THE INTERACTION OF CHLOROQUINE WITH NUCLEIC ACIDS AND ... fileTHE INTERACTION OF CHLOROQUINE WITH...

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THE INTERACTION OF CHLOROQUINE WITH NUCLEIC ACIDS AND NUCLEOPROTEINS* BY FRANK S. PARKERt AND J. LOGAN IRVIN1 (From the Department of Physiological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland) (Received for publication, July 11, 1952) The interaction of chloroquine’ with the ribonucleic acid of yeast was reported in a preliminary paper (1). The present paper is concerned with a study of the binding of chloroquine by the nucleoprotein and desoxy- pentose nucleic acid of spleen and with the presentation of additional data for the interaction of chloroquine with yeast ribonucleic acid. Interactions of this type may be important in the antimalarial activity of chloroquine and in the distribution of this compound in the tissues and fluids of patients receiving this drug. Such interactions also provide information of value in the study of nucleic acids. EXPERIMENTAL The ionization exponents of chloroquine have been reported previously (2). The interactions with nurleate anions were studied in the range of pH 5.7 to 6.0, in which chloroquine exists solely as the species +HB-B’H+; H represents the ring nitrogen and 13’ the diethylamino nitrogen of the side chain. Hereafter this species of chloroquine will be designated as the ligand and will be symbolized as L. Additional symbols used in the equa- tions are defined as follows: 01 = the maximum number of ligand molecules which can combine reversibly with 1 molecule of t,he polyvalent nucleate anion V = the average number of ligand molecules bound per polyvalent nucleate anion under some stated experimental condition P = the number of atoms of phosphorus in each polyvalent nucleate anion ‘I’* = the total molar concentration of the nucleate 7 = the total concentration of the nucleate when the latter is aDnsidered to be a mixture of “monovalent” nucleate anions. The monovalent nucleate * Aided by grants from the Penrose Fund of the American Philosophical Society and from the Permanent Science Fund of the American Academy of Arts and Sciences. t Submitted by Frank S. Parker to the Board of University Studies of The Johns Hopkins University in partial fulfilment. of t.he requirements for the degree of Doctor of Philosophy, 1950. Present address, Department of Biology, Bryn Mawr College, Bryn Mawr, Pennsylvania. $ Present address, Department of Biological Chemistry and Nutrition, School of Medicine, University of North Carolina, Chapel Hill, North Carolina. 1 Chloroquine is 7-chloro-4-(1’-methyl-4’-diethylaminobutylamino)quinoline. 897 by guest on December 30, 2019 http://www.jbc.org/ Downloaded from

Transcript of THE INTERACTION OF CHLOROQUINE WITH NUCLEIC ACIDS AND ... fileTHE INTERACTION OF CHLOROQUINE WITH...

Page 1: THE INTERACTION OF CHLOROQUINE WITH NUCLEIC ACIDS AND ... fileTHE INTERACTION OF CHLOROQUINE WITH NUCLEIC ACIDS AND NUCLEOPROTEINS* BY FRANK S. PARKERt AND J. LOGAN IRVIN1 (From the

THE INTERACTION OF CHLOROQUINE WITH NUCLEIC ACIDS AND NUCLEOPROTEINS*

BY FRANK S. PARKERt AND J. LOGAN IRVIN1

(From the Department of Physiological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland)

(Received for publication, July 11, 1952)

The interaction of chloroquine’ with the ribonucleic acid of yeast was reported in a preliminary paper (1). The present paper is concerned with a study of the binding of chloroquine by the nucleoprotein and desoxy- pentose nucleic acid of spleen and with the presentation of additional data for the interaction of chloroquine with yeast ribonucleic acid. Interactions of this type may be important in the antimalarial activity of chloroquine and in the distribution of this compound in the tissues and fluids of patients receiving this drug. Such interactions also provide information of value in the study of nucleic acids.

EXPERIMENTAL

The ionization exponents of chloroquine have been reported previously (2). The interactions with nurleate anions were studied in the range of pH 5.7 to 6.0, in which chloroquine exists solely as the species +HB-B’H+; H represents the ring nitrogen and 13’ the diethylamino nitrogen of the side chain. Hereafter this species of chloroquine will be designated as the ligand and will be symbolized as L. Additional symbols used in the equa- tions are defined as follows:

01 = the maximum number of ligand molecules which can combine reversibly with 1 molecule of t,he polyvalent nucleate anion

V = the average number of ligand molecules bound per polyvalent nucleate anion under some stated experimental condition

P = the number of atoms of phosphorus in each polyvalent nucleate anion ‘I’* = the total molar concentration of the nucleate 7 = the total concentration of the nucleate when the latter is aDnsidered to be a

mixture of “monovalent” nucleate anions. The monovalent nucleate

* Aided by grants from the Penrose Fund of the American Philosophical Society and from the Permanent Science Fund of the American Academy of Arts and Sciences.

t Submitted by Frank S. Parker to the Board of University Studies of The Johns Hopkins University in partial fulfilment. of t.he requirements for the degree of Doctor of Philosophy, 1950. Present address, Department of Biology, Bryn Mawr College, Bryn Mawr, Pennsylvania.

$ Present address, Department of Biological Chemistry and Nutrition, School of Medicine, University of North Carolina, Chapel Hill, North Carolina.

1 Chloroquine is 7-chloro-4-(1’-methyl-4’-diethylaminobutylamino)quinoline. 897

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898 CHLOROQUINE AND NUCLEIC ACIDS

unit is defined ae that unit of the polyvalent nucleate anion which can combine with 1 molecule of the ligand under conditions of maximum binding. T = mT*

P = gm.-atoms of nucleate phosphorus per liter of solution. P = pT* [IV] = the “molar” concentration of free nucleate in terms of the monovalent unit [NL] = the molar concentration of the ligand-nucleate complex in which the nucleate

is the monovalent unit; this also corresponds to the molar concentration of bound ligand

[Ll = the molar concentration of the free ligand S = the molar concentration of the total ligand. S = [L] + [NL]

Klotz (3) and Scatchard (4) have derived equations for the multiple interaction of small molecules with a large molecule, which with modifica- tion can be applied in the present case. At constant temperature and ionic strength and in the absence of a change in the electrostatic factor involved in the progressive binding of ligand molecules to the nucleate, the interac- tion corresponding to Process A can be evaluated in terms of an apparent intrinsic association constant, k’, as expressed in Equation 1.

N+Li=NL (A)

[NLI k’ s - P

li WI [Ll (m - 6) WI

(1)

Scatchard (4) has pointed out the advantages of casting this equation in the form

When the electrostatic factor remains constant, a plot of o/[L] against B should yield a straight line; the intercept on the tr axis is m and the intercept on the til[L] axis is k’m. However, application of this equation would require knowledge of the molecular weight of the nucleic acid. In the present case the molecular weights of the nucleic acids are not known with certainty, but k’ can be evaluated by means of Equation 5, which is based upon Equations 1, 3, and 4.

(N] = T - [NL] (3)

mP T--

P

[NL1 a~ k’ - mP - k’[NL]

[Ll P

(4)

(6)

When varying amounts of ligand are added to a constant concentration of total nucleate (constant value of P), a plot of [iVL]/[L] against [NL] should

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F. 8. PARKER AND J. L. IRVIN 899

give a straight line in the absence of a change in the electrostatic factor. The intercept on the [NL]/[L] axis is k’mP/p, and the intercept on the [NL] axis is mP/p. Ideally, this procedure permits the calculation of k’ and of m/p since the value of P can be determined accurately by analysis of the nucleic acid phosphate. The values of [NL] and [L] can be deter- mined spectrophotometrically as described below (Equations 7 and 8).

I I I I I I

315 325 335 345 35! 5 FIG. 1. Spectrophotometric absorption curves for chloroquine at pH 5.8. Curve

1, nucleates absent; Curve 2, in the presence of excess ribonucleate of yeast (415 y of nucleate phosphorus per ml.) with I’/2 = 0.1; Curve 3, in the presence of excess desoxypentosenucleate of beef spleen (44 y of nucleate phosphorus per ml.) with r/2 = 0.02.

The interaction of chloroquine with nucleates at constant pH results in a shift in the absorption spectrum of chloroquine (Fig. 1). At some selected wave-length at which the absorption by the species L and NL of chloro- quine differs considerably and at which spectrophotometric absorption bJ the nucleate is negligible, the optical density, D, of a solution containing both free and bound ligand is given by Equation 6

D = e&T,]1 + e2[NL]1 (6)

in which I? is the length of the optical path through the solution and cl and

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900 CHLOROQUINE AND NUCLEIC ACIDS

t2 are the molar absorption coefficients of the species L and NL, respec- tively. From Equation 6 and the definition of S, the following equations can be obtained.

ad?1 - D

(7)

(8)

After the evaluation of k’ and m/p by the above method, which will be designated as Procedure 1, it was advantageous to verify the determination of k’ by a slightly different set of measurements, which will be designated as Procedure 2. In Procedure 2, S was maintained constant, while the concentration of total nucleate was varied and the optical density was de- termined at a fixed wave-length and fixed length of optical path through the solution. Equations 9 and 10, which are applicable to this procedure, can be derived from the preceding equations.

D - D, - log IN = log k’ + log D--D I

(10)

In these equations, Dl is the optical density of the solution of chloroquine in the absence of nucleate when the ligand is present solely as the species L, and D2 is the optical density when nucleate is present in such excess that the chloroquine is present solely as the bound species NL. When the electrostatic factor concerned with the interaction is constant, a symmet- rical sigmoid curve is obtained when -log [N] is plotted versus D. For purposes of showing the fraction of the total chloroquine which is in the free condition at various concentrations of nucleate, it is convenient to define a degree of dissociation, (Y = [L]/S. When expressed in terms of the degree of dissociation, Equation 9 becomes

- log [Nj = log k’ + log f- (11) a

The yeast ribonucleic acid used in this study was a preparation obtained from the Schwarz Laboratories, which was purified by adaptations of the procedures of Fletcher el al. (5) and Vischer and Chargaff (6). Prior to the final precipitation with ethyl alcohol, the nucleic acid was dialyzed against several changes of distilled water to remove inorganic salts and

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F. s. PARKER AND J. L. IRVIN 901

nucleotides of low molecular weight. Microanalysis of a sample dried at 110” gave N (Dumas) 16.1 per cent, P (Pregl-Lieb) 8.8 per cent. Relative viscosities were determined for a series of concentrations of the sodium salt of this ribonucleate in 1.0 M sodium chloride as solvent to minimize electro-viscous effects. The intrinsic viscosity (lim,,,, Q,/c) was found to be 5.0, and from this value the axial ratio was calculated to be 6.6 by ap- plication of the equation of Kuhn (7, 8),

a 2 lim ‘?! = 2.3 + 2~ b c-0 c 0

(12)

in which q and q. are the relative viscosities of solution and solvent, re- spectively, 7sp is the specific viscosity, c is the volume fraction occupied by the solute (the nucleate), and a and b are the lengths of the long and short axes of the nucleate. The value for the axial ratio of this prepara- tion of yeast ribonucleic acid, when compared with data for yeast ribonu- cleic acid isolated by special methods (9), indicates that the material is somewhat degraded. However, for our present purpose this specimen of nucleic acid is u&eful, inasmuch as it permits a comparison between the strengths of interaction of chloroquine with this nucleate of low molecular weight and with a desoxypentose nucleate of high molecular weight.

Desoxypentose nucleoprotein was isolated from beef spleen essentially as described by Petermann and Lamb (10). The best one of three prepara- tions contained 12.9 per cent nitrogen and 3.56 per cent phosphorus (N:P ratio by weight, 3.62). These analyses were based upon dry weight ob- tained by lyophilization of a small sample to constant weight, a procedure which probably does not remove all of the bound water. The interaction with chloroquine was studied with solutions of the nucleoprotein which had not been subjected to lyophilization. The specific viscosity of solu- tions of the nucleoprotein in 1.0 M sodium chloride containing 0.01 M so-

dium citrate was measured with a modified Ostwald viscosimeter at 25”. Extrapolation of the data of Fig. 2 yields a value of 1300 for the intrinsic viscosity and a value of 144 for the axial ratio by Equat,ion 12. These values from measurements with a relatively crude viscosimeter probably are subject to more than the usual objections, but they indicate the order of magnitude. Dialysis of solutions of the nucleoprotein in 1.0 M sodium chloride in cellophane bags against repeated changes of 0.001 M sodium citrate solution first caused precipitation of the nucleoprotein, which was followed by re-solution of the precipitate when the ionic strength of the solution became small. Such dialyzed aqueous solutions of the nucleopro- t,ein were used for the study of the interaction with chloroquine at low

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902 CHLOROQUINE AND NUCLEIC ACIDS

ionic strength. A salt-free aqueous solution of the best preparation of the nucleoprotein gave a relative viscosity of 8.6 at a concentration of 0.193 mg. of nucleoprotein phosphorus per ml.

Desoxypentose nucleic acid was isolated from the desoxypentose nucleo- protein of beef spleen by removal of the protein by the method of Sevag et al. (11, 12). The protein-free aqueous solution of the sodium desoxy-

FIG. 2. Viscosity of solutions of desoxypentose nucleoprotein of beef spleen in 1.0 M sodium chloride containing 0.01 M sodium citrate; c is the volume fraction of the nucleoprotein in the solution.

pentose nucleate was dialyzed repeatedly against distilled water before use in the study of the interaction with chloroquine. A portion of the sodium desoxypentose nucleate was isolated in the dry state by lyophilization, repeated washing with ethyl alcohol, and drying at 70”. Analysis of the dry preparation gave P 8.8 per cent, N 15.1 per cent. The ultraviolet absorption spectrum is presented in Fig. 3. An axial ratio of 230 was cal- culated from the viscosity data of Fig. 4.

Spectrophotometry was performed with a Beckman photoelectric quartz spectrophotometer, model DU, the cuvette compartment of which was equipped with plates through which water was circulated from a thermo- stat to maintain the solutions at 30” f 0.5”. Blank solutions were identi-

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F. S. PARKER AND J. L. IRVIN 903

cal with the corresponding test solutions except for the omission of chloro- quine. The absorption data in Figs. 1 and 2 are reported as molar absorp- tion coefficients, 8, which are defined by the equation - log T = D - ccl, in which T is the transmittancy, D is the optical density, c is the concen-

210 230 250 270 290 310 FIQ. 3 Fro. 4

FIG. 3. Absorption spectrum of sodium desoxypentose nucleate of beef spleen at pH 6. e(P) s 30.98 D/cl as defined by Chargaff and Zamenhof (16) ; c is the concen- tration of nucleate expressed as gm. of nucleate phosphorus per liter of solution and 30.98 is the atomic weight of phosphorus.

FIG. 4. Viscosity of solutions of sodium desoxypentose nucleate of beef spleen in 1.0 M sodium chloride containing 0.01 M sodium citrate; c is the volume fraction of the nucleate in the solution.

tration of chloroquine in moles per liter, and I is the length (cm.) of the light path through the solution. The pH of each solution was determined at 30” with a glass electrode and electronic potentiometer, the design and standardization of which have been described previously (I 3).

DISCUSSION

The change in the absorption spectrum of the species +HB-B’H+ of chlo- roquine, which results from the binding of this species to nucleates, is de-

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904 CHLOROQUINE .4ND NUCLEIC ACIDS

pendent in part upon the molecular size of the nucleate. Thus, the shift in absorption spectrum is greater when chloroquine is bound to the highly polymerized desoxypentose nucleate of spleen than when it is bound to the partially degraded ribonucleate of yeast (Fig. 1). The absorption spectra of the species +HB-B’H+ bound to the desoxypentose nucleoprotein and to the corresponding desoxypentose nucleate are identical. In all of these cases the change in absorption spectrum involves a small shift to higher wave-lengths and a large decrease in the absorption coefficients.

Data for the interaction of chloroquine with yeast ribonucleate have been presented in a previous publication (1) in terms of Procedure 2. Addi- tional data are given in Fig. 5 in terms of Procedure 1. The data could

3.2 -5

[NL] x IO’

3*o0 2 I 4 I 6 I 8 I IO FIG. 5. Data for the interaction of chloroquine with yeast ribonucleate

of Procedure 1; PH 5.8; r/2 = 0.1; t,emperature, 30”; P = 1.0 X 1C4 M. in terms

not be extended beyond [NL] = 8.2 X lO-‘j (at this point [NL]/[N] = 0.196) because of precipitation of the complex and because the concentra- tion of chloroquine required for greater binding would have been too large for spectrophotometric observation. At the highest concentrations of chlo- roquine, 1 mm. cuvettes were used. The intercept on the [NL]/[L] axis could be determined with precision, but the intercept on the [NL] axis

could not be calculated accurately, inasmuch as a long extrapolation was involved. However, the value m/p = 0.5, which was estimated from the latter intercept, is in agreement with analytical data on the composition of a chloroquine-ribonucleate complex which was precipitated under condi- tions (high concentration of chloroquine) that would result in maximum binding of chloroquine. With this value of m/p, k’ = 795 and log k’ = 2.90 at pH 5.8, 30”, and ionic strength 0.1. Electrostatic corrections did not seem to be required in treating the data of Fig. 5. It is possible that

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F. S. PARKER AND J. L. IRVIN 905

the binding of chloroquine ions to the nucleate is accompanied by release of some of the bound sodium ions; this would tend to minimize the change in the electrostatic factor which would be expected from successive binding of chloroquine ions. It should be emphasized that we have interpreted

08 -

0.7 -

0.6 - a

0.5 -

0.4 -

0.3-

0.2 -

o.fd++WLk.o FIG. 6. Data for the interaction of chloroquine with the desoxypentose nucleo-

protein of beef spleen in terms of Procedure 2; pH 5.8; temperature, 30”. 0, experi- mental values for r/2 = 0.02; l , for r/2 = 0.07. Curve 1, theoretical curve for Equation 11 with m/p = 0.5 and log k’ = 3.73; Curve 2, theoretical curve for Equa- tion 11 with m/p = 0.5 and log k’ = 3.18.

the data in the simplest possible manner in terms of a single apparent association constant. It is possible, of course, that there are several types of binding sites on the nucleate and that these sites have different intrinsic binding strengths, but this point cannot be decided with present techniques and data. In particular, it might be expected that at higher pH values terminal phosphate groups would possess double charges which might be expected to produce tighter binding of chloroquine at these sites. How-

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906 CHLOROQUINE AND ;“iUCLEIC ACIDS

ever, at the pH values at which we have studied the interaction, this second ionization of the terminal phosphate groups would not be very important. We have not studied the interactions above pH 6, inasmuch as the proton dissociations of chloroquine complicate the direct spectrophotometric method in that range.

0.9 -

0.0 -

0.7 -

0.6 -

0.5 -

0.4 -

0.3 -

0.2 -

0.1 -

0.0 I I 5.5

FIQ. 7. Data for the interaction of chloroquine with the desoxypentose nucleate of beef spleen in terms of Procedure 2; pH 5.85; temperature, 30”; r/2 = 0.02. The points are experimental values. The curve is drawn according to Equation 11 with m/p = 0.5 and log k’ = 3.77.

Data for the interaction of chloroquine with the highly polymerized desoxypentose nucleoprotein and desoxypentose nucleate of beef spleen are presented in Figs. 6 and 7 in terms of Procedure 2, and the apparent in- trinsic association exponents are compiled in Table I. Data for the inter- action of chloroquine with the nucleoprotein had to be confined to a narrow range of concentrations because of the limited solubility of the nucleopro- tein, particularly at ionic strength 0.07. It is of considerable interest that

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F. S. PARKER AND J. L. IRVIN 907

the binding of chloroquine to the nucleoprotein is almost as strong as the binding to the nucleate derived from the nucleoprotein. This result was unexpected, inasmuch as competition between the protein and chloroquine for attachment to the polyvalent nucleate anion was anticipated in view of the generally accepted idea that the bond between the protein and the nucleate is principally an electrostatic one. The results of these experi- ments on the binding of chloroquine suggest that the bond between the nucleate and the protein may involve some other type of linkage, at least in part. It is of course possible that our preparation of nucleoprotein may have been somewhat denatured. The procedure for the isolation involved extraction with 1.0 M sodium chloride, and Gajdusek (14) has presented

TABLE I

Apparent Association Exponents for Interaction of Chloroquine with Various Nucleates and Nudeoproteins

Temperature, 30”; pH 5.86.

Log k’

P/2 - 0.02

r/2 = 1.10

-- -- __- Ribonucleate of yeast. 2.90 Desoxypentose nucleate of beef spleen

Preparation I. “

1.36

3.77 3.74 II. . .

Desoxypentose nucleoprotein of beef spleen Preparation I.

I‘ II.................................... 3.73 3.48

3.25

3.18

evidence recently that this method may produce some dissociation of the nucleoprotein. We intend to study the interaction of chloroquine with nucleoprotein isolated by methods which avoid the use of 1.0 M sodium chloride.

The data of Table I demonstrate that chloroquine interacts more strongly with the highly polymerized desoxypentose nucleate of spleen than with the partially degraded ribonucleate of yeast. That this effect is largely due to the difference in electrical charge which accompanies the difference in the size of these polyvalent anions is indicated by the fact that partial degrada- tion of the desoxypentose nucleate with alkali causes a decrease in the strength of binding of chloroquine. A similar effect has been described (1) in the case of ribonucleate. This relationship between the size and charge of nucleates and the strength of binding of chloroquine suggests that such binding studies might be useful in comparing the sizes and electrical charges

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908 CHLOROQUINE AND NUCLEIC ACIDS

of various naturally occurring nucleic acids. Both the effect of the charge of the nucleates and the large effect of ionic strength (r/2) on the inter- action of chloroquine with these polyvalent anions (Table I) can be inter- preted in terms of the interionic attraction theory of Debye and Hiickel (15). However, a quantitative application of this theory to the present problem does not appear feasible at this time, since we do not have ade- quate information concerning the molecular sizes and shapes of these nu- cleates. It seems probable that supplementary bonds such as hydrogen bonds and van der Waals’ forces are involved in the interaction of chloro- quine, and of certain acridine derivatives, with nucleates, inasmuch as one of the acridine derivatives has been shown (1) to interact with ribonucleate more strongly than chloroquine, although both compounds were doubly charged cations under the conditions of the measurements. Evidently electrostatic bonds are not the only ones involved.

The strong interaction of chloroquine with nucleates and nucleoproteins provides a plausible explanation for the accumulation of this chemothera- peutic compound in liver, spleen, white blood cells, and parasitized red blood cells of patients treated with this antimalarial. Such interactions should be considered in any attempt to explain the chemotherapeutic mode of action of this compound.

SUMMARY

A spectrophotometric method has been described for the study of the reversible interaction of chloroquine and similar compounds with nucleic acids. Chloroquine is bound more strongly by the highly polymerized desoxypentose nucleate and nucleoprotein of beef spleen than by a some- what degraded preparation of yeast ribonucleate. The strength of the interactions decreases with increasing ionic strength. The nucleate and nucleoprotein of beef spleen bind chloroquine with similar strengths. It is suggested that such interactions may be important in the distribution of chloroquine among various tissues and in the chemotherapeutic mode of action of this 4aminoquinoline derivative.

BIBLIOGRAPHY

1. Irvin, J. L., Irvin, E. M., and Parker, F. S., Science, 110,426 (1949). 2. Irvin, J. L., and Irvin, E. M., J. Am. Chem. Sot., 69,1091 (1947). 3. Klotz, I. M., Arch. Biochem., 9. 109 (1946). 4. Scatchard, G., Ann. New York Acad. SC., 61,660 (1949). 5. Fletcher, W. E., Gulland, J. M., and Jordan, D. O., J. Chem. Sot., 30 (1944) 6. V&her, E., and Chargaff, E., J. Biol. Chem., 176, 715 (1948). 7. Kuhn, W., 2. physik. Chem., Abt. A, 161, 1, 427 (1932). 8. Kuhn, W., Kolloid-Z., 62, 269 (1933) ; 68, 2 (1934) ; 76, 258 (1936). 9. Delcambe, L., and Desreux, V., Bull. Sot. chim. Beiges, 69, 521 (1950).

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F. S. PARKER AFD J. L. IRVIN 909

10. Petermann, M. L., and Lamb, C. M., J. Biol. Chem., 176, 685 (1948). 11. Sevag, M. G., Biochem. Z., 273. 419 (1934). 12. Sevag, M. G., Smolens, J., and Lackman, D. B., J. Biol. Chem., 134, 523 (1940). 13. Irvin, J. L., and Irvin, 8. M., J. Am. Chem. Sot., 72, 2743 (1950). 14. Gajdusek, D. C., Biochim. et. biophys. acta, 6. 397 (1950). 15. Debye, P., and Hlickel, E., Phys. Z., 24, 185 (1923). 16. Chariaff, E., and Zamenhof, S., J. Biol. Chem., 173,327 (1948).

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Frank S. Parker and J. Logan IrvinACIDS AND NUCLEOPROTEINS

CHLOROQUINE WITH NUCLEIC THE INTERACTION OF

1952, 199:897-909.J. Biol. Chem. 

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