1]118 BIOCHEMISTRY: KAMRIN ET AL. PROC. N. A. S.

18 McLaren, A. D., and D. Shugar, Photochemistry of Proteins and Nucleic Acids (Oxford:Pergamon Press, 1964), pp. 174-209.

19 Griffith, J. D., and R. B. Setlow, unpublished observations.20 Wierzchowski, K. L., and D. Shugar, Photochem. Photobiol., 1, 21 (1962).21 Setlow, R. B., and W. L. Carrier, Photochem. Photobiol., 2, 49 (1963).22 Cohn, W. E., and D. G. Doherty, J. Am. Chem. Soc., 78, 2863 (1956).23 Green, M., and S. S. Cohen, J. Biol. Chem., 228, 601 (1958).24 Swenson, P. A., and R. B. Setlow, Photochem. Photobiol., 2, 419 (1963).25 Butanol-acetic acid-water, butanol-water (86: 14), and saturated ammonium sulfate-M sodium

acetate-isopropyl alcohol (80:18:2).26 Uracil dimers made in ice differ from those found in the polynucleotide in that 50% of them

are decomposed by formic acid hydrolysis. The remainder of the dimers are not destroyed byfurther hydrolysis-a result indicating that the dimer made by irradiating uracil in ice is nothomogeneous in structure.

27 Setlow, R. B., W. L. Carrier, and F. J. Bollum, Biochim. Biophys. Acta, 91, 446 (1964).28 Inman, R. B., J. Mol. Biol., 9, 624 (1964).29 Setlow, R. B., W. L. Carrier, and F. J. Bollum, Abstracts, 9th Annual Meeting of the Biophys-

ical Society, San Francisco, 1965, p. 111.30 Setlow, J. K., M. E. Boling, and F. J. Bollum, these PROCEEDINGS, in press.31 Haug, A., and W. Sauerbier, Photochem. Photobiol., in press.32 Sauerbier, W., J. Mol. Biol., 10, 551 (1964).

ANILIDES AND PHENYLUREAS: CORRELATIONBETWEEN CALCULATED PI-ELECTRON STRUCTURE

AND INHIBITION OF PHOTOSYNTHESIS*

BY MICHAEL KAMRIN, WALTER F. BERTSCH, AND R. F. WOODt

BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY

Communicated by William Arnold, March 18, 1965

In the 14 years since 3-(4-chlorophenyl)-1,1-dimethylurea (CMU) was firstsynthesized and discovered to be a potent herbicide,1 substituted chlorophenylureassuch as CMU and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) havecome intogeneral use as specific poisons of an early step in photosynthetic energy conversion.The evidence suggests that these poisons, and others in the phenylurea and anilidefamilies, block an electron transfer step near the oxygen-evolving photoreaction(system II).2, 3 However, the nature of the interaction between these poisons andthe photosynthetic system is unclear. It is thus important to determine whichproperties of these molecules are related to their biological activity.

Good,4 in 1961, investigated the poisoning potency of a series of about 100 knownand newly synthesized anilides and phenylureas, but no clear-cut correlationsbetween the structure of the molecules and their potency as inhibitors could bemade. Good suggested, in agreement with Wessels and van der Veen,3 that thepoisoning process involved a physical obstruction by the poison of some site on itsbiological substrate. However, Good reported that 2-chloroanilides were in-effective as poisons, and we now present evidence that the pi-electron structure of thepoison at the 2 position of its phenyl ring is involved in the poisoning process. Fromour calculations it appears that chemical reactivity at this site is important, and

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we suggest that covalent bond formation between the 2 position and the biologicalsubstrate is a part of the poisoning process.We present calculations on 12 electronic structures representing 67 anilides and

6 phenylureas which were investigated by Good.4 Good found that these com-pounds covered a range of about 105 in effectiveness as poisons of the ferricyanideHill-reaction. We attempted to find correlations between 5 calculated electronicproperties (described below) and potency of the molecules as poisons. We foundno correlations between potency and those electronic properties associated with themolecule as a whole. We also found no positive correlation with those propertiesassociated with any of the local sites on the molecule, except at the 2 position of thephenyl ring. At the 2 position in both the anilides and the phenylureas we foundthat two of the calculated properties (free valence and electron density) correlatedwith poisoning potency.

Description of the Huckel Molecular Orbital Method.-The present quantum-mechanical calcula-tions were carried out within the framework of molecular orbital theory, using the basic approxima-tions introduced by Huckel for his calculations on the benzene molecule.5 The Huckel molecularorbital method is a semiempirical approximation procedure for obtaining the one-electron wave-functions and energies of a complex many-electron system. Calculations of this type are notcapable of giving exact results for the electronic structure of any particular molecule, but theymay be used to establish relative values for certain electronic properties of a given series of similarmolecules.6-8 Correlations between such a set of calculated properties and the biological activitiesof a series of molecules are useful in relating chemical (electronic) properties to biological function.For conjugated systems, one assumes that pi-electrons (mobile electrons) may be considered

separately from sigma-electrons (localized electrons), and that many chemical properties of thesystem can be predicted from wave functions of the pi-electrons alone. One-electron wave func-tions of molecular systems are termed molecular orbitals. In order to solve the Schrodingerequation approximately for the energies of these molecular orbitals, one assumes that their form isthat of a linear combination of the individual atomic orbitals located on atoms making up theconjugated molecular system. The set of molecular orbitals for the pi-electrons may thus bewritten 'i' = ccjrr, where Ij is the jth molecular orbital, 0, is the rth atomic orbital, cjr isthe coefficient of the rth atomic orbital in the jth molecular orbital, in n is the number of atomicorbitals (which we take to be the number of atoms in the conjugated system).Our problem is to find appropriate values for the set of coefficients, c;,, and for the energies of the

set of molecular orbitals. This can be done by a variation method, which leads to a set of simul-taneous equations. A secular equation whose roots are the energies of the molecular orbitals canbe obtained by converting the simultaneous equations to matrix form. Each matrix element ofthe secular equation connects two atomic orbitals, all possible pairs of orbitals being representedin the complete secular equation.Huckel introduced two procedures which, when taken together, allow an approximate solution

of the secular equation for the energies of the molecular orbitals. First, the secular equation issimplified by assuming that the only nonzero matrix elements are the diagonal elements (whichconnect each atomic orbital to itself) and the immediately off-diagonal elements (which connectatomic orbitals on adjacent atoms). These elements contain so-called "overlap integrals,""Coulomb integrals," and "resonance integrals." Second, empirical values are assigned to allthese integrals, in order to solve the secular equation for energies of the molecular orbitals.

In order to gain some insight into the assumptions implied by the above procedures, one mustexamine the structure of the individual matrix elements. The general form of these elements is.fo4rHot dT - Ef ,4. dT, where H is an Hamiltonian operator, E is the energy of the molecularorbitals, and dT indicates integration over spatial coordinates. The first term of each matrixelement describes interactions between the rth and sth atomic orbitals, and (through the operator,H) all electrons and nuclei in the conjugated system. Thus, Huckel's drastic simplification ofthe secular equation does not involve complete neglect of interactions between nonadjacent partsof the conjugated system.

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1120 BIOCHEMLSTRY: KAMRIN ET AL. PROC. N. A. S.

TABLE 1 The second term of each matrix elementPARAMETERS USED IN CALCULATIONS contains an integral as a coefficient of the

Atom in energy. This overlap integral describes theBond ', bond a extent to which the rth and sth atomic

-C=(mC- 1.0 C 0.0 orbitals occupy the same region of space. The(aro )

C1.1 C 0.0 overlap integral of an atomic orbital with itself(double bond) is always chosen as one, in order to normalize

the atomic orbitals. Thus the diagonal-C-N 0.9 N 1.0 matrix elements of the secular equation may

-C=O 2.0 0 1.2 be writtenJf rHt7dr -EaEa- E, where-C-Cl 2.0 Cl 0.5 a denotes the "Coulomb integral." We follow

az- ac Huckel's assumption that the overlap in-?-v ax-

#C tegral of an atomic orbital with any otherwhere x = atom or bond of interest, atomic orbital is zero. Thus the off-diagonal

c = carbon atom or bond in benzene. matrix elements may be written fq0tHq>s drus ,3, where jB denotes the "resonance integral."

With these assumptions the simplified secular equation can thus be written in terms of the energiesand two empirically determinable parameters: a and #. We follow the customary procedure ofintroducing two new parameters, 5 and a, which allows us to choose the # of carbon (#c) as theunit of energy, and the a of carbon (ax) as the zero of energy (see Table 1).

Given a set of empirical values for a and j3, it is a straightforward matter to solve the simplifiedsecular equation and obtain energies of the molecular orbitals. Two calculated properties aredetermined directly from the energies of the molecular orbitals: energy of the highest occupiedmolecular orbital (HOMO), and energy of the lowest empty molecular orbital (LEMO). We

I0 0 0 0 0 0III U N IIHN4.R HN 0R HN-C.>R HNNCR HNNA-R HN-.' R

ANILIDES 2 6 t C C

CI CI

CONJUGATED IO m ZzSTRUCTUREI I

AVERAGE POISONING 10-2.6 -74.4 -F4.7 -C5.0 -F5.1 -5.9POTENCY 10 10 10 10 10 10

POTENCY (10 -.10ICF) (10 -7_10 5°) (10-403-152) (1O045 10-57) (10-4510755) (10-5°10-70

90 0 0 0.CH3 ;',CH CC3 .C..CH NN4N G3 ~HN *N N.NHN HNN"3 HN`N. .NH3HN&NH3H

oI CH3 HoN;CH3 HoNfCH3 H~~~CH3 CH~sr3 CHo~t3PHENYLUREAS 2

CI CI

CONJUGATED m :MSTRUCTURE m

POISONING -33 -5.2 o-6.0 o6.3 1-6.3 10-75POTENCY ~ * 1 01 01

FIG. 1.-The 12 conjugated structures on which calculations were performed and the poisoningpotency associated with each structure. R indicates an unconjugated organic group. Dottedlines separate unconjugated groups from the conjugated structure. Poisoning potencies (molarconcentration for 50% inhibition of Hill reaction) were taken from data reported by Good.4Average poisoning potency9 of the anilides represents data from a different number of moleculeswhich had each conjugated structure. The range of potencies covered by the various moleculeswhich had each conjugated structure shows that the least potent anilide conjugated structure (I)did not contain any molecules which were as potent as those with the most potent anilide con-jugated structure (VI), although there was a great deal of overlap in the ranges covered by con-jugated structures with intermediate potency (II, III, IV, V).

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VOL. 53, 1965 BIOCHEMISTRY: KAMRIN ET AL. 1121

begin with the lowest-energy orbital, and fill each of the orbitals with two electrons until all of thepi-electrons are used. The HOMO is considered to be a measure of the ionization potential of themolecule as a whole. Conversely, the LEMO is considered to be a measure of the electron affinityof the molecule.The coefficients of the atomic orbitals, c;, may be obtained by substituting the corresponding

energies into the system of simultaneous equations. From the coefficients, cr, it is possible tocalculate a third property, electron density, which can be considered to be related to the proba-bility of finding a pi-electron associated with a given atom. We define q,= 2z4 Nici,2, whereqr is the electron density of the rth atom, and Ni is the number of electrons in the jth molecularorbital: 2, 1, or 0.The set of coefficients, cr, may also be used to calculate another property, bond order, which has

been found empirically to be related to bond length, and to ability of a bond to undergo additionreactions. We define pre 2j = Njcjcj8, where pTa is bond order, and r and s refer to adjacentatomic orbitals.From bond orders we define another calculated property, free valence, which has been found

empirically to be related to the ability of an atom to undergo free radical and substitution reactions.We first define an empirically determined quantity, maximum free valence, which is considered tobe the total binding power for an unbound atom. The sum of the bond orders of all bonds attached

8

to a given atom is subtracted from this maximum free valence, Fr-Fr, max - 2; adjacent pra, wherer

F, is the free valence of the rth atom and Fr, max is the maximum free valence of the rth atom.Application of the Method to Photosynthetic Poisons.-The five calculated properties defined

above were determined for the 12 conjugated structures shown in Figure 1. The parametersemployed in these calculations were taken from Pullman6 and Streitwieser8 and are shown inTable 1. These 12 structures represent the pi-electron systems of 67 anilides and 6 phenylureaswhose poisoning potency had previously been reported by Good.4 Good measured the poisoningpotency of each conjugated structure by determining the molar concentration of poison required toinhibit the ferricyanide Hill-reaction by 50% (Fig. 1). In the case of the anilides this poisoningpotency represents an average of from 5 to 31 individual molecules, which we calculated fromGood's data.9 In the case of the phenylureas, the poisoning potency associated with each con-jugated structure was taken from Good's data for a single molecule: a substituted phenyl-1,1-dimethylurea. As a first and crude approximation we ignored in our calculations the influence ofthe nonconjugated portions ("R-group") of the molecules on the conjugated structures. Subse-quent calculations showed this approximation to be justified, since a given "R-group" had similareffects on all the conjugated structures.

Results.-The complete results of molecular orbital calculations on the 12 con-jugated structures are shown in Tables 2-5. Table 2 contains the values of theHOMO and LEMO for each of the 12 conjugated structures. Neither the anilidesnor the phenylureas showed a correlation between poisoning potency and HOMOor LEMO. Table 3 contains the calculated free valence, F., of each atom in the

TABLE 2HOMO AND LEMO

Conjugated Poisoningstructure potency HOMO LEMO

Anilides9I 10-2.6 0.0278 1.0669

II 10-4.4 -0.5870 1.0000III 10-4.7 0.0280 1.0000IV 10-5o -0.1008 1.3038V 10-5.' -0.1246 1.0590VI 10-5.9 0.4335 1.4683

PhenylureasVII 10-3.3 0.0359 1.0770VIII 10-5.2 -0.5785 1.0000IX 10-6.0 -0.0992 1.3430X 10-6.3 -0.1229 1.0666XI 10-6.3 0.0360 1.0000XII 10-7.5 0.4466 1.5249

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1122 BIOCHEMISTRY: KAMRIN ET AL. PROC. N. A. S.

000000 00 0 000000000

00 0 r 1t t 0 =IttN t 't .414r- 00 It- I-I-J . I-I--I-to--Jr-I-0000c 0000 =c~000000 000000~ t

oooSSS~ Stoomoo

0o CoXcaom~ m m ooo so,cor

0000000 000000 1 - ,4"ubOCD N 0 t-OO +C4inmo O~tn t wEO0 '

Lo k 0 t=Q<9 r~-e -c =e to+ <'t-nIn CDcoI- ~t_" di 4"-___ 4_ w I-t-t-l-tt- t-t-t- t-. .. . . . . .. .

o coooo 000000 000000 000000

0. 0-C o0 0 40 - 0Cq~~~xt-n1)-~~~1t- r-t:" m N o r- C t- N rOO£

oooSSS4AC><~ S~oooom mo-ott0 o ocoa

sz :C0 -0eD Co- 0-~ 00te9 bcb0-t'-4t-C-1t-

. . . . . . . . . . .O OO)O--O-O-~O~O 1_4___ _

CD LnO sCTS~ms ea-4coCe9-

+ +oCJeoettcJ2 Q s00S 00CoO)n5555,ooooooooo --o___ __H

CoL 3 o0C o= 0N to 0re4tO-)NcO ) t-0=08x0= o= oxtOO m t- tOF C5CT( m" mLO mco CYDt- toM re- tuM N=N~m" N c mN ca

0Clt NNk L) 00=NCo4 >4-eirR44t--t-o0Q)oowecscO

CQZ N OCTO-C50°Ot- eq 00 Ci CrOi0 tOeq VD 0_O co C14 m 11 00 co-D__a CD 0 o N O )O O (m N 4

5555550000005555 -_0_0 _00-0-14 r14

;>;

09)

cq0encoo .v o ca- cco co

.~~~~000000000000 ~~~~~~~~~~~~~~~~~~~~~ =000000m 00000

......~~~~~~~ ~ ~ ~ ~~~~~~~~~~.. .. .. .... . .. .. .

c0. -c ,-40.4; -DD CD' - r-4 r-4 ~-4 -4 '-4 -) "-4 ,-4 r--4

_4 OC g t-slC) m t- C LO o" N I" c_-Xc -400 CO CD COD C

)0 _ ccO0-e-o0 es0°0 e0oorl r'Re1o~ oco C o .4

U*~_O - SOnC< DC CZ e +

. 000 ,

.o o ° oooo oo oo o o o 0)o C°

A4 )Vbe

e

rr _ =~~~~~~~~~~~~~~~~~~~~~~~~~~~~-

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VOL. 53, 1965 BIOCHEMISTRY: KAMRIN ET AL. 1123

0 12 conjugated structures. The only correla-. tion between free valence and poisoning

potency in either the anilides or the phenyl-O - N LO co r r N _4 CD _ ureas was at the 2 position of the ring. In

o a)o t- t- t-to ° C 00°° Figure 2 free valence of the 2 position of the000 00o 000 555; anilides has been plotted as a function of

their poisoning potency in order to illustrateM 10 M co N: X = =$ a typical case which we interpret as showing0o m- 0m- t-sco> t- co t-

. .I

a correlation. Figure 3 gives an example of000000 000000

a case which we interpret as showing noMM = 0 0 1, _ , correlation.

1-4 >4xt>>¢.,XLOe-°cLo <, Table 4 contains the calculated electron000000oo o ooo o o o densities, qt, of each atom in the 12 con-

jugated structures. The only correlationo- It- It°° N W = co co between electron density and poisoning po-

.ss s s < 9 > s s se 9 tency in either the anilides or the phenylureasis at the 2 position.

XXbe< s ><> Table 5 contains the calculated bondc, MCD IL - CD CO°L°O order, pra, of each bond in the 12 conjugated.555500 0 structures. In both the anilides and the

phenylureas the only correlation is a negative4 0 to LO N 0 co co 0 one between poisoning potency and bond

SSSR+.co.$Lo CDC ,-.1° order at the 3,4 bond.

000o°°°°°°° Discussion.-Our calculations show that a

¢z u,00,., , ,,, >O positive correlation between poisoning po-00 LO N t- L>o ,1,N 0 tency and electronic structure in both the555555oo o 0 0 anilides and the phenylureas exists only at

the 2 position of the phenyl ring. The pi--c 0 " r M

° , electron structure at this site must thereforeco csooactroc_ . ..0NI-=

cos r- '` t M c M " be involved in the poisoning process. The55555° ° ° ° ° ° ° ° ° positive correlation appears in two calculated--4CoM

properties: free valence and electron den-2 No M r -4 C 4 M O r sity. The increase in free valence at the 2Lo66o55; 65 56 666 position with increasing poisoning potency

(Table 3, Fig. 2) suggests that chemical re-activity (i.e., ability to form covalent bonds)at this position determines potency. The in-

44 04 ~ ~ ~ ~^ ^~creasein electron density at the 2 positionwith increasing poisoning potency (Table 4)suggests that the moiety with which thisatom reacts is positively charged. Fromthese data taken together it seems reasonableto suppose that the importance of pi-electron

2 structure in the poisoning process is due tocovalent bond formation at the 2 positionbetween the poison and a positively charged

P-4;biological substrate.

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1124 BIOCHEMISTRY: KAMRIN ET AL. PROC. N. A. S.

'1r6- io-6

>10-5s 0j*o-5 *i

Z

o * 0a. a.

z z

5 5w w

4 4

o10-3 <04_0 0

ict2 o-v0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8

FREE VALENCE AT THE 2 POSITION FREE VALENCE AT THE 4 POSITION

FIG. 2.-orrelation between calculated free FIG. 3.-Lack of correlation between cal-valence at the 2 position of the anilides, and culated free valence at the 4 position of thetheir average poisoning potency.9 Poisoning anilides, and their average poisoning potency.9potency was the molar concentration of the Poisoning potency was the molar concentra-compound which resulted in 50% inhibition of tion of the cornpound which resulted in 50%the ferricyanide Hill-reaction. Data taken inhibition of the ferricyanide Hill-reaction.from Table 3. Data taken from Table 3.

A negative correlation is present between inhibitor potency and bond order atthe 3,4 bond (Table 5). This correlation suggests that increased ability of themolecule to undergo addition reactions across the 3,4 bond results in reducedeffectiveness as a poison.ethis r t consistent with our conclusion that the2 position is a reactive site in poisoning, since addition reactions across the 3,4bond would probably hinder any reaction at theneighbtorig 2 position and therebyreduce poisoning potency.The atoms (except number 2) and bonds in the phenyl ring exhibit definite varia-

tion which does not correlate the poisoning potency (Tables 3, 4, 5). We thereforeconclude that these atoms (1, 3, 4, 5, 6), and all bonds in the ring, are not involvedin chemical reactions which are related to poisoning. However, since the nonringatoms and bonds (positions 7, 8, 9, 10) were calculated to have about the sameproperties inl the various conjugated structures, we cannot determine whether thepoisoning process involves chemical reactions at these positions.

Good's results4 shed some light on the possible role of the nonring positions.Data on molecules with the same conjugated structure showed that polar and non-polaru tR-groups6of comparable size have about the same efect on poisoningpotency. Polar "R-groups" would affect the electronic structure of the nonringpositions, whereas nonpolar groups would have ltle effect. These data thereforesuggest that the electronic properties, i.e., chemical reactivities, of these positionsare not involved in the poisoning process. Nevertheless, it is clear from Good'sdata that the nonring positions are somehow involved in poisoning. Substitutionof a sulfur atom for the oxygen results in a decrease ranging from 50 to 5000 inpoisoning potency. Good's experiments on hydrogen-bonding in solution showthat the oxygen and the nitrogen can both form hydrogen-bonded complexes, sothat this property could explain the importance of these sites. Good's data alsoshow that poisoning potency is diminished when the unconjugated "R-group"

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VOL. 53, 1965 BIOCHEMISTRY: KAMRIN ET AL. 1125

consists of only an hydrogen atom. Since the size of this group seems more im-portant than its chemical composition, Good suggests that the "R-group" may beinvolved in a steric interaction with the biological substrate.

It is interesting to speculate about the events involved in the poisoning reaction,and about the peculiar importance of the number 2 carbon atom of the phenyl ring.Apparently the unconjugated "R-group" must be of a size and shape to fit stericallyinto some particular pocket on the surface of the unknown biological substrate.The nitrogen and the oxygen presumably become hydrogen-bonded to other specificsites on the biological surface. These events would position the phenyl ring of thepoison rather rigidly, since the pi-electron system extends into the nonring portionof the molecule. We suspect that this relatively rigid positioning of the ring resultsin a configuration in which the number 2 carbon atom is near a positively chargedreactive site on the biological substrate. The poisoning reaction would be com-pleted by covalent bond formation between the 2 position and the reactive site.There would be essentially no possibility of other atoms or bonds of the poisonmolecule becoming involved in such covalent bond formation, because of the par-ticular configuration between the poison and its biological substrate. This modelof the poisoning reaction would explain our finding that a positive correlationbetween poisoning potency and calculated electronic structure occurred only at the2 position of the phenyl ring, and is in good agreement with Good's data about thefunction of the nonring portion of the poison molecule.

* Research sponsored by the U.S. Atomic Energy Commission under contract with the UnionCarbide Corporation.

t Solid State Division, Oak Ridge National Laboratory.l Bucha, H. C., and C. W. Todd, Science, 114, 493 (1951).2 Bertsch, W. F., J. B. Davidson, and J. R. Azzi, in Photosynthetic Mechanisms of Green Plants,

NAS-NRC Pub. 1145 (1963), p. 701; Bishop, N. I., Biochim. Biophys. Acta, 27, 205 (1958);Bishop, N. I., Biochim. Biophys. Acta, 41, 323 (1961); Duysens, L. N. M., in PhotosyntheticMechanisms of Green Plants, NAS-NRC Pub. 1145 (1963), p. 1; Forti, G., and B. Parisi, Biochim.Biophys. Acta, 71, 1 (1963); Gingraf, G., C. Lemasson, and D. C. Fork, Biochim. Biophys. Acta,69, 438 (1963); Jagendorf, A. T., in The Photochemical Apparatus-Its Structure and Function,Brookhaven Symposia in Biology, No. 11 (1958), p. 236; Kok, B., in Photosynthetic Mechanisms ofGreen Plants, NAS-NRC Pub. 1145 (1963), p. 35; Kroll, A. R., N. E. Good, and B. C. Mayne,Plant Physiol., 36, 44 (1961); Rumberg, B., P. Schmidt-Mende, J. Weikard, and H. T. Witt, inPhotosynthetic Mechanisms of Green Plants, NAS-NRC Pub. 1145 (1963), p. 18; Sweetser, P. B.,C. W. Todd, and R. T. Hersh, Biochim. Biophys. Acta, 51, 509 (1961); Vernon, L. P., and W. S.Zaugg, J. Biol. Chem., 235, 2728 (1960); Weaver, E. C., and H. E. Weaver, Photochem. Photobiol.,2, 325 (1963); Witt, H. T., A. Muller, and B. Rumberg, Nature, 197, 987 (1963).

3 Wessels, J. S. C., and R. van der Veen, Biochim. Biophys. Acta, 19, 548 (1956).4Good, N. E., Plant Physiol., 36, 788 (1961).5Huckel, E., Z. Physik, 70, 204 (1931); Huckel, E., Z. Physik, 72, 310 (1931); Huckel, E.,

Z. Physik, 76, 628 (1932).6 Pullman, B., and A. Pullman, Quantum Biochemistry (New York: Interscience, 1963).7Pullman, B., ed., Electronic Aspects of Biochemistry (New York: Academic Press, 1964);

Coulson, C. A., Valence (Oxford Univ. Press, 1961), 2nd ed.8 Streitwieser, A., Jr., Molecular Orbital Theory for Organic Chemists (New York: John Wiley

and Sons, 1961).9 In arriving at an average poisoning potency for each conjugated structure of the anilides, we

averaged the exponents to the base 10 of the molar concentration reported by Good4 to inhibit theferricyanide Hill-reaction by 50%. We excluded Good's data on compounds of questionablesolubility, as well as data on those compounds in which the unconjugated "R-group" consisted of

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1126 BIOCHEMISTRY: RABINOW1TZ ET AL. PROC. N. A. S.

only an hydrogen atom. Compounds of the latter type were about 100 times less potent thanother compounds representing that structure and were not represented in every conjugatedstructure.

ISOLATION OF DEOXYRIBONUCLEIC ACID FROM MITOCHONDRIA OFCHICK EMBRYO HEART AND LIVER*

BY MURRAY RABINOWITZ,t JOHN SINCLAIRt, Louis DESALLE,ROBERT HASELKORN,t AND HEWSON H. SWIFT

DEPARTMENTS OF MEDICINE, BIOCHEMISTRY, ZOOLOGY, AND BIOPHYSICS,

AND THE ARGONNE HOSPITAL FOR CANCER RESEARCH,§ UNIVERSITY OF CHICAGO

Communicated by Dwight J. Ingle, March 11, 1965

Studies on algae, yeast, and higher plants strongly indicate that the structureand function of mitochondrial' 2 and chloroplasts3-7 may be altered by ultra-nuclear genetic changes. Cytoplasmic heritable changes can be produced byultraviolet irradiation and by dyes which interact with nucleic acids,8" 9 impli-cating cytoplasmic nucleic acid as their site of action. Cytoplasmic DNA hasbeen localized in chloroplasts and mitochondria10-'2 and measured by chemicalmeans, but contamination with nuclear DNA has always been possible. Cytolog-ical and autoradiographic studies have also supported the mitochondrial localiza-tion of DNA.13-19 The recent isolation from chloroplasts of DNA having a buoyantdensity different from that in the nucleus,20-24 and the demonstration that mito-chondrial DNA from Neurospora crassa also has a unique buoyant density25 suggestthat such organelles have an independent store of genetic information.26

This paper presents evidence that mitochondria of avian heart and liver containDNA which differs in its base composition from that of nuclear DNA. The chickembryo system was chosen for study because rapid mitochondrial multiplicationmakes it particularly suitable for subsequent investigations concerning the mecha-nism of mitochondrial nucleic acid and protein synthesis.

Materials and Methods.-Mitochondria were prepared from hearts and livers of 16-19-daychick embryos by the method of Crane et al.'7 Prior to purification by isopycnic density gradientcentrifugation, the mitochondrial pellet was suspended in 0.25 M sucrose, 0.005 M MgCl2, and in-cubated for 45 min at 250C with 200 ,ug/ml of pancreatic DNase (Worthington, recrystallized).With this treatment, more than 98% of P32-labeled T4-phage DNA added to the mitochondriawas rendered TCA-soluble. After washing the mitochondria three times with 0.25 M sucrose,0.002 M EDTA, they were suspended in a small volume and layered over a sucrose gradientcontaining 10-3M EDTA (density 1.138-1.242). The gradients were centrifuged for 2 hr at25,000 rpm in the Spinco SW25.1 or SW25.2 rotors. The narrow mitochondrial band was collectedeither by puncture of the bottom of the tube or by careful removal of successive layers from thetop of the gradient. The mitochondrial fraction was diluted with four volumes of 0.25 M sucrose,10-3 M EDTA, and centrifuged for 10 min at 20,000 g.

Isolation of DNA: DNA was isolated by modification of the method of Marmur.28 Mitochon-dria isolated from 50-100 dozen chick hearts and livers were suspended in 2.5-5.0 ml of buffer atpH 8.0 containing 1% sodium deoxycholate, 0.15 M NaCl, and 0.1 M EDTA. The mixture wasthen shaken for 20 min at room temperature with an equal volume of phenol which had beenequilibrated with the same buffer. Treatment with phenol was repeated until deproteinization wascomplete as indicated by the absence of a precipitate at the interface. The aqueous phase was

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