Structural requirements for the binding of ethidium to nucleic acids

234

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95322

STRUCTURAL R E Q U I R E M E N T S FOR T H E BINDING OF E T H I D I U M TO NUCLEIC ACIDS

M. J. W A R I N G

Sub-Department o[ Chemical Microbiology, Department o/ Biochemistry, University o[ Cambridge, Cambridge (Great Britain) (Received May i4th, 1965)

SUMMARY

I. Ethidium bromide, a phenanthridine drug, forms soluble metachromatic complexes with nucleic acids. The mechanism of the interaction has been investigated by studying the ability of substances related to nucleic acids to shift the visible absorption band of the drug in solution. 2. Purine and pyrimidine nucleotides produce slight shifts to longer wavelengths and the purine compounds are the more effective. However, neither purine nor pyrimidine bases in DNA act as specific binding sites for the drug since complete removal of either type of base from DNA leads to diminished interaction. 3. Although ethidium forms metachromatic complexes with homopolymers, binding curves indicate that the interaction with these materials is quite different from the strong primary binding seen with DNA or RNA. The curves suggest a facilitated binding process which probably represents the weaker secondary binding to nucleic acids. 4. From measurements of the interaction between ethidium and mixtures of homopolymers a correlation is described between the ability of synthetic polynucleotides to bind the drug strongly and the possession of base-paired helical structure. The helical structure need not necessarily be stabilised by purine-pyrimidine contacts. 5. These results provide further evidence that the primary binding of ethidium to nucleic acids occurs by a process of intercalation between adjacent base-pairs while secondary binding occurs by a "stacking" mechanism.

INTRODUCTION

The trypanocidal drug ethidium bromide (2,7-diamino-9-phenyl-Io-ethylphenanthridinium bromide) inhibits the synthesis of nucleic acid by living organisms 1,z Abbreviations: poly-A, polyadenylic acid; poly-C, polycytidylic acid; poly-I, polyinosinic acid; poly-U, polyuridylic acid; poly-(A+U), poly-(I+C) and poly-(A+I), two-stranded intermolecular complexes of the indicated homopolymers; poly-(A+Uz), three-stranded complex of one poly-A strand with two poly-U strands.

Biochim. Biophys. Acta, 114 (1966) 234-244

BINDING OF ETHIDIUM TO NUCLEIC ACIDS

235

and also by cell-free enzyme systems a& Moreover the drug forms metachromatic complexes with DNA or RNA (refs. 3, 5) and it is probable that the effects of ethidium on nucleic acid synthesis in vivo and in vitro are direct consequences of its physical binding to these substances l& Previous papersS, ~ reported quantitative data on the interaction between ethidium bromide and nucleic acids. It was found that a strong primary binding, saturated when I drug molecule was bound for every 4-5 nucleotides, could be followed by much weaker secondary binding leading to the precipitation of a I : I complex. The primary binding was not significantly influenced by the base-composition nor by denaturation of DNA but was sensitive to increases in ionic strength, particularly when magnesium salts were added. This communication describes experiments designed to elucidate the nature of the primary sites on nucleic acids to which ethidium is strongly bound. The criterion of specific binding of the drug is taken to be the metachromatic shift in the visible absorption band of ethidium. While the absence of a metachromatic effect does not necessarily indicate lack of interaction between the drug and a given substance the spectral shift provides a convenient and sensitive test for interaction of the type characteristic of ethidiumnucleic acid complexes. The results, obtained by studying the interaction with nucleic acid derivatives and synthetic homopolymers, indicate that the principal requirement for primary binding of ethidium is that the polynucleotide possess some form of base-paired secondary structure.

MATERIALS

Ethidium bromide was a gift from Dr. G. WOOLFE of Boots Pure Drug Co. Ltd., Nottingham. The drug was dissolved in water at a concentration of I mg/ml and stored at 0-4 ° in the dark. All experiments were performed in the presence of 0.04 M Tris-HC1 buffer (pH 7.9) prepared with recrystallised primary standardgrade Tris (Sigma Chemical Co.) and doubly distilled water. Ribonucleoside monophosphates were obtained from Nutritional Biochemicals Corporation. Deoxyribonucleoside monophosphates were products of California Corporation for Biochemical Research. RNA "core", the non-dialysable residue of yeast RNA after treatment with ribonuclease (EC 2.7.7.I6) 7, was purchased from Worthington Biochemical Corporation. The base-composition of the RNA "core" was" 27 % adenine, 53 ~o guanine, 12 ~o cytosine, 8 % uracil. These values were kindly determined by Dr. A. SCHEIN. Ribitol teichoic acid was isolated from Bacillus subtilis according to the procedure of ARMSTRONG et al.S; before use the product was freed from traces of nucleic acid by incubation with 5 ~g/ml each of ribonuclease and deoxyribonuclease (EC 3.I.4.5), followed by deproteinisation and recovery of the ethanol-precipitable material. Apurinic DNA was prepared from calf-thymus DNA (British Drug Houses) by the method of LALAND9. 5 0 0 - # g samples of the product and of untreated DNA were hydrolysed with 72 ~o HC1Q at IOO° for I h followed by chromatography in the isopropanol-HC1 solvent of WYATT10. Adenine and guanine were not detectable in the hydrolysate from the treated DNA. Apyrimidinic DNA was obtained from calf-thymus DNA by treatment with hydrazine 11. The product was shown to contain no detectable cytosine or thymine by hydrolysis and chromatography as described for apurinic DNA. Biochim. Biophys. Acta, 114 (1966) 234-244

236

M.J. WARING

Synthetic homopolymers (poly-A, poly-C, poly-I and poly-U) were obtained as the potassium salts from Miles Chemical Co. They were dissolved at 2 mg/ml in the Tris-HC1 buffer and stored frozen. For the preparation of intermolecular complexes 12, solutions were mixed in equimolar proportions with respect to nucleotides and allowed to stand at room temperature for at least 15 h before use. The ultraviolet absorption spectra of the mixtures were then measured and compared with those predicted for non-interacting systems. When the spectra were measured at polymer concentrations 6o #M with respect to nucleotides it was clear that p o l y - ( A + U ) and p o l y - ( I + C ) complexes had formed; with both these mixtures a substantial hypochromic effect was evident. However, all other mixtures, including the combination poly-A plus poly-I, gave spectra corresponding to non-interacting systems. The apparent lack of formation of a poly-(A + I) complex was probably due to dissociation of the complex at 6o ~M, since at o.6 mM using a i - m m light path the spectrum of the mixture showed an absorption m a x i m u m at 254 m¢, and a hypochromicity at that wavelength of 17 % compared with the calculated non-interacting spectrum. These values are in good agreement with those reported for the poly- (A + I) complex la. When the spectra of the remaining mixtures (poly-A plus poly-C, poly-I plus poly-U, and poly-C plus poly-U) were checked at o.6 mM with the I - m m light path again no evidence for interaction was found.

METHODS

Concentrations of polynucleotides are expressed in terms of molarity with respect to nucleotides, based on phosphorus determinations by the method of IhSKE AND SUBBARow 14 after digestion with conc. H2SO 4 and conc. HNO~. The binding of ethidium to polynucleotides was estimated from measurements of the absorbance of the drug at 46o, 47o and 48o m# as previously described ~. The parameters of interaction, i.e. the dissociation constant K and number of binding sites per nucleotide n, were derived from the relation Y

c --

I

n

Kr+K

where r is the number of drug molecules bound concentration of free drug. In the experiments measurements are used to estimate the fraction permitting calculation of r and c since the total acid are known.

per nucleotide and c is the molar reported here, spectrophotometric of ethidium in a complexed form, concentrations of drug and nucleic

RESULTS

Spectral shifts produced by nucleotides In the presence of high concentrations of nucleoside monophosphates the visible absorption band of ethidium is slightly shifted to longer wavelengths (Fig. i). The quality of the spectral shift is similar to that produced b y DNA (ref. 5), with an isosbestic point in the region of 500 me, (see Fig. 3). Purine nucleotides give a more Biochim. Biophys. Acta, I 1 4 (1966) 2 3 4 - 2 4 4

237

BINDING OF ETHIDIUM TO NUCLEIC ACIDS

I

CMP

I UMP

GMP

I

.E o

0.05

de AMP

-~ U

~0 @

~400

500

I de CMP

600 400

I de GMP

I

500

600 400

I (de) TMP

I

500

600 400

I

500

600

Wavelength (m~) Fig. I. E f f e c t s of n u c l e o t i d e s o n t h e a b s o r p t i o n s p e c t r u m of e t h i d i u m b r o m i d e (o.125 mM) N u c l e o t i d e s were p r e s e n t a t 1.5 m g / m l e x c e p t for G M P (0.9 m g / m l ) a n d d e G M P (o. 3 m g / m l ) . A b s o r b a n c e w a s m e a s u r e d as a difference s p e c t r u m u s i n g a i - c m l i g h t p a t h .

marked effect than the pyrimidine compounds, and allowing for the fact that guanosine nucleotides were tested at lower concentrations because of their insolubility it is probable that the purine compounds are about equally effective. Nevertheless the shifts produced by nucleotides are much less pronounced than those seen with nucleic acids; a spectral shift as large as that given by deAMP in Fig. I can be achieved with a concentration of DNA almost Ioo-fold lower. Binding to R N A "core" The larger effect of purine nucleotides on the spectrum of ethidium compared with that given by pyrimidine nucleotides suggests the possibility that purines act as sites for attachment of ethidium to nucleic acids. This suggestion would be consistent with the observation that the drug interacts equally well with DNA samples having guanine plus cytosine contents ranging from 35 ~o to 72 ~o (ref. 5)- To test the effect of nucleic acid containing a high proportion of purines the interaction between ethidium bromide and RNA "core" was measured (Fig. 2). Compared with the binding to ribosomal RNA, the drug is bound much less tightly and to a greatly reduced extent by the "core", indicating that a high purine content does not lead to enhanced interaction with ethidium. However, the interpretation of this experiment is not unambiguous since RNA "core" consists of small fragments of nucleic acid which might bind the drug less effectively simply as a consequence of their short chain length. Interaction with modi/ied DNA and teichoic acid The question whether purinb or pyrimidine bases form a specific part of the Biochim. Biophys. Acta, 114 (1966) 234-244

238

M.J. WARING

0.2--

0.1

RNA "core"

0

~ - -

1

2

Free drug concn.(M x 105)

Fig. 2. B i n d i n g of e t h i d i u m to R N A " c o r e " , c a l c u l a t e d f r o m m e a s u r e m e n t s m a d e w i t h a 4-cm l i g h t p a t h on s o l u t i o n s c o n t a i n i n g 25 # M e t h i d i u m b r o m i d e . T h e c u r v e for b i n d i n g to R N A is t a k e n f r o m p r e v i o u s r e s u l t s 6.

ethidium-binding sites was investigated more directly by measuring the ability of DNA to form a metachromatic complex with the drug after complete removal of either type of base. Fig. 3 shows results obtained with mixtures containing I drug molecule per 3.12 phosphate groups or I per 15.6 phosphate groups. At the higher drug concentration both apurinic and apyrimidinic DNA produce considerable spectral shifts although the apurinic nucleic acid is clearly more effective. However, at the lower drug concentration the shifts are smaller and apyrimidinic DNA gives the greater effect. These results show that neither purines alone nor pyrimidines alone can be considered as primary requirements for binding of ethidium; on the contrary, removal of either type of base from DNA leads to diminished interaction. As an example of a polymeric compound containing phosphodiester linkages but otherwise unrelated to nucleic acids, the ribitol teichoic acid from Bacillus subtilis was tested for metachromatic interaction with ethidium (Fig. 3). This substance is a polymer of nine alanylglucosylribitol 5-phosphate units joined through phosphodiester linkages at positions I and 5 of the ribitol 8. There was no detectable interaction between teichoic acid and ethidium bromide, showing that the ability to form a metachromatic complex with the drug is not shared by all polymers containing secondary phosphoryl groupings.

Interaction with homopolymers Further attempts to elucidate the nature of the interaction between ethidium and nucleic acids were made using synthetic homopolymers. These substances were expected to provide estimates of the affinity of ethidium for each of the four bases in polynucleotide combination. The interaction of the drug with GMP residues could not be tested directly because the homopolymer of GMP is not readily synthesised by polynucleotide phosphorylase; however, the analogous compound poly-I was investigated. Fig. 3 includes results obtained v~ith the four homopolymers. Apart Biochim. Biophys. Acta, 114 (1966) 234 244

239

BINDING OF ETHIDIUM TO NUCLEIC ACIDS

0.4J DNA

Teichoic acid

o[ [

-0.4~J

,

o °41P°'Y' 4~ - 0 . 4 a 400 J~ a c

500

Po,yC 600 400

04[oNA

500

600 400 500 Wavelength (rnu)

Apurlnic DNA

600 400

yr'm'd'n,o

500

600

Telchoic acid

1

10.4

0.4 [Poly I

Poly C

Poly U

_oO 400

500

600 400

500

600 400 500 Wavelength (mu)

600 400

500

600

Fig. 3. E f f e c t s of nucleic acid d e r i v a t i v e s a n d h o m o p o l y m e r s o n t h e a b s o r p t i o n s p e c t r u m of e t h i d i u m b r o m i d e , E a c h p o l y p h o s p h a t e c o m p o u n d w a s p r e s e n t a t 0,39 m M w i t h r e s p e c t to p h o s p h a t e residues. U p p e r c u r v e s : s o l u t i o n s c o n t a i n e d o. 125 m M e t h i d i u m b r o m i d e a n d difierence s p e c t r a were m e a s u r e d u s i n g a i - c m l i g h t p a t h , Lower c u r v e s : s o l u t i o n s c o n t a i n e d 25/zM e t h i d i u m b r o m i d e a n d difference s p e c t r a were m e a s u r e d u s i n g a 4 - c m l i g h t p a t h .

from poly-C, which shows virtually no interaction at either drug concentration, these materials produce spectral shifts which are qualitatively almost identical to the shift in the presence of DNA although differing in magnitude. The effects of poly-A and poly-U parallel quite closely those of apyrimidinic and apurinic DNA, respectively, while poly-I is as effective as native DNA at the higher drug concentration. In order to determine binding curves for the interaction of ethidium with homopolymers the absorbance of the drug at 460, 470, 480 and 54 ° m/~ was measured in the presence of varying homopolymer concentrations. Fig. 4 shows the effects at 460 and 540 mkt. Even at the highest concentrations of homopolymers the absorbance does not attain a constant value, making direct estimation of the values for the fully bound drug impossible. However, if it is assumed that the absorbance of ethidium Biochim. Biophys. Acta, 114 (1966) 2 3 4 - 2 4 4

240

M.J.

WARING

I

o PolyC

--,o Poly U 0.4 o PolyA

-o Poly I o

--------ePoly I

~ 0V

Poly A

0.2

I

0

I

5

10

Poly

1

15

Polynucleotldeconch.(M x 104) Fig. 4. A b s o r b a n c e of e t h i d i u m b r o m i d e in t h e presence of h o m o p o l y m e r s . T h e d r u g c o n c e n t r a t i o n w a s 25 # M a n d a b s o r b a n c e w a s m e a s u r e d u s i n g a 4-cm l i g h t p a t h . H o m o p o l y m e r c o n c e n t r a t i o n s are e x p r e s s e d in t e r m s of m o l a r i t y w i t h r e s p e c t to nucleotide p h o s p h o r u s . © - © , a b s o r b a n c e a t 460 m ~ ; 0 - 0 , a b s o r b a n c e at 54 ° m # .

bound to homopolymers is the same as that of the drug when bound to natural nucleic acids, binding curves can be calculated ~. Some justification for this assumption is provided by the close similarity of the difference spectra for homopolymers and DNA in Fig. 3. The resulting curves for binding to poly-A, poly-I and poly-U are shown in Fig. 5. Clearly the interaction between ethidium and homopolymers follows

0.10 0.08

I

I

I

Polly i ~>--)~

-

/

0.06-

/~oly

r

0.040.02

A

/~Po~y

-

U

I

0

0

I 1

L

I'a~ 2

Free drug ¢oncrt(Mx

-

1

105)

Fig. 5. B i n d i n g of e t h i d i u m to h o m o p o l y m e r s , c a l c u l a t e d f r o m 46o-m/~ d a t a in Fig. 4 a n d corres p o n d i n g m e a s u r e m e n t s a t 47 ° a n d 480 mkt.

Biochim. Biophys. ,4cta, 114 (1966) 234 244

241

BINDING OF ETHIDIUM TO NUCLEIC ACIDS

a pattern very different from that observed with nucleic acids. Instead of rising rapidly to r ---- 0.2 the binding curves show very little interaction at low drug concentrations, revealing that the strong primary interaction seen with nucleic acids does not occur with homopolymers. The increasing slope of the curves is characteristic of facilitated binding processes in which the presence of bound drug molecules leads to an increased affinity for further binding. In all probability this process represents secondary binding of ethidium seen in isolation instead of superimposed upon the stronger primary binding. While the calculations leading to Fig. 5 were based on assumed values for the absorbance of bound ethidium the results would not be greatly influenced by errors in the assumed values unless the absorbance of the bound drug varies widely as the level of binding changes. Only small variations in the isosbestic point are apparent in the difference spectra of Fig. 3.

Interaction with homopolymer mixtures The intermolecular complexes formed between pairs of homopolymers provide excellent model systems for investigating the effect of secondary structure on the binding of ethidium to polynucleotides. With the four readily available homopolymers there are six possible combinations in pairs, but of these only three mixtures form base-paired helical complexes: poly-(A+U), poly-(I+C) and p o l y - ( A + I ) 12. Fig. 6 shows the effect of homopolymer mixtures on the absorbance of ethidium at 460 and 540 m~. When compared with the spectral changes produced by the separate 0.6

I 1 Poly (I + C)

I I Poly (A + U)

I I Poly ( A + I)

0.3 ~

c

0

_~ 0.~ <

I

I

1

] I Poly A+ poly C

I I Poly I +poly U

o

I

l

]

I

0.3

f 1

5

1

10

15

0

I

5

I

10

15

I

0

1

5

1

10

15

Polynucleotlde concn. (M x 104) Fig. 6. A b s o r b a n c e of e t h i d i u m b r o m i d e in t h e presence of h o m o p o l y m e r m i x t u r e s . T h e d r u g c o n c e n t r a t i o n w a s 25/zM a n d a b s o r b a n c e w a s m e a s u r e d u s i n g a 4 - c m l i g h t p a t h . C o n c e n t r a t i o n s of h o m o p o l y m e r m i x t u r e s are e x p r e s s e d in t e r m s of m o l a r i t y w i t h r e s p e c t to t o t a l n u c l e o t i d e p h o s p h o r u s . ( D - Q , a b s o r b a n c e at 460 m/z; 0 - 0 , a b s o r b a n c e at 54 ° m/~.

Biochim. Biophys. Acta, 114 (1966) 234-244

242

M.J. WARING

homopolymers (Fig. 4) it is clear that mixtures which interact to form helical structures also show a markedly enhanced capacity to shift the spectrum of ethidium to longer wavelengths. On the other hand the three homopolymer mixtures which do not form intermolecular base-paired structures produce spectral changes which are simply intermediate between those given b y the separate components. Moreover, the absorbance of the drug at 46o, 47o and 48o met in the presence of p o l y - ( A + U ) , p o l y - ( I + C ) and p o l y - ( A + I ) attains constant values on the basis of which binding curves can be calculated (Fig. 7a). These curves are of the same type as those given I

12 (a)

1

I

I

(b)

Po,y (A +

3.2

Poly ( A + U',

8 p/c

x 10- 4

I

l 1

I

I 2

0

0. I

0.2

~:ree drug concn.(M x 105 )

Fig. 7- Binding of ethidium to homopolymer mixtures, calculated from 46o-m/~ data in Fig. 6 and corresponding measurements at 47 ° and 48o m#.

b y natural nucleic acids, showing the strong primary interaction at low concentrations of free drug. It is evident that there are quantitative differences between the binding to the different homopolymer complexes. Binding to p o l y - ( A + U ) up to r = o.125 appears to be so strong that free ethidium could not be detected, but the binding to p o l y - ( I + C ) and p o l y - ( A + I ) is weaker and gives rise to curves very similar to those observed with DNA and RNA although indicative of less powerful interaction. These differences are reflected in plots of r/c versus r (Fig. 7b); as found for the interaction with DNA and RNA (ref. 5) the plots are not linear but decrease in slope as the level of binding rises. Straight lines fitted to the points yielded the approximate binding parameters n ---- o.21 and K = o.4/~M for p o l y - ( A + U ) , n ~ o.17 and K 6/,M for p o l y - ( I + C ) , and n = 0.20 and K = 4/~M for p o l y - ( A + I ) .

DISCUSSION

The most striking feature of the present results is the clear-cut correlation between the ability of synthetic polynucleotides to exhibit strong primary interaction with ethidium and the possession of base-paired helical structure. While the Biochim. Biophys. Acta, 114 (1966) 234-244

BINDING OF ETHIDIUM TO NUCLEIC ACIDS

243

experiments with apurinic and apyrimidinic DNA showed an apparent requirement for both types of base the results could in principle have been explained in terms of three alternative hypotheses, namely that primary binding of ethidium requires (a) the presence of purines and pyrimidines without necessary interaction between them giving rise to secondary structure, or (b) the presence of both types of base in hydrogenbonded contact, or (c) an organised form of secondary structure stabilised by hydrogen-bonding between bases which need not necessarily be purine-pyrimidine contacts. Hypothesis (a) can be eliminated since mixtures of poly-A plus poly-C and of poly-I plus poly-U did not interact with the drug any differently from the separate homopolymers. A distinction between (b) and (c) is provided by the strong binding found with poly-(A + I ) , showing that helical structures stabilised by purinepurine hydrogen bonding are also able to support primary interaction with ethidium. The suggestion has been made that ethidium binds to DNA by intercalation 15 of drug molecules between adjacent base-pairs of the double helix 5. That conclusion, which was based on the similarity between the binding of ethidium 5 and that of proflavinel~, 16, is strongly supported by the present results since a prerequisite for intercalation must be that the nucleic acid have a base-paired secondary structure. X-ray diffraction studies on the ethidium-DNA complex have been shown to agree with the proposal of binding by intercalation and a molecular model for the interaction has been described 17. It is, however, of interest to consider what special features of nucleic acid secondary structure permit primary binding (i.e. intercalation) of ethidium. A significant finding is that the interaction between the drug and poly-I shows no sign of strong primary binding, yet poly-I is believed to have an ordered triple helical conformation at neutral pH (ref. 12). It would seem therefore that certain forms of secondary structure such as three-stranded structures do not allow binding by intercalation. LERMANis reached a similar conclusion in a study of the binding of acridines to mixtures of poly-A and poly-U; he showed that the acridines stabilised the double-helical poly-(A + U ) complex with respect to the three-stranded poly-(A +U2) combination. It may well be that in the present experiments ethidium was similarly stabilising the two-stranded poly-(A + U ) structure. On the other hand, primary binding of ethidium is not restricted to interaction with double-helical molecules stabilised by AT and GC base-pairing since the drug binds strongly to poly( A + I ) . Furthermore, primary interaction occurs with RNA and denatured DNA (ref. 5) which do not have highly ordered secondary structure. As pointed out earlier 5, the helical regions which these materials are believed to contain may account for strong binding of ethidium up to r ~- 0.2-0.25 (the limit for primary interaction) but the fact remains that long stretches of helical structure cannot be absolute requirements for binding. In this connexion it may be noted that RNA "core" is known to have at most an extremely small degree of secondary structure 19, to which its very weak interaction with ethidium may be ascribed. These considerations show that there is some specificity involved in the interaction of ethidium with molecules possessing different helical structures. The basis for this specificity is not yet clear; perhaps the requirement is simply for twostranded structures rather than three-stranded forms but there may be additional requirements such as opposite polarity of the complementary strands. It is also possible that at low levels of drug binding there may exist preferences for sites adjacent to AT or GC base-pairs as suggested for acriflavine 2°. Although ethidium is Biochim. Biophys. Acta, 114 (1966) 234-244

244

M.J.

WARING

more strongly bound by poly-(A+U) than by poly-(I+C) these results cannot be taken as definitive evidence that sites adiacent to AT pairs are preferred because of the uncertainty about the extent of analogy between IC and GC base-pairing. Another important question concerns the reason for lack of primary interaction above binding ratios of 0.2-0.25. None of the results at present available provide a clear explanation for this effect. A final comment may be made with reference to the metachromatic effects of nucleic acid derivatives and homopolymers on the spectrum of ethidium. The curves for binding of the drug to homopolymers clearly represent some kind of facilitated binding process, very probably the "stacking" interaction seen with acridine orange ~1, and it is likely that the metachromatic effects of nucleotides, apurinic DNA and apyrimidinic DNA also represent interactions of this type. Thus while results with these materials throw light on the secondary binding of ethidium to nucleic acids they add little to an understanding of the stronger primary binding. The distinction between the two modes of interaction is complicated by the apparent similarity between the spectra of ethidium bound by the primary (intercalation) and secondary (stacking) processes.

ACKNOWLEDGEMENTS

I am indebted to Dr. K. McQuILLEN for much valuable discussion and to the staff of the Carnegie Institution of Washington, Department of Terrestrial Magnetism, for assistance in the preparation of the manuscript. This work was supported by a Scholarship from the Medical Research Council. REFERENCES I B. A. NEWTON, in R. M. HOCHSTER AND J. H. QUASTEL, Metabolic Inhibitors, Vol. 2, Academic Press, New Y o r k - L o n d o n , 1963, p. 285. 2 R. TOMCHICK AND H. G. MANDEL, J. Gen. Microbiol., 36 (1964) 225. 3 W. H. ELLIOTT, Biochem. J., 86 (1963) 562. 4 M. J. WARING, Biochim. Biophys. Acta, 87 (1964) 3585 M. J. SVARING, J. Mol. Biol., 13 (1965) 269. 6 M. J. WARING, Mol. Pharmaeol., i (1965) i. 7 R. J. HILMOE, dr. Biol. Chem., 235 (196o) 2117. 8 J. J. ARMSTRONG, J. BADDILEY AND J. G, BUCHANAN, Biochem. J., 76 (196o) 61o. 9 S. G. LALAND, Acta Chem. Scan&, 8 (1954) 449. I O G . R. WYATT, Biochem. J., 48 (1951) 584 . I I V. HABERMANN, Collection Czech. Chem. Commun., 26 (1961) 3147 . 12 R, F. STEINER AND R. F. BEERS, Polynucleotides, Elsevier, A m s t e r d a m , 1961, p. 186. 13 A. RICH, Nature 181 (1958 ) 521. 14 C. H. FISKE AND Y. SUBBAROW J. Biol. Chem., 66 (1925) 375. 15 L. S. LERMAN, J. Mol. Biol., 3 (1961) 18. 16 A. ]),. PEACOCKE AND J. N. H. SKERRETT, Trans. Faraday Soc., 52 (1956) 261. 17 W. FULLER AND M. J. WARING, Ber. Bunsenges. Physik. Chem., 68 (1964) 805. 18 L. S. LERMAN, J. Cellular Comp. Physiol. Suppl., 64, i (1964) i. 19 P. DOTY, H. BOEDTKER, J. R. FRESCO, R. HASELKORN AND M, LITT, Proc. Natl. Acad. Sci. U.S., 45 (1959) 482. 20 R. K. TUBBS, W. E. DITMARS, Jr. AND Q. VAN WINKLE, J. Mol. Biol., 9 (1964) 545. 21 A. L. STONE AND D. F. BRADLEY, J. Am. Chem. Soc., 83 (1961) 3627 . 22 J.-B. LE PECQ, P. YOT AND C. PAOLETTI, Compt. Rend., 259 (1964) 1786.

Biochim. Biophys. Acta, 114 (1966) 234-244