaining the data to this point. In order to distinguish between them, we tested sodium diethyl phosphate which lacks a donatable proton. Like phosphate, this c o m p o u n d produced no spectral change in anthramycin, indicating that no complex affecting the chromophore is produced. Unlike phosphate, diethyl phosphate did not catalyse the hydrolysis of ll-MeO-anthramycin (Fig. 5). This indicates that phosphate acts by donating a proton and facilitating an acid-catalysed reaction (Fig. 12, III).
295 1.0 '
I.O
'
I
'
i
,
I
,
I
'
F
i
0.8
'•0.5
~. 0 . 6
5 z z <
•
•
4;
0.4
0
0.I
0.2
~0.05 L
I
i
IO
I
20
MINUTES
,
I
30
,
I
i
20
I
40
J
60
MINUTES
Fig. 5, C o m p a r i s o n b e t w e e n e f f e c t s o f p h o s p h a t e (D) and d i e t h y l p h o s p h a t e ( A ) o n rate of l l - M e O a n t h r a m y c i n h y d r o l y s i s , relative t o c o n t r o l ( o ) . C o n c e n t r a t i o n s o f p h o s p h a t e and d i e t h y l p h o s p h a t e w e r e 0 . 5 M. 1 1 - M e O - a n t h r a m y e i n w a s f r e s h l y dissolved in d i m e t h y l f o r m a m i d e . Final c o n c e n t r a t i e s : 1 1 - M e O a n t h r a m y c i n , 1 . 2 5 • 1 0 -4 M; N - t r i s - ( h y d r o x y m e t h y l ) - m e t h y l - 2 - a m i n o s u l f o n a t e b u f f e r , 0 . 1 2 M; t o t a l Na+, 1 . 0 M ( m a d e u p w i t h N a C 1 ) : d i m e t h y l f o r m a m i d e , 1%. p H = 7 . 0 0 -+ 0 . 0 2 , 2 3 . 2 ° C . Fig. 6. E f f e c t o f 1 1 - M e O - a n t h r a m y c i n p r e - i n c u b a t i o n o n r e a c t i o n w i t h D N A . ( e ) no p r e - i n c u b a t i o n ; ( A ) p r e - i n c u b a t i o n for 1 8 0 r a i n a t 2 2 . 5 ° C i n 0 . 0 6 M t r i e t h a n o l a m i n e - - H C 1 b u f f e r ( p H 7 . 3 4 ) , 0 . 0 2 M triethan o l a m i n e , 0 . 0 2 M N a C 1 , 0 . 4 m M E D T A . T h e r e a c t i o n w i t h c a l f - t h y m u s D N A w a s carried o u t u n d e r the s a m e c o n d i t i o n s e x c e p t that t h e b u f f e r w a s d i l u t e d to 7 5 % o f t h e a b o v e c o n c e n t r a t i o n s . P r e - i n c u b a t i o n w a s b e g u n b y r a p i d l y dissolving solid 1 1 - M e O - a n t h r a m y c i n in b u f f e r as d e s c r i b e d u n d e r Materials and M e t h o d s . T h e h a l f - t i m e f o r t h e h y d r o l y s i s o f l l - M e O - a n t h r a m y c i n u n d e r t h e s e c o n d i t i o n s (in t h e a b s e n c e o f D N A ) w a s d e t e r m i n e d to b e 2 7 , 3 rain.
Relation between I1-MeO-anthramycin hydrolysis and DNA reaction kinetics In order to determine the relative reactivities of 11-MeO-anthramycin and anthramycin with DNA, 11-MeO-anthramycin was pre-incubated in neutral aqueous solution for various times and then mixed with DNA. Pre-incubation of 11-MeO-anthramycin was found to increase the rate of reaction with DNA. As the pre-incubation time was increased, the initial rate of reaction with DNA increased up to a maximum beyond which longer pre-incubation had no effect. The extreme cases, no pre-incubation and maximal pre-incubation, are shown in Fig. 6. Intermediate times of pre-incubation produced a family of curves between the t w o extremes shown. The initial rate of reaction with DNA was correlated with the fraction of the 11-MeO-anthramycin hydrolysed at the time of the earliest DNA rate measurement (about 2 rain after mixing with DNA) (Fig. 7). The fraction of 11-MeO-anthramycin hydrolysed was determined under similar conditions in the absence of DNA by following the change in absorbance at 375 nm. The linear relation in Fig. 7 indicates that the increase in reaction rate with DNA is due to the same change in 11-MeO-anthramycin that is responsible for the spectral change of 11-MeO-anthramycin in aqueous solution. It is seen that hydrolysed 11-MeO-anthramycin reacts about 5 times as fast with DNA as does
296 I
I
7
8
I
p
9
I0
6
80
0
g E
4 60
o
uJ
I
z w :L
4O
2
20 2'
0.5 FRACTION
OF
AME
1.0 HYDROLYZED
pN
Fig. 7. R e l a t i o n b e t w e e n initial r e a c t i o n rate w i t h D N A a n d e x t e n t o f h y d r o l y s i s o f 1 1 - M e O - a n t h r a m y e i n ( A M E ) . D a t a d e r i v e d f r o m e x p e r i m e n t s like t h o s e d e s c r i b e d in Fig. 6. T h e h a l f - t i m e ( i n rain) c a n be c a l c u l a t e d f r o m t h e o r d i n a t e v a l u e s ( k ) b y , tl/2 = 0 . 6 9 3 / ( k [ D N A ] ) , w h e r e [ D N A ] is in m o l e s o f n u c l e o t i d e u n i t s p e r I. ( S m a l l c o r r e c t i o n s w e r e a p p l i e d t o k f o r c h a n g e s in a b s o r b a n c e at 3 2 1 n m d u e t o l l - M e O a n t h r a m y c i n h y d r o l y s i s . T h e s e c o r r e c t i o n s d i d n o t m a t e r i a l l y a f t e r t h e r e s u l t s a n d n e v e r e x c e e d e d 15%.) Fig. 8. p H t i t r a t i o n o f a n t h r a m y c i n ( ~ ) a n d a n t h r a m y c i n / D N A c o m p l e x ( o ) . A n t h r a m y c i n c o n t e n t in e a c h c a s e w a s 4 . 1 # m o l e s at a c o n c e n t r a t i o n o f a p p r o x . 1 0 - 3 M . A n t h r a m y c i n / D N A n u c l e o t i d e ratio = 0 . 1 0 ; t h e c o m p l e x w a s p r e p a r e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s . ( o ) s o l v e n t ( 0 . 0 1 M N a C 1 , 2 • 1 0 - 5 M N a 3 E D T A ) ; a d d i t i o n o f D N A d i d n o t alter t i t r a t i o n c u r v e o f s o l v e n t . ( A slight d i f f e r e n c e w a s n o t e d between the titration curves of ll-MeO-antbxamycin before and after hydrolysis, indicating a small i n c r e a s e in p Ka u p o n h y d r o l y s i s . T h e c u r v e s h o w n is f o r f u l l y h y d r o l y s e d 1 1 - M e O - a n t h r a m y c i n .
unhydrolysed 11-MeO-anthramycin. Unhydrolysed 11-MeO-anthramycin also, however, reacts at a rate significantly greater than zero, as is seen by the zero-time intercept in Fig. 7. Hence, ll-MeO-anthramycin (or a product to which it is very rapidly converted} is able to react with DNA directly without first undergoing the hydrolysis reaction that produces the spectral change at 375 nm.
Role o f I-£ in the reaction with DNA The proportionality of the DNA reaction rate to H ÷ concentration (Fig. 3b) indicates that H ÷ is kinetically involved in the rate-limiting step of the reaction. In order to determine whether H ÷ is actually consumed, the reaction was carried out in a pH-stat (see Materials and Methods}. It was found that there is neither consumption nor loss of H ÷ in the reaction. Therefore, the kinetic dependance on H ÷ concentration must be in the rate-limiting formation of a transient intermediate. In order to determine whether there is a change in dissociable protons during the reaction, the pH titration curves of anthramycin and anthramycin-DNA complex were compared. Free anthramycin was found to have a pKa at about 8.7. In the complex, however, this pKa was completely absent (Fig. 8). Hence, the dissociable proton of anthramycin either is missing or is stabilized in the complex.
297
Kinetics of the reaction of anthramycin with DNA We have previously noted [4] l l - M e O - a n t h r a m y c i n that the reaction of anthramycin (fully hydrolysed ll-MeO-anthramycin) with DNA slows down with time more than would be expected on the basis of depletion of free anthramycin or DNA. It is as if the reactivity of either the anthramycin or the DNA decreased with time. In order to distinguish between these two possibilities, either fresh anthramycin or fresh DNA was added to a reaction mixture after it had reached the slow phase (Fig. 9). If one of the two reactants has lost reactivity, then addition of fresh reactant should increase the reaction rate more than in proportion to the total concentration of free reactant. At the time of addition of fresh reactant, the extent of binding, r, was approx. 0.03, far from the saturation value of r = 0.1 bound anthramycin per nucleotide [4]. Therefore, about 70% of the DNA sites were still available for reaction. Anthramycin could n o t have been depleted by reaction with DNA because its initial concentration was fax in excess of the available DNA sites. Addition of fresh anthramycin to give a 3-fold increase in total anthramycin concentration (Fig. 9A) produced approx, a 3-fold increase in reaction rate, indicating that fresh anthramycin was no more reactive than t h a t present in the reaction mixture. Addition of fresh DNA to give a 2-fold increase in concentration of total DNA (equivalent to approx, a 3-fold increase in unreacted DNA sites), however, produced far more than a 3-fold increase in reaction rate (Fig. 9B). The kinetics were in fact identical to the original reaction. This confirms that the reactivity of the anthramycin has n o t changed, and demonstrates that the reduced reaction rate is due to reduced reactivity of the DNA sites remaining after a partial reaction.
to
i A
0
i
i
I
i
i
~
i
I
i
L
I
t
I
I
I
I
i
i
~
FRESH ANTHRAMYCIN
~
I
I
I
I
I
i
I
Z
i IO REACTION
I
[
I
20 TIME
(minutes)
Fig. 9. E f f e c t s o f a d d i t i o n o f f r e s h a n t h r a m y c i n ( A ) o r f r e s h D N A (B) d u r i n g t h e slow p h a s e o f a n a n t h r a m y c i n - - D N A r e a c t i o n . (a) I n i t i a l c o n c e n t r a t i o n : a n t h r a m y c i n , 80/~M; D N A , 1 2 0 ~M. A n t h r a m y e i n a d d e d at 5 m i n : 1 7 0 JzM. (B) I n i t i a l c o n c e n t r a t i o n s : a n t h r a m y c i n , 87/~M; D N A , 1 2 5 / ~ M . D N A a d d e d at 5 m i n : 1 2 5 ~zM. R e a c t i o n m e d i u m : 0 . 0 1 M s o d i u m p h o s p h a t e b u f f e r ( p H 7 . 3 3 ) , 2 3 . 0 - - 2 3 . 5 ° C. " A n t h r a mycin" was derived from 1 1 - M e O - a n t h r a m y c i n by complete hydrolysis prior to use in the DNA reactions. Bound
anthramycin
was determined
by isopropanol
extraction
(see Materials and Methods).
298 Partial reaction of DNA with anthramycin therefore reduces the reactivity of the remaining DNA sites to further reaction with anthramycin. When reaction rates were compared at fixed values of r, however, the rates were found to be proportional to both anthramycin and DNA concentration, as expected for a bimolecular reaction. The reaction can therefore be expressed in terms of a bimolecular reaction rate constant (k') that is a function of the extent of reaction, r: dr
- - = k'(r)AC(~---r) dt
where A is the concentration of unreacted anthramycin, C is the total concen. tration of DNA, and v is the fraction of nucleotide residues that are potential binding sites. A plot of k ' ( r ) = ( d r / d t ) / a C ( v - - r ) against r should therefore obey a consistant function of r for various reaction times and reactant concentrations. This is demonstrated in Fig. 10. The apparent reaction rate constant, k ' ( r ) , is seen to decrease exponentially with r. Specificity of the reaction
Previous studies, based on spectral measurements, have shown that anthramycin reacts with p o l y ( d G ) , poly(dC), but not with poly[d(A--T)] (alternating) or p o l y ( r G ) , poly(rC) [4]. It was also found that although anthramycin reacts preferentially with double-stranded DNA, there is a substantial reaction rate with single stranded DNA at moderate ionic strength. We have therefore tested the reactivity with single-stranded poly(dG) and poly(dC) in order to determine which of these bases is required for reaction (Table II). Poly(dG) reacted to an extent similar to denatured calf-thymus DNA (Table II). A significant extent of reaction was, however, observed also with poly(dC) (Sample 1). This sample of poly(dC) was therefore analysed by acid hydrolysis and high-voltage paper electrophoresis, and was found to be contaminated with
&
•
[
r
t
I
r
•,
107
~
10s
~
10 5
AAA~ I
,
l
I
I
1
0.02 0.04 0.06 O.OS 0.I0 MOLES ANTHRAMYCIN BOUND PER MOLE DNA NUCLEOTIOES (r)
Fig, 10. D e p e n d a n c e o f a p p a r e n t r e a c t i o n r a t e c o n s t a n t ( k ' ) o n e x t e n t o f r e a c t i o n w i t h D N A (r). k ' w a s c a l c u l a t e d b y t h e e q u a t i o n g i v e n in t h e t e x t , a s s u m i n g ~ = 0.1 [ 4 ] . R e a c t i o n s w e r e c a r r i e d o u t in 0.01 M s o d i u m p h o s p h a t e b u f f e r . ( p H 6 . 7 - - 7 . 4 ) , A p p a r e n t r a t e c o n s t a n t s w e r e a d j u s t e d t o p H 7.0, b a s e s o n proportionality between reaction rate and H÷ concentration [ 4 ] . Reactant concentrations were anthram y c i n , 50---970 ~zM; D N A n u e l e o t i d s , 60---1800 /~M. R e a c t i o n s w e r e t e r m i n a t e d at v a r i o u s t i m e s . A n t h r a m y c i n b i n d i n g w a s d e t e r m i n e d e i t h e r s p e c t r o s c o p i c a l l y (o) or b y e x t r a c t i o n w i t h i s o a m y l a l c o h o l (A).
299 T A B L E II EXTENTS OF REACTION OF ANTHRAMYCIN
WITH POLYNUCLEOTIDES
1.5 X 1 0 - ~ M p o l y n u c l e o t i d e w a s r e a c t e d w i t h 1 0 _ 3 M a n t h r a m y c i n in 0.1 M s o d i u m p h o s p h a t e b u f f e r , ( p H 6 . 1 ) a t 2 3 ° C f o r 8 h. T h e e x t e n t o f b i n d i n g (r) w a s d e t e r m i n e d b y r e p e a t e d d i a l y s i s f o l l o w e d b y s p e c tral determination of anthramycin nucleotide ratio [4]. Polynucleotide Calf-thymus DNA (native) Calf-thymus DNA (denatured) p o l y ( d G ) : p o l y (dC) poly(dG) poly(dC) (Sample I ) p o l y ( d C ) ( S a m p l e 2) poly (dI)• poly(dC) poly [d(A--T)] (alternating) poly(dA) • poly(dT) poly(rG) poly(rC)
E x t e n t o f b i n d i n g (r) 0.099 0.032 0.099 0.031 0.012 0.006 <:0.002 <:0.002 <:0.002 <:0.002 <:0.002
about 10% poly(dG). A second, more highly purified, sample of poly(dC) exhibited substantially lower reactivity, consistant with the view that poly(dC) itself does not react. This was confirmed by the finding that poly(dI) • poly(dC) exhibited no detectable reaction.
Competition with actinomycin The finding that the reaction of anthramycin with DNA requires guanine suggested that anthramycin should compete with actinomycin for some of the same binding sites, since actinomycin also requires guanine for binding to DNA [14]. In order to test this, a series of anthramycin-DNA complexes of various extents of reaction were prepared, and the actinomycin-binding abilities of these cOmplexes were determined. Since the binding of anthramycin to DNA is firmer than the binding of actinomycin, it is possible to consider the anthramycin--DNA bonds to be essentially irreversible while the actinomycin--DNA reaction is a reversible equilibrium [4]. It is seen from Fig. l l a that the actinomycin binding capacity varied inversely with the extent of anthramycin binding. When the DNA was saturated with anthramycin (r = 0.10), the actinomycin binding capacity was nil. At lower extents of anthramycin binding, the capacity for actinomycin binding increased in proportion to the fraction of the anthramycin binding capacity that was unfilled. The binding constant of actinomycin with DNA, however, was unaffected by the presence of anthramycin, since the slopes in the Scatchard plots were similar for DNA and anthramycin--DNA complex (Fig. l l a ) . Discussion
The hydrolysis of ll-MeO-anthramycin is accompanied by a small change in absorption spectrum (Fig. 1) which was utilized to study the hydrolysis
300 O. I0
I
I
I
•
~
f
•
=o
[
I
Anthromycin: DNA 000
~c
0
~ o.os ~ru
~-~ :,= o
0.05 r (mol. onthromYcin bound per tool. DNA nucleoflde)
0.10
002
0.04 0.06 r ~Actinomycin)
0.08
Fig. 11. E f f e c t o f b o u n d a n t h r a m y c i n o n t h e i n t e r a c t i o n of D N A w i t h a c t i n o m y c i n . ( A ) A c t i n o m y c i n b i n d i n g c a p a c i t y ; (B) S c a t c h a r d p l o t . A n t h r a m y c i n - - D N A c o m p l e x e s w e r e d i a l y s e d to r e m o v e a n y u n b o u n d a n t h r a m y c i n . T h e c o m p l e x e s w e r e t i t r a t e d s p e c t r o p h o t o m e t r i c a l l y b y successive a d d i t i o n s o f a c t i n o m y c i n , a n d t h e d a t a w e r e a n a l y s e d as d e s c r i b e d b y H y m a n a n d D a v i d s o n [ 1 3 ] . T h e t i t r a t i o n s w e r e c a r r i e d o u t in 0 . 0 0 1 M s o d i u m p h o s p h a t e , ( p H 7.0).
' C
OH
H
Jg
N~/
OR
O
3
~/
\CONH 2
I
H
I
O--R OH
H ~)/
~"
O
\CONH 2
"rr O~.~
~O
o/P"~O H
e
O
-"
\CONH 2
"n'r
Fig. 12. S t r u c t u r e of a n t h r a m y c i n (R~----I-I) a n d l l - M e O - a n t h r a m y c i n (R--~CH3). ( I I ) P r o p o s e d p r o t o n a t e d i n t e r m e d i a t e . ( I I l ) Possible m o d e o f c a t a l y s i s b y p h o s p h a t e a c t i n g as a p r o t o n d o n o r .
301 kinetics. The hydrolysis obeys first-order kinetics and is acid-catalysed (Fig. 2). The pH-dependance of the kinetics (Fig. 3a) indicates that a protonated intermediate determines the reaction rate. Protonation of the oxygen atom at Position 11 would favor displacement of the m e t h o x y group and is a likely intermediate in the hydrolysis (Fig. 12, II). The fact that the reaction of anthramycin with DNA also is acid-catalysed (Fig. 3b) suggests that protonation of the oxygen at Position 11 may be an intermediate step in this reaction as well. Position 11 may, in fact, be the site through which anthramycin becomes covalently linked to DNA. This would be analogous to the case of alkylating agents, where reactivity with nucleophiles and ready hydrolysis have a c o m m o n chemical basis. The chemistry differs from alkylation, however, in that hydrolysis of alkylating agents inactivates them, whereas hydrolysis of 11-MeOanthramycin does not inactivate. The relationship of H ÷ to the interaction of anthramycin with DNA may be summarized as follows: (1) H ÷ (above pH 4) stimulates the reaction with DNA; (2) H ÷ is n o t consumed during the reaction; (3) the dissociation of H ÷ from the phenolic group of anthramycin is n o t seen in the titration curve of the complex; (4) below pH 4, H ÷ stimulates the dissociation of the complex [4]. A possible interpretation of these data is that the interaction involves both the C , , group and the d e p r o t o n a t e d phenolic group. This conforms with the finding that the 9-methoxy and l l - k e t o derivatives of anthramycin do not react with DNA and are biologically inactive [9] (Leimgruber, W., personal communication). The evidence that ll-MeO-anthramycin can react directly with DNA b u t at a slower rate than anthramycin (Fig. 7) is compatible with a displacement reaction at Position 11, since either a hydroxyl or a m e t h o x y group could undergo displacement by the same mechanism (Fig. 9, II) although the rates may differ. The slowness of the interaction of ll-MeO-anthramycin with DNA is one of the factors that suggested that the binding involves formation of a covalent bond [4]. An alternative possibility, however, would be that ll-MeO-anthramycin is slowly converted to a reactive species that then reacts rapidly, and perhaps non-covalently, with DNA. The increased rate of reaction of 11-MeOanthramycin after pre-incubation {Figs 6 and 7) tended to support this suggestion. Closer examination, however, reveals that the pre-incubation effect is limited: b e y o n d an optimal pre-incubation time, the reaction rate is not further increased and still remains slow compared to non-covalent DNA interactions with agents such as acridines or actinomycin, which occur on a time scale of seconds or milliseconds [ 1 0 , 1 1 ] . It may still be argued however that even after complete hydrolysis, anthramycin still must undergo a rate-limiting conversion to a reactive species normally present in very low concentration. If this were so, the rate of binding of anthramycin should be governed by the rate of its conversion to the reactive form and should b e c o m e independent of DNA concentration. Since DNA concentration did affect the rate of binding, this argument t o o is ruled out. We therefore conclude that the slowness of the reaction of anthramycin with DNA is n o t due to a rate-limiting conversion of anthramycin to a rapidly reactive species. Similarly, the possibility that the reaction rate is limited by a requirement for a slow conformational change in the DNA is ruled o u t by the finding that the reaction rate does n o t b e c o m e independent
302
of anthramycin concentration. These findings therefore support our previous inference that the slow reaction is due to covalent bond formation [ 4 ] . The site of anthramycin reaction apparently is guanine, since reaction occurs only with guanine-containing deoxypolynucleotides, and since there is competition with actinomycin for bindings sites. The exacting structural requirements for the anthramycin reaction are shown by the loss of reactivity when dG is replaced by dI, the absence of reactivity with helical ribo-polymers, and by the greatly reduced reactivity with single-stranded D N A [4]. A characteristic of the reaction of anthramycin with D N A is the marked slowing of the reaction with time. This apparently is not due to heterogeneity of sites with preferential reaction at more reactive sites, because the slowing was also seen in the reaction with poly(dG) • poly(dC} [4]. The most likely explanation is that reacted sites interfere with further reaction at nearby sites. Acknowledgement We thank Dr V.H. Bono, Jr for assistance with the pH stat and pH titration experiments. References 1 2 3 4 5 6 7 S 9 10 11 12 13 14
Tendler, M.D. and Korman, S. (1963) Nature 199, 501 Adamson, R.H., Hart, L.G. Devita, V.T. and Oliverio, V.T. (1968) Cancer Res. 28, 343--347 Kohn, K.W., Bono, Jr, V.H. and Kann, Jr, H.E. (1968) Biochim. Biophys. Acta 155, 121--129 Kohn, K.W. and Spears, C.L. (1970) J. Mol. Biol. 51, 551--572 Bates, H.M., Kuenzig, W. and Watson, W.B. (1969) Cancer Res. 29, 2 1 9 5 - - 2 2 0 5 Horwitz, S.B. and Grollman, A.P. (1968) Antimicrob. Agents Chemother. 21--24 Leimgruber, W., Stefanovic, V., Schenker, F., Karr, A. and Berger, J. (1965) J. Am. Chem. Soc. 87, 5791--5793 Leimgruber, W., Bateho, A.D. and Schenker, F. (1965) J. Am. Chem. Soc. 87, 5793--5795 Leimgruber, W., Batcho, A.D. and Czajkowski, R.C. (1968) J. Am. Chem. Soc. 90, 5641--5643 Miiller, W. and Crothers, D.M. (1968) J. Mol. Biol. 35, 251--290 Li, H.J. and Crothers, D.M. (1969) J. Mol. Biol. 39, 461--477 Leimgruber, W., Batcho and Schenker, F. 4th Int. Syrup. Chem. Nat. Prod. IUPAC Congr. Stockholm, 1966, Abstr. p. 106 Hyman, R.W. and Davidson~ N. (1971) Biochim. Biophys. Acta 228, 3 8 - 4 8 Jain, S.C. and Soben, H,M~ (1972) J. Mol. Biol. 68, 1--20