Energy transfer in the DNA-chloroquine complex

545

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Energy transfer in the DNA-chloroquine complex* Chloroquine (7-chloro-4(4-diethylamino-I-methylbutylamino)quinoline) binds to double-stranded DNA 1. We have studied energy transfer in this complex b y photochemical and luminescence techniques. Low quantum yields for emission at room temperature make quantitative measurement of sensitized fluorescence imprecise;we show that photochemical techniques can be used to demonstrate energy transfer when luminescence techniques are inappropriate. We report the first demonstration of triplet-triplet transfer from DNA to a non-acridine molecule. 7 6 5

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Fig. I. I n h i b i t i o n of p y r i m i d i n e d i m e r f o r m a t i o n b y chloroquine, iSH~thymidine-labeled Escherichia coli D N A (about 2. lO 5 c o u n t s / m i n per/~g) in I m M p h o s p h a t e b u f f e r (pH 7.o) in t h e presence of chloroquine u p to r = 1. 4 w a s e x p o s e d to 4" l ° s J "m-2 of 254-nm radiation. I r r a d i a t i o n t i m e s were corrected for s e l f - a b s o r p t i o n b y D N A a n d for a b s o r p t i o n of u l t r a v i o l e t l i g h t b y chloroquine. D e t a i l e d m e t h o d s for a s s a y i n g a n d i d e n t i f y i n g p y r i m i d i n e d i m e r s h a v e been described 2 ~ Briefly, t h e s a m p l e s were h y d r o l y z e d in formic acid, c h r o m a t o g r a p h e d on cellulose t h i n layers in n - b u t a n o l - a c e t i c a c i d - w a t e r ( 4 o : 6 : 1 5 , b y vol.) 9, c o u n t e d in a scintillation c o u n t e r a n d a n a l y z e d for t h y m i n e a n d t h y m i n e - c o n t a i n i n g dimers. Chloroquine did n o t interfere w i t h h y d r o l y s i s , c h r o m a t o g r a p h y or c o u n t i n g .

We have shown that energy transfer from DNA to bound molecules (with appropriate energy levels) plays an important role in the inhibition of cyclobutylpyrimidine dimer formation in ultraviolet-irradiated DNA 2. The inhibition of dimer formation b y chloroquine is shown in Fig. I. For small chloroquine to DNA phosphate ratios, there is no overlap of the segments of the DNA in which each molecule affects dimer formation. Therefore, /5, the effective number of base pairs over which each chloroquine inhibits dimer formation, is given b y N = N O (I--2flr)

(I)

where N o is the percent dimers formed in the absence of chloroquine and N is the percent at a chloroquine to DNA phosphate ratio of r. fl is calculated from the initial * P r e s e n t e d in p a r t before t h e B i o p h y s i c a l Society in L o s Angeles, Calif., F e b r u a r y , 1969.

Biochim. Biophys Acta, 19o (1969) 545-548

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slope of the plot of d i m e r yields vs. r. Fig. I gives fl of a b o u t 8 base pairs. These results i n d i c a t e t h a t in solution at room t e m p e r a t u r e , D N A can transfer energy to chloroquine. B o t h the lowest e x c i t e d singlet s t a t e (S1) a n d lowest t r i p l e t state (T1) of chloroquine are lower t h a n the corresponding energy levels of the D N A bases (see Fig. 2). H o w e v e r , in c o n t r a s t to the other cationic molecules which have been shown to inhibit d i m e r f o r m a t i o n a, the S 1 of chloroquine is a b o v e the T 1 of the D N A bases. Thus, as we discussed elsewhere 3, D N A t r i p l e t - c h l o r o q u i n e singlet transfer is energ e t i c a l l y forbidden. -----$3

S2

>34 Sl I T:sc~~~~SS'.~Tsc ~ --~isc T~ T~ Sl LI.Iw~IZz' ABSORPTO I N PH~/LUDFI [

0 So i DNA

CHLOROQUN IS Eo

Fig. 2. Energy levels of the DNA bases and chloroquine. Tile energy of the lowest singlet ($1) and lowest triplet (Ti) of DNA relative to the ground state (So) are taken from LAMOLAel al. TM. The energies of the excited singlet states (S~,S2,Sa) of chloroquine relative to the ground state (So) were determined from the red edge of the corresponding absorption bands; the energy of chloroquine's Ta was determined from the blue edge of its phosphorescence (see Fig. 3). The singlet state of chloroquine may be populated either by direct absorption or by singlet-singlet (SS) transfer; the triplet state can be populated by intersystenl crossing (ISC) or triplet-triplet (TT) transfer. The wavelengths used in the sensitized phosphorescence experiment (28o and 3o4 nm) excited only the S~ of chloroquine.

E n e r g y transfer from D N A to a b o u n d molecule can also be d e m o n s t r a t e d b y observing the l i g a n d ' s fluorescent emission of energy a b s o r b e d b y D N A (sensitized fluorescence) 4,5. The fluorescence of chloroquine b o u n d to D N A was e n h a n c e d for wavelengths below 3o0 niu where D N A absorbs. However, the low q u a n t u m yield for fluorescence of b o u n d chloroquine u n d e r these conditions m a d e q u a n t i t a t i v e meas u r e m e n t s impractical. These results p o i n t out the value of p h o t o c h e m i c a l techniques in d e m o n s t r a t i n g energy transfer where emission of the donor or a c c e p t o r is difficult or impossible to observe. A t 77°K a n d in a rigid n l a t r i x (5o o/o glycerol) the fluorescent emission increases a n d phosphorescence is also observable. U n d e r these conditions, triplet D N A - t r i p l e t chloroquine transfer can be observed. Direct a b s o r p t i o n of a photon b y chloroquine or transfer from the D N A singlet p o p u l a t e s the S l and, b y i n t e r s y s t e m crossing, the T 1 of chloroquine (see Fig. 2). Thus the r a t i o of phosphorescence to fluorescence ( p / / ) is the same for either e x c i t a t i o n p a t h . However, t r i p l e t - t r i p l e t transfer c o n t r i b u t e s o n l y to phosphorescence a n d t h u s increases the p / / r a t i o . Fig. 3 shows t h a t t h e phosphorescence of b o u n d chloroquine increases relative to its fluorescence when the exBiochim. lJiophys. Acla, 19o (t969) 545-548

SHORT COMMUNICATIONS

i

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Fig. 3. Emission spectrum of chloroquine bound to DNA in a rigid matrix (5o % glycerol) at 77°K. The maximum intensities of fluorescent and phosphorescent emission are at 36o and 45 ° nm, respectively. Excitation was by 3o4-nm (- - -) and 28o-nm ( - - ) radiation. Before mixing with glycerol the solution contained 41/~M chloroquine, o.33 mM calf-thymus DNA (in phosphate) and I mM phosphate buffer (pH 5.7). The spectra are not corrected for tile spectral sensitivity of the emission monochromator and photomultiplier (IP28) of the Aminco spectrophotofluorimeter (equipped with a low-temperature Dewar). The scales on which the emission spectra were recorded are normalized so that their fluorescent intensities coincide. Spectra were corrected for emission due to scattered light, DNA and impurities. The emission from chloroquine was much stronger than that from DNA.

citing w a v e l e n g t h is a b s o r b e d b y D N A . F o r free chloroquine, the p/] r a t i o is t h e same at 280 a n d 304 nm, i n d i c a t i n g t h a t t h e increased phosphorescence is n o t due to increased i n t e r s y s t e m crossing at s h o r t e r wavelengths. GALLEY 6'7 o b s e r v e d e n h a n c e d p/] in the 9 - a m i n o a c r i d i n e - D N A c o m p l e x a n d p o i n t e d out t h a t t r i p l e t - t r i p l e t t r a n s f e r proceeds t h r o u g h short range interactions. T h u s "sensitized phosphorescence" implies close p h y s i c a l p r o x i m i t y of donor a n d acceptor. Fig. 3 shows t h a t the increase in p/] is a b o u t 50 % for e x c i t a t i o n at 280 nm, smaller t h a n t h a t o b s e r v e d for 9-aminoacridine. This does n o t i m p l y less efficient t r i p l e t - t r i p l e t transfer, as t h e r e l a t i v e e n h a n c e m e n t of p/] is i n v e r s e l y p r o p o r t i o n a l to t h e p r o b a b i l i t y of i n t e r s y s t e m crossing (~ise). Fig. 3 shows t h a t t h e p h o s p h o r e s c e n t i n t e n s i t y of chloroquine is t h e same order of m a g n i t u d e as the fluorescent i n t e n s i t y (i.e. ~Ise ~ 0-5), while t h e p h o s p h o rescence of 9-aminoacridine c a n n o t be d e t e c t e d in the fluorescent s p e c t r u m (i.e. ~blse << o.5) 7. Sensitized phosphorescence of chloroquine is f a v o r e d b y t h e absence of t r i p l e t D N A - s i n g l e t chloroquine t r a n s f e r (energetically impossible, see Fig. 2) which would reduce the n u m b e r of D N A t r i p l e t s a v a i l a b l e for t r i p l e t - t r i p l e t transfer. The value of p// d i d not change significantly for r ' s from 0.05 to o.125. W e h a v e d e m o n s t r a t e d energy transfer from D N A to chloroquine b o t h in solution at r o o m t e m p e r a t u r e a n d in a rigid m a t r i x at 77°K. W e find t h a t p h o t o c h e m i c a l techniques are useful not only as a p r o b e of D N A - l i g a n d i n t e r a c t i o n s b u t also as a m e a n s of d e m o n s t r a t i n g energy t r a n s f e r when emission of t h e donor a n d a c c e p t o r are difficult to observe. Since t r i p l e t - t r i p l e t t r a n s f e r requires p h y s i c a l p r o x i m i t y of d o n o r a n d a c c e p t o r 7, our o b s e r v a t i o n of sensitized phosphorescence at 77°K is c o m p a t ible w i t h t h e h y p o t h e s i s of O'BRIEN et al. s t h a t chloroquine i n t e r c a l a t e s b e t w e e n a d j a c e n t D N A base pairs. W e t h a n k F. E. H a h n for his i n t e r e s t a n d e n c o u r a g e m e n t , D a v i d Ginsberg for the use of his J a g g e r meter, L. K a y z a k for his gift of h i g h l y purified chloroquine, a n d

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W. C. Galley, McGill U n i v e r s i t y , for helpful discussions. Dr. B.M. S u t h e r l a n d was a p o s t d o c t o r a l fellow of the N a t i o n a l I n s t i t u t e of General Medical Sciences, U.S., Public H e a l t h Service, Fellowship I-F2-GM-36, 62o-o2.

Department o~ Molecular Biology, Walter Reed Army Institute o/Research, Washington, D.C. 20012 (U.S.A.)

J . C . SUTHERLAND* B . M . SUTHERLAND**

i s. N. COHEN AND K. L. YIELDIN~, Proc. Natl. Acad. Sci. U.S., 54 (1965) 521. 2 13. M. SUTHERLAND AND J. C. SUTHERLAND, Biophys.

J., 9 (1969)

292.

3 ]3. M. SUTHgRLANDANO J. C. SUTHERLAND,Biophys. J., 9 (1969) lO45. 4 G. ~¢VEILLAND M. CALVIN, Biopolymers, i (1963) 4Ol. 5 J.-13. LEPECQ AND C. PAOLETTI, J. Mol. Biol., 27 (1967) 87.

6 W. C. GALLEY, Biopolymers, 6 (1968) 1279. 7 W. C. GALLEY, Triplet Energy Delocalization in Polynucleotide-Acridine Complexes, Ttlesis, California Institute of Technology, Pasadena, Calif., (1967). 8 R. L. O'BRIEN, J. L. ALLISONAND F. E. HAHN, Biochim. Biophys. Acta, 129 (1966) 622. 9 I{. C. SMITH, Photochem. Pholobiol., 2 (1963) 503IO A. A. LAMOLA, M. GUERON, T. YAMANE, J. EISINGER AND R. G. SHULMAN. J. Chem. Phys., 47 (1967) 221o. R e c e i v e d J u l y 25th, I969 " Present address: Laboratory of Chemical 13iodynamics, Lawrence Radiation Laboratory, University of California, Berkeley, Calif. 9472o, U.S.A. ** Present address: Department of Molecular 13iology and Virus Laboratory, University of California, 13erkeley, Calif. 9472o, U.S.A.

Biochim. Biophys. Acla, 19° (1969) 545-548

BBA 93453 Preparation of [ct-32p]nucleoside and deoxynucleoside 5"-triphosphates from 32Pi and protected ond unprotected nucleosides I n a r a p i d a u d simple m e t h o d described p r e v i o u s l y 1,2, nucleoside 5'-I32PJ m ° n ° p h o s p h a t e s a n d d e o x y t h y m i d i n e 5'- a n d 3'-[a2P~monophosphates were p r e p a r e d in high yield with a specific a c t i v i t y g r e a t e r t h a n I mC/pmole s t a r t i n g with a2Pl a n d a s u i t a b l y p r o t e c t e d nucleoside. Methods are now described for the extension of the original m e t h o d to the use of u n p r o t e c t e d nucleosides a n d t h e selective e n z y m i c conversion of nucleoside 5 ' - m o n o p h o s p h a t e s to the t r i p h o s p h a t e s . The chemical m e t h o d used for the synthesis of nucleoside C32P_~monophosphates i n v o l v e d t h e coupling of I.o ~ m o l e of 32P1 ( I - 5 m e ) to an excess (5o ~moles) of a s u i t a b l y p r o t e c t e d nucleoside using trichloroacetonitrile as condensing agent in the presence of t r i e t h y l a m i n e a n d with d i m e t h y l s u l p h o x i d e as solvent 2. A f t e r r e m o v a l of the p r o t e c t i n g g r o u p a n d purification of the reaction m i x t u r e b y p a p e r c h r o m a t o g r a p h y or p a p e r electrophoresis, m o n o n u c l e o t i d e s were o b t a i n e d in a yield of 70-9 ° ~; relative to a2Pl added. W h e n this m e t h o d was e x t e n d e d to the use of u n p r o t e c t e d nucleosides, all hyd r o x y l groups were susceptible to p h o s p h o r y l a t i o n . However, if the consequent mixture of nucleotides can be r e a d i l y s e p a r a t e d b y c h r o m a t o g r a p h y or the required nucleotide selected b y e n z y m i c means, then the use of u n p r o t e c t e d uucleosides has the

Biochim. Biophys. Acta, 19o (1969) 548-55 °