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The interaction of polynucleotides with metal ions, amino acids, and polyamines
BIOCHIMICA ET BIOPHYSICA ACTA
425
THE INTERACTION OF POLYNUCLEOTIDES WITH METAL IONS, AMINO ACIDS, AND POLYAMINES GARY FELSENFELD AND SYLVIA HUANG Department o/Biophysics*, University of Pittsburgh, Pa. (U.S.A .) (Received April 2ISt, 1959)
SUMMARY The formation of the two-stranded complex between the sodium salts of polyadenylic acid and polyuridylic acid requires the presence in solution of additional cations. I t is found that the effect of magnesium ion is characteristic of a strong interaction between metal ion and polynucleotide, while the effect of sodium ion is characteristic of weak interaction. I t is also found that the formation of the two-stranded structure requires neutralization of the charge on the phosphate backbone. This information makes it possible to use the extent of the poly A-poly U interaction as a measure of binding of cations to polynucleotide. The method is applied to charged amino acids and polyamines. I t is shown that the amino acids are weakly bound and the polyamines strongly bound to polynucleotides.
INTRODUCTION The interaction of the synthetic polyribonueleotides, polyadenylic acid and polyuridylic acid, with each other is dependent upon the nature and concentration of cations present in the solution 1-s. Previous studies 4 have shown that both poly A and poly U interact strongly with Mg++ and Mn++ and that about one equivalent of divalent ion is bound per mole of phosphate. In this paper we describe further studies of the ionic conditions necessary for the combination of poly A and poly U, and use this information to provide a simple measure of the strength of interaction between polynucleotides and both charged amino acids and polyamines. EXPERIMENTAL The polynucleotides were prepared enzymicaUy from nucleoside diphosphates with polynucleotide phosphorylase isolated from Azotobacter agile according to a method kindly provided b y Prof. S. OCHOA. Preparations were dialyzed in succession against ethylenediaminetetra-acetic acid, sodium chloride, and redistilled water before * Contribution No. 64 from the Department of Biophysics. Abbreviations: Polyadenylic acid = poly A; polyuridylic acid = poly U; deoxyribonucleic acid = DNA; ribonucleic acid = RNA. The materials referred to by these names a r e t h e sodium salts.
Biochim. Biophys. Acta, 37 (I96o) 425-433
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G. FELSENFELD, S. HUANG
freeze-drying. Experiments involving mixing of poly A and poly U were performed so that the pH of the final reacting mixtures was generally between 6. 7 and 7.4. Where necessary the pH of solutions was adjusted with NaOH, and the reacting mixtures of polymer were stored in dessicators over saturated NaOH solution to eliminate absorption of atmospheric CO,. These precautions were necessary to avoid the formation of the ordered poly A complexS, 6 which occurs in distilled water below pH 6.5. The upper limit in pH is necessary to avoid metal ion-hydroxyl ion interactions in certain cases. The amino acids were commercial preparations (glycine, Fisher; L-histidine, California Corporation for Biochemical Research; L-histidine monohydrochloride, Eastman; imidazole, Eastman; L-lysine monohydrochloride, Interchemical). Putrescine hydrochloride was obtained from California Corporation for Biochemical Research, and was analyzed for chloride ion by silver nitrate titration. Diaminodecane and diaminopentane (cadaverine) were obtained from Aldrich Chemical Company, and were analyzed for titratable amino groups by dissolving weighed quantities in an excess of standard hydrochloric acid and back-titrating with standard sodium hydroxide solution. The diaminopentane and diaminodecane were also analyzed for nitrogen colorimetrically with Nessler's reagent. The results agreed with the titration within 3 %. Solutions of sodium, manganese and magnesium chloride were made from analytical grade reagents. The divalent ion chlorides were standardized by titration with silver nitrate using dichlorofluorescein as an indicator. O.D. measurements were made with the Cary Model 14 recording spectrophotometer. All O.D. reported were measured at 259 m/%. In the case of the most dilute solutions, cylindrical cells with a path length of IO cm were used. In the case of the most concentrated solutions, rectangular cells of path length 0.5 cm were used, and when the O.D. exceeded 2.0, readings were made against standardized blanks of sufficiently high O.D. to bring the readings within the range of the spectrophotometer. Ultracentrifuge studies were made with the Spinco Model E, equipped with u.v. optics. RESULTS
Metal ions
The decrease in O.D. in the 26o-m~ region which accompanies the combination of poly A and poly U in solution has already been used to obtain qualitative information about the relative effectiveness of various cations in promoting the combination *. In order to relate the fractional decrease in O.D. to the fractional completion of the reaction, it is naturally necessary to establish that the entire drop in O.D. which occurs when cations are added is attributable to the reaction. Investigation of the effect of the addition of MgC1, on the O.D. of poly A alone shows that this is not the case (Fig. I). With the pH carefully adjusted above 7.0 so that the "ordered" poly A structureS, e is unstable, poly A shows a marked hypochromic effect upon addition of small amounts of divalent ion. The molar O.D. is independent of polymer concentration for a given ratio of equivalents of divalent ion added to moles of polymer phosphate, and the limiting O.D. is reached when about one equivalent of divalent ion has been added/mole of polymer phosphate. The total hyp0chromic effect is about Biochim. Biophys. Acta, 37 (196o) 425-433
POLYNUCLEOTIDES AND METAL IONS, AMINO ACIDS, POLYAMINES
427
1.09 o 7.85.10 "~ /%.4 . 0 6 • 10.-4 1.1~
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1.05
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i
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cA Fig. I. O.D. (divided by limiting O.D.) of poly A solutions at various concentrations plotted against equivalents of magnesium ion added] mole ofpolymerphosphate. Measured at 259 m/~.
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Fig. 2. O.D. (divided by limiting O.D.) of poly A solutions at two concentrations plotted against the negative logarithm of NaC1 concentration.
I6 % of the O.D. in distilled water. The limiting molar O.D., about 8840 at 259 m~, is somewhat lower than that obtained in o.I M NaC1 at p H 6.72. Poly U does not show such strong dependence of O.D. upon ionic concentration. A small decrease of about 3 % in O.D. is observed in going from distilled water to one equivalent of divalent ion. The effect of the addition of sodium chloride to poly A in distilled water is characteristically different from that obtained with divalent ions. Though there is a drop in O.D. (Fig. 2), the change occurs much more gradually as the salt concentration is increased, beginning at IO-s M NaC1, but not complete until the concentration is raised to lO -1 M, so that NaC1 is in large excess. The total drop in O.D. is only about half that observed when divalent ions are added. With the use of these corrections it is possible to interpret the O.D. decrease of a mixture of poly A and poly U in terms of formation of poly (A + U). If we assume that the O.D. contributed b y partly formed poly (A + U) is directly proportional to the number of combined sites, it is easy to show that F, the fraction of completion of combination, is given b y F
D'o - - D D'o - - DM
(X)
where D is the O.D. of the poly A + poly U solution in question. D'o is the sum of the O.D. of two solutions containing poly A and poly U separately at the concentration used in the mixed solution, and with the same amount of salt added, and DM is the limiting O.D. density of the mixture at high salt concentration. In Fig. 3 (D'o--D)]Do is plotted as a function of magnesium ion concentration for a series of x : I poly A + poly U mixtures over a hundred-fold range of polynucleotide concentration. With a knowledge* of Do, it is possible to compute F. * The molar O.D. at 259 m# in distilled water is.about 11,6oo for poly A and 97oo for poly U. Biochim. Biophys. Acta, 37 (I96o) 425-433
428
G. FELSENFELD, S. HUANG
o ~
o 7.4 •10-4
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Fig. 3. Fractional O.D. change (D'o--D)/D o for a series of I :i p o l y A - poly U m i x t u r e s as a function of Mg ++ concentration. P o l y m e r p h o s p h a t e concentrations are s h o w n a t t h e u p p e r right.
At the highest polynucleotide concentrations, the amount of magnesium ion necessary to cause poly A and poly U to interact increases as the polynucleotide concentration increases. Within about io %, the reaction requires one equivalent of divalent ion/mole of phosphate to go to completion. At the lowest concentration there appears to be a limiting curve. Similar experiments involving the addition of Mn++ rather than Mg++ give the same results. The addition of NaC1 to a I : x poly A - poly U mixture also causes combination of the two polymers, but in this case the degree of reaction for a given sodium chloride concentration is independent of polynucleotide concentration (Fig. 4). As in the case of the O.D. drop of poly A alone, about a IOO fold increase in NaCI concentration is required to go from F = o.oI to F ----- 0.99.
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Fig. 4. Fractional O.D. change (D'o--D)/D o for a series of z :I poly A - poly U m i x t u r e s as a function of t h e negative l o g a r i t h m of NaC1 concentration. P o l y m e r p h o s p h a t e concentrations are s h o w n a t t h e u p p e r right.
Amino acids and diamines Having established the behavior of two classes of ions, those which are bound strongly and those which are bound weakly (see DlSCUSSlOlq), we can use the poly A poly U interaction as a measure of cationic binding. In Fig. 5 are shown the effects upon the O.D. of a I : I poly A - poly U mixture of the addition of various amino acids, compared with the effect of NaC1 upon the Biochim. Biophys. Acta, 37 (196o) 425-433
POLYNUCLEOTIDES AND METAL IONS, AMINO ACIDS, POLYAMINES
429
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Fig. 5. Decrease in O.D. divided by maximum decrease in O.D. (uncorrected for changes in poly A alone) plotted against the negative logarithm of the molar concentration of hydrochlorides of glycine, lysine and histidine, compared with the effect of NaC1. Polymer phosphate concentration = 7.2' I o-6.
lOconc" . lOs
Fig. 6. Decrease in O.D. of poly (A + U) divided by maximum decrease in O.D. (uncorrected for changes in poly A alone) plotted against concentration of putrescine, cadaverine and diaminodecane hydrochlorides, compared with the effect of MgC12. Polymer phosphate concentration = 6.7. i o'-5.
same solution. Glycine, which has no net charge, produces essentially no 0 . D . change even in I M solution. Positively charged lysine, (lysine hydrocMoride) on the other hand, does cause an interaction to exactly the same extent as sodium ion at the same concentration. I n the case of histidine it is necessary to add a mixture of the free amino acid and the hydrochloride to keep the p H at 7 or above, since at lower p H there is danger of formation of the ordered, non-reactive poly A structure. Because the p K of histidine is 6.o5, approximately a ten-fold excess of neutral histidine must be present in solution to keep the p H at 7. Since glycine does not cause poly A and poly U to interact, we assume t h a t no other dipolar ion has the ability to do so. The concentration of positively charged histidine is c o m p u t e d from the measured p H and the total histidine concentration (assuming t h a t a negligible a m o u n t is b o u n d to the polymer). W h e n the O.D. drop is plotted against the negative logarithm of concentration of the charged histidine species, it is found t h a t these points also fall on the curve for sodium chloride. The effect of imidazole has been studied in a similar manner, though in this case the p K of the ring nitrogen is higher, so t h a t only half the imidazole need be in the TABLE I S=,
Poly A Poly U Poly (A + U)
o.o5 M NaCl
xi .o 5.7 8.9
o.oJ M iralda~ol~ kydrocMoride
+ o.osM~i4em~
x1.4 5.9 9.2
Biochim. Biophys. Acta, 37 (I96o) 4a5-433
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G. FELSENFELD, S. HUANG
neutral form to bring the pH to 7. Imidazohum ion also behaves hke sodium ion in O.D. studies. Comparative ultracentrifuge sedimentation studies (Table I) of poly A, poly U, and poly A( + U) in o.o5 M imidazohum chloride (with 0.05 M imidazole) and 0.05 M sodium chloride show slightly higher values for the mean value of S~o in imidazole in each case. We cannot attach any significance to such small differences at present. We have also studied the influence of the dihydrochlorides of some aliphatic diamines upon the poly A - poly U interaction. Fig. 6 shows the effect of three compounds having the formula +H3N (CH~)nNH3+. The compounds used are putrescine (n = 4), cadaverine (n = 5) and diaminodecane (n -----IO). The experimental points for all three compounds lie essentially on one curve, and this curve is close in position to that for the addition of Mg ++. DISCUSSION
Metal ions The O.D. drop which occurs when Mg++ and Na + are added to poly A cannot be explained by the formation of the "ordered" poly A structure of FRESCO AND DOTY~ and BEERS AND STEINERe, since the formation of that structure is reported to be inhibited by the addition of salts, particularly those of divalent ions. In addition, the O.D. maximum in our experiments remains at 257 mt~ as salts are added. This is the position of the maximum reported for the "random coil" form of poly A. We do not observe the shift of the maximum to 252 mt~ which should occur if the more rigid poly A structure were being formed. The work of FREsco AND DOTY5 indicates that poly A in o.I M NaC1 at pH 7.5 is a random coil, so that, in the case of addition of NaC1 to poly A, we can be reasonably certain that the hypochromic effect does not involve formation of any multiple-stranded poly A structure. It appears that the hypochromic effect is in this case attributable to the increased flexibihty* of the single-stranded poly A molecule as it is neutralized by addition of excess sodium ion. The increased flexibility may lead to more satisfactory opportunities for adjacent bases on a given chain to "stack", and this may in turn increase the hypochromic effectL The same argument apphes to divalent ion, except that the neutralization occurs upon the addition of equivalent amounts of ion. The fact that the hypochromic effect is larger for Mg ++ than for Na + may be explained if the "random coil" is more compact, with better opportunity for stacking, in the presence of the very low magnesium chloride concentrations than it is in the presence of the rather high sodium chloride concentrations which are necessary to reduce the charge on the polymer chain. Measurements of the radius of gyration in each case have not yet been made. It should be pointed out that there are other explanations of the hypochromic effect which do not involve formation of multiple strands. Dr. E. P. GEIDUSCHEK (personal communication) has suggested that the polarization of the bases in the field of the phosphate ions is largely abolished when the backbone is neutrahzed, and that this could account for the change in absorption of the bases, and all of the * P o l y e l e c t r o l y t e s in general i n c l u d i n g p o l y U (H. EISENBERG AND G. FELSENFELD, unpublished results) s h o w a d r a m a t i c drop in v i s c o s i t y w h e n NaC1 is a d d e d to a distilled w a t e r solution.
Biochim. Biophys. Acta, 37 (196o) 425-433
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431
above results. Either hypothesis will also account for ,themuch smaller effect observed in the case of the polymer involving the smaller base, uracil. Whatever the cause of the hypochromic effect in poly A, the results shown in Fig. I indicate once again that there is a strong interaction between divalent ions :and polynucleotides, and that the sites on the polynucleotide are saturated when one equivalent of divalent ion has been added/mole of phosphate. The results of SHACK,JENKINS AND THOMPSETT8 bear an interesting resemblance to those reported here for poly A. These investigators added salts of both monovalent and divalent ions to solutions of DNA in distilled water, and observed the O.D. drop. It was subsequently realized9 that DNA in distilled water has a large portion of its hydrogen bonded system disrupted. In view of our results, it is not necessary to postulate the partial reconstitution of the DNA hydrogen bonded structure to account for the increased hyperchromicity. The disrupted DNA structure may behave somewhat like two single strands over considerable lengths. The effect of divalent ion upon the formation of poly (A + U) is also characteristic of strong interaction. At the highest polynucleotide concentrations one equivalent of divalent ion/mole of phosphorus is required to complete the reaction. The excess concentration of divalent ion required to complete the reaction at lower polynucleotide concentrations, and the existence of a limiting curve, suggest a partial dissociation of the poly (A + U) - Mg++ complex at these low concentrations. It is not possible to account quantitatively for the results of Fig. 3 on the basis of any simple reaction scheme of the kind which was used 8 in the study of threestranded poly (A + 2U). The widely-varying conditions of ionic strength in the experiments described here are sufficient to explain this difficulty. On the other hand, any one of a number of reaction schemes involving partial dissociation of the poly (A + U) - magnesium complex at low concentrations will qualitatively account for the results. Despite the partial dissociation of the complex at low concentrations the interaction is a "strong" one (i.e., the reaction goes to completion with a very small excess of Mg++) at phosphate concentrations of IO-4 M and above. It should be pointed out that partial dissociation of the poly (A + U) - metal complex is not necessarily inconsistent with a stronger affinity of single polynucleotide strands for divalent ion, since the unit which combines with divalent ion in the two-stranded case may be an uncombined, but neighboring, pair on a partly-formed two-stranded structure. This structure could be somewhat less flexible than the single-stranded polymers, with a consequent decrease in affinity for metal ion x°. Unlike divalent ions, the effect of NaC1 upon the polynucleotide combination is characteristic of a weak interaction. Both the large excess of ion required and the absence of polynucleotide concentration dependence in Fig. 4 are consistent with this interpretation. Amino acids and polyamines The contrast between the effects of "strong" and "weak" cation interaction on formation of two-stranded poly (A + U) provide a useful method for studying cation binding to polynucleotides. This is particularly true in the case of strong binding, which can be studied at low ionic strength, thus providing complementary information to that obtained by means of dialysis equilibrium at high ionic strength. At high ionic strength, there is competition for the polynucleotide sites from the sodium Biochim. Biophys. Avta, 37 (I96o) 4z5-433
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G. FELSENFELD, S. HUANG
ion or other cation used to raise the ionic strength. At low ionic strength the interaction between the cation of interest and the polynucleotide is enhanced by the absence of this competition. The application of this technique to amino acid binding shows conclusively that even under optimal conditions, charged amino acids are not bound in a selective manner to the polynucleotides studied. Earlier studies n-is of the binding of amino acids to DNA and RNA at high ionic strength have resulted in the same conclusion. It is particularly interesting that the large base of the histidine molecule, which might be expected to stabilize the binding through "stacking", does not give histidine any advantage. The diamines on the other hand are obviously bound strongly to the polynucleotides. The fact that slightly higher concentrations of the diamines than of Mg++ are required to cause the poly A - poly U interaction suggests that the diamines are a little less strongly bound than the metal ion, but the affinities are certainly of the same order of magnitude, and it is likely that diamines would compete strongly with divalent ions for sites on polynucleotide chains under conditions of equal concentration, even at higher ionic strength. Naturally, the presence of monovalent ions such as Na+ under physiological conditions tends to reduce the absolute amount of diamine bound, but in systems which involve diamine concentrations of the order of divalent ion concentration, as much diamine as divalent ion will be bound. Preliminary studies of binding of putrescine to poly U by means of conductometric titration show that there is also strong interaction between single-stranded polynucleotides and diamines. Otheg titrations involving the tetra-amine spermine indicate that in distilled water spermine tetrahydrochloride is capable of displacing divalent barium stoichiometrically from poly U, precipitating a spermine-poly U complex. Thus, it appears that in distilled water the affinity of single-stranded polynucleotides for polyamines is strong, and it is probable that the affinity increases as the number of amine groups per molecule increases. In a physiological situation such as that encountered by the DNA of certain bacteriophages 14, which involves high polyamine concentrations, it is likely that nucleic acids will carry significant amounts of polyamine. The quantity which is carried will of course depend upon the particular polyamines present, their concentration, and the concentration of small ions which can compete for sites on the nucleic acid. In addition, the presence of strongly bound protein chains may block the combination with polyamines. Our preliminary experiments with spermine indicate a behavior strongly resembling that of polylysine*. If the more highly charged polyamines do approach polylysine in their affinity for polynucleotide, they may exert a marked influence upon the physical properties of the polynucleotide to which they are attached. This possibility must be considered whenever a nucleic acid has been isolated from an environment containing polyamines. ACKNOWLEDGEMENTS
We are indebted to Dr. B. AMES for helpful discussions. This work was supported by Grant C-3883, National Cancer Institute, Public Health Service.
Biochim. Biophys. Acta, 37 (196o) 425-433
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REFERENCES I R. C. WARNER, J. Biol. Chem., 229 (I957) 711. 2 G. FELSENF~LD AND A. RICH, Biochim. Biophys. Acta, 26 (1957) 457. 8 G. FELSESFELD, D. R. DAVmS AND A. RICH, ]. Am. Chem. Soc., 79 (1957) 2023. 4 G. FELSENFELD AND S. HUANG, Biochim. Biophys. Acta, 34 (1959) 234. 8 j . R. FRESCO AND P. DorY, J. Am. Chem. Soc., 79 (1957) 3928. 6 R. F. BEERS AND R. F. SrEINER, Nature, 179 (1957) lO76. 7 A. M. MICH~LSON, Nature, 182 (1958) 15o2. s j . SHACK, D. J. JENKINS AND J. M. THOMPSETT, ]. Biol. Chem., 203 (I953) 373. 9 R. THOMAS, Biochim. Biophys. Acta, 14 (1954) 231. t0 F. E. HARRIS AND S. A. RICE, J. Phys. Chem., 61 (1957) 136o. 11 C. D. JARDErZKY, J. Am. Chem. Soc., 80 (1958) 1125. 18 j . S. WIBERG AND W. F. NEUMAN, Arch. Biochem. Biophys., 72 (1957) 66. 18 G. ZUBAYAND P. DoTe, Biochim. Biophys. Acta, 29 (1958) 47. It B. N. AMES, D. T. DUBIN AND S. M. ROSENXHAL, Science, 127 (1958) 814.
Biochim. Biophys. Aaa, 37 (196o) 425-433
STUDIES ON T H E ENZYMIC OXIDATION OF AMINOPURINES F E L I X BERGMANN, HANNA KWIETNY*, GERSHON LEVIN* AND HANNA E N G E L B E R G
Department o[ Pharmacology, The Hebrew University, Hadassah Medical School, Jerusalem (Israel) (Received April 24th, I959)
SUMMARY
I. 8-Aminopurine is oxidised by mammalian xanthine oxidase along the following pathway: 8-Aminopurine ---> 6-hydroxy-8-aminopurine ~
2,6-dihydroxy-8-aminopurine
It is thus evident that oxidation of all three aminopurines takes a course different from the corresponding hydroxypurines. 2. Adenine and 6-methylaminopurine are oxidised along the same pathway, but all other methyl derivatives of adenine are refractory. 3-2-Aminopurine is converted by a bacterial enzyme system into 2-amino8-hydroxypurine. These results are discussed in terms of specific enzyme--substrate complexes.
INTRODUCTION WYNGAA~DEN AND DUNN I h a v e r e c e n t l y e s t a b l i s h e d t h a t a d e n i n e is a t t a c k e d b y m a m m a l i a n x a n t h i n e o x i d a s e (XO) first a t p o s i t i o n 8 a n d t h e r e a f t e r a t c a r b o n a t o m ~. T h e o r d e r of s t e p s is t h u s a t v a r i a n c e w i t h t h e s e q u e n c e , a p p l y i n g t o t h e e n z y m i c o x i d a t i o n of h y p o x a n t l f i n e : Hypoxanthine --~ xanthine ~ uric acid Adenine ~ 8-hydroxyadenine ~ 2,8-dihydroxyadenine. * The results of this investigation are parts of Ph.D. theses, submitted to the Faculty of Science, The Hebrew University, Jerusalem.
Biochim. Biophys. Aaa, 37 (196o) 433-441
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THE INTERACTION OF POLYNUCLEOTIDES WITH METAL IONS, AMINO ACIDS, AND POLYAMINES GARY FELSENFELD AND SYLVIA HUANG Department o/Biophysics*, University of Pittsburgh, Pa. (U.S.A .) (Received April 2ISt, 1959)
SUMMARY The formation of the two-stranded complex between the sodium salts of polyadenylic acid and polyuridylic acid requires the presence in solution of additional cations. I t is found that the effect of magnesium ion is characteristic of a strong interaction between metal ion and polynucleotide, while the effect of sodium ion is characteristic of weak interaction. I t is also found that the formation of the two-stranded structure requires neutralization of the charge on the phosphate backbone. This information makes it possible to use the extent of the poly A-poly U interaction as a measure of binding of cations to polynucleotide. The method is applied to charged amino acids and polyamines. I t is shown that the amino acids are weakly bound and the polyamines strongly bound to polynucleotides.
INTRODUCTION The interaction of the synthetic polyribonueleotides, polyadenylic acid and polyuridylic acid, with each other is dependent upon the nature and concentration of cations present in the solution 1-s. Previous studies 4 have shown that both poly A and poly U interact strongly with Mg++ and Mn++ and that about one equivalent of divalent ion is bound per mole of phosphate. In this paper we describe further studies of the ionic conditions necessary for the combination of poly A and poly U, and use this information to provide a simple measure of the strength of interaction between polynucleotides and both charged amino acids and polyamines. EXPERIMENTAL The polynucleotides were prepared enzymicaUy from nucleoside diphosphates with polynucleotide phosphorylase isolated from Azotobacter agile according to a method kindly provided b y Prof. S. OCHOA. Preparations were dialyzed in succession against ethylenediaminetetra-acetic acid, sodium chloride, and redistilled water before * Contribution No. 64 from the Department of Biophysics. Abbreviations: Polyadenylic acid = poly A; polyuridylic acid = poly U; deoxyribonucleic acid = DNA; ribonucleic acid = RNA. The materials referred to by these names a r e t h e sodium salts.
Biochim. Biophys. Acta, 37 (I96o) 425-433
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G. FELSENFELD, S. HUANG
freeze-drying. Experiments involving mixing of poly A and poly U were performed so that the pH of the final reacting mixtures was generally between 6. 7 and 7.4. Where necessary the pH of solutions was adjusted with NaOH, and the reacting mixtures of polymer were stored in dessicators over saturated NaOH solution to eliminate absorption of atmospheric CO,. These precautions were necessary to avoid the formation of the ordered poly A complexS, 6 which occurs in distilled water below pH 6.5. The upper limit in pH is necessary to avoid metal ion-hydroxyl ion interactions in certain cases. The amino acids were commercial preparations (glycine, Fisher; L-histidine, California Corporation for Biochemical Research; L-histidine monohydrochloride, Eastman; imidazole, Eastman; L-lysine monohydrochloride, Interchemical). Putrescine hydrochloride was obtained from California Corporation for Biochemical Research, and was analyzed for chloride ion by silver nitrate titration. Diaminodecane and diaminopentane (cadaverine) were obtained from Aldrich Chemical Company, and were analyzed for titratable amino groups by dissolving weighed quantities in an excess of standard hydrochloric acid and back-titrating with standard sodium hydroxide solution. The diaminopentane and diaminodecane were also analyzed for nitrogen colorimetrically with Nessler's reagent. The results agreed with the titration within 3 %. Solutions of sodium, manganese and magnesium chloride were made from analytical grade reagents. The divalent ion chlorides were standardized by titration with silver nitrate using dichlorofluorescein as an indicator. O.D. measurements were made with the Cary Model 14 recording spectrophotometer. All O.D. reported were measured at 259 m/%. In the case of the most dilute solutions, cylindrical cells with a path length of IO cm were used. In the case of the most concentrated solutions, rectangular cells of path length 0.5 cm were used, and when the O.D. exceeded 2.0, readings were made against standardized blanks of sufficiently high O.D. to bring the readings within the range of the spectrophotometer. Ultracentrifuge studies were made with the Spinco Model E, equipped with u.v. optics. RESULTS
Metal ions
The decrease in O.D. in the 26o-m~ region which accompanies the combination of poly A and poly U in solution has already been used to obtain qualitative information about the relative effectiveness of various cations in promoting the combination *. In order to relate the fractional decrease in O.D. to the fractional completion of the reaction, it is naturally necessary to establish that the entire drop in O.D. which occurs when cations are added is attributable to the reaction. Investigation of the effect of the addition of MgC1, on the O.D. of poly A alone shows that this is not the case (Fig. I). With the pH carefully adjusted above 7.0 so that the "ordered" poly A structureS, e is unstable, poly A shows a marked hypochromic effect upon addition of small amounts of divalent ion. The molar O.D. is independent of polymer concentration for a given ratio of equivalents of divalent ion added to moles of polymer phosphate, and the limiting O.D. is reached when about one equivalent of divalent ion has been added/mole of polymer phosphate. The total hyp0chromic effect is about Biochim. Biophys. Acta, 37 (196o) 425-433
POLYNUCLEOTIDES AND METAL IONS, AMINO ACIDS, POLYAMINES
427
1.09 o 7.85.10 "~ /%.4 . 0 6 • 10.-4 1.1~
[ ] 5.90.10 "s • L6-10 "4
O.D.
01.2"10 .5
0.[2 O.D. M
1.10
1.05
1.05
1.0
o
i
i
,
,
I
o
°o i
i
1.00 4
,
cA Fig. I. O.D. (divided by limiting O.D.) of poly A solutions at various concentrations plotted against equivalents of magnesium ion added] mole ofpolymerphosphate. Measured at 259 m/~.
i
i
i
I
I
3
2 -Log NuCI
Fig. 2. O.D. (divided by limiting O.D.) of poly A solutions at two concentrations plotted against the negative logarithm of NaC1 concentration.
I6 % of the O.D. in distilled water. The limiting molar O.D., about 8840 at 259 m~, is somewhat lower than that obtained in o.I M NaC1 at p H 6.72. Poly U does not show such strong dependence of O.D. upon ionic concentration. A small decrease of about 3 % in O.D. is observed in going from distilled water to one equivalent of divalent ion. The effect of the addition of sodium chloride to poly A in distilled water is characteristically different from that obtained with divalent ions. Though there is a drop in O.D. (Fig. 2), the change occurs much more gradually as the salt concentration is increased, beginning at IO-s M NaC1, but not complete until the concentration is raised to lO -1 M, so that NaC1 is in large excess. The total drop in O.D. is only about half that observed when divalent ions are added. With the use of these corrections it is possible to interpret the O.D. decrease of a mixture of poly A and poly U in terms of formation of poly (A + U). If we assume that the O.D. contributed b y partly formed poly (A + U) is directly proportional to the number of combined sites, it is easy to show that F, the fraction of completion of combination, is given b y F
D'o - - D D'o - - DM
(X)
where D is the O.D. of the poly A + poly U solution in question. D'o is the sum of the O.D. of two solutions containing poly A and poly U separately at the concentration used in the mixed solution, and with the same amount of salt added, and DM is the limiting O.D. density of the mixture at high salt concentration. In Fig. 3 (D'o--D)]Do is plotted as a function of magnesium ion concentration for a series of x : I poly A + poly U mixtures over a hundred-fold range of polynucleotide concentration. With a knowledge* of Do, it is possible to compute F. * The molar O.D. at 259 m# in distilled water is.about 11,6oo for poly A and 97oo for poly U. Biochim. Biophys. Acta, 37 (I96o) 425-433
428
G. FELSENFELD, S. HUANG
o ~
o 7.4 •10-4
I:l 3.6 .I0 "~
AQI~
O.[Xo al
a2
, , l l
0
10
2 0 Conc.. 10 s 3 0
Fig. 3. Fractional O.D. change (D'o--D)/D o for a series of I :i p o l y A - poly U m i x t u r e s as a function of Mg ++ concentration. P o l y m e r p h o s p h a t e concentrations are s h o w n a t t h e u p p e r right.
At the highest polynucleotide concentrations, the amount of magnesium ion necessary to cause poly A and poly U to interact increases as the polynucleotide concentration increases. Within about io %, the reaction requires one equivalent of divalent ion/mole of phosphate to go to completion. At the lowest concentration there appears to be a limiting curve. Similar experiments involving the addition of Mn++ rather than Mg++ give the same results. The addition of NaC1 to a I : x poly A - poly U mixture also causes combination of the two polymers, but in this case the degree of reaction for a given sodium chloride concentration is independent of polynucleotide concentration (Fig. 4). As in the case of the O.D. drop of poly A alone, about a IOO fold increase in NaCI concentration is required to go from F = o.oI to F ----- 0.99.
,~o.[;).
• • •
O.D.,
6.7 .tO -(= 1.2.10 "s 7.1 "10 -s
0 3.4"10-'~ 0.10
0.20
i
4
i
i
i
I
3
i
J
i
J
[
2
I
i
i
-Log No
i
T
1
Fig. 4. Fractional O.D. change (D'o--D)/D o for a series of z :I poly A - poly U m i x t u r e s as a function of t h e negative l o g a r i t h m of NaC1 concentration. P o l y m e r p h o s p h a t e concentrations are s h o w n a t t h e u p p e r right.
Amino acids and diamines Having established the behavior of two classes of ions, those which are bound strongly and those which are bound weakly (see DlSCUSSlOlq), we can use the poly A poly U interaction as a measure of cationic binding. In Fig. 5 are shown the effects upon the O.D. of a I : I poly A - poly U mixture of the addition of various amino acids, compared with the effect of NaC1 upon the Biochim. Biophys. Acta, 37 (196o) 425-433
POLYNUCLEOTIDES AND METAL IONS, AMINO ACIDS, POLYAMINES
429
0 ~ /~ O.D. M
Q |
0.2
O MG "+
~-
• PUT Q CAD • DAD
&.
Z~O.D. ~. O.D.M
0.4 0.~ 0,5
0.8
• NoCI
o GLY • LYS • HIST
1.0
-Log C
2
LC
1
0
Fig. 5. Decrease in O.D. divided by maximum decrease in O.D. (uncorrected for changes in poly A alone) plotted against the negative logarithm of the molar concentration of hydrochlorides of glycine, lysine and histidine, compared with the effect of NaC1. Polymer phosphate concentration = 7.2' I o-6.
lOconc" . lOs
Fig. 6. Decrease in O.D. of poly (A + U) divided by maximum decrease in O.D. (uncorrected for changes in poly A alone) plotted against concentration of putrescine, cadaverine and diaminodecane hydrochlorides, compared with the effect of MgC12. Polymer phosphate concentration = 6.7. i o'-5.
same solution. Glycine, which has no net charge, produces essentially no 0 . D . change even in I M solution. Positively charged lysine, (lysine hydrocMoride) on the other hand, does cause an interaction to exactly the same extent as sodium ion at the same concentration. I n the case of histidine it is necessary to add a mixture of the free amino acid and the hydrochloride to keep the p H at 7 or above, since at lower p H there is danger of formation of the ordered, non-reactive poly A structure. Because the p K of histidine is 6.o5, approximately a ten-fold excess of neutral histidine must be present in solution to keep the p H at 7. Since glycine does not cause poly A and poly U to interact, we assume t h a t no other dipolar ion has the ability to do so. The concentration of positively charged histidine is c o m p u t e d from the measured p H and the total histidine concentration (assuming t h a t a negligible a m o u n t is b o u n d to the polymer). W h e n the O.D. drop is plotted against the negative logarithm of concentration of the charged histidine species, it is found t h a t these points also fall on the curve for sodium chloride. The effect of imidazole has been studied in a similar manner, though in this case the p K of the ring nitrogen is higher, so t h a t only half the imidazole need be in the TABLE I S=,
Poly A Poly U Poly (A + U)
o.o5 M NaCl
xi .o 5.7 8.9
o.oJ M iralda~ol~ kydrocMoride
+ o.osM~i4em~
x1.4 5.9 9.2
Biochim. Biophys. Acta, 37 (I96o) 4a5-433
430
G. FELSENFELD, S. HUANG
neutral form to bring the pH to 7. Imidazohum ion also behaves hke sodium ion in O.D. studies. Comparative ultracentrifuge sedimentation studies (Table I) of poly A, poly U, and poly A( + U) in o.o5 M imidazohum chloride (with 0.05 M imidazole) and 0.05 M sodium chloride show slightly higher values for the mean value of S~o in imidazole in each case. We cannot attach any significance to such small differences at present. We have also studied the influence of the dihydrochlorides of some aliphatic diamines upon the poly A - poly U interaction. Fig. 6 shows the effect of three compounds having the formula +H3N (CH~)nNH3+. The compounds used are putrescine (n = 4), cadaverine (n = 5) and diaminodecane (n -----IO). The experimental points for all three compounds lie essentially on one curve, and this curve is close in position to that for the addition of Mg ++. DISCUSSION
Metal ions The O.D. drop which occurs when Mg++ and Na + are added to poly A cannot be explained by the formation of the "ordered" poly A structure of FRESCO AND DOTY~ and BEERS AND STEINERe, since the formation of that structure is reported to be inhibited by the addition of salts, particularly those of divalent ions. In addition, the O.D. maximum in our experiments remains at 257 mt~ as salts are added. This is the position of the maximum reported for the "random coil" form of poly A. We do not observe the shift of the maximum to 252 mt~ which should occur if the more rigid poly A structure were being formed. The work of FREsco AND DOTY5 indicates that poly A in o.I M NaC1 at pH 7.5 is a random coil, so that, in the case of addition of NaC1 to poly A, we can be reasonably certain that the hypochromic effect does not involve formation of any multiple-stranded poly A structure. It appears that the hypochromic effect is in this case attributable to the increased flexibihty* of the single-stranded poly A molecule as it is neutralized by addition of excess sodium ion. The increased flexibility may lead to more satisfactory opportunities for adjacent bases on a given chain to "stack", and this may in turn increase the hypochromic effectL The same argument apphes to divalent ion, except that the neutralization occurs upon the addition of equivalent amounts of ion. The fact that the hypochromic effect is larger for Mg ++ than for Na + may be explained if the "random coil" is more compact, with better opportunity for stacking, in the presence of the very low magnesium chloride concentrations than it is in the presence of the rather high sodium chloride concentrations which are necessary to reduce the charge on the polymer chain. Measurements of the radius of gyration in each case have not yet been made. It should be pointed out that there are other explanations of the hypochromic effect which do not involve formation of multiple strands. Dr. E. P. GEIDUSCHEK (personal communication) has suggested that the polarization of the bases in the field of the phosphate ions is largely abolished when the backbone is neutrahzed, and that this could account for the change in absorption of the bases, and all of the * P o l y e l e c t r o l y t e s in general i n c l u d i n g p o l y U (H. EISENBERG AND G. FELSENFELD, unpublished results) s h o w a d r a m a t i c drop in v i s c o s i t y w h e n NaC1 is a d d e d to a distilled w a t e r solution.
Biochim. Biophys. Acta, 37 (196o) 425-433
POLYNUCLEOTIDES AND METAL IONS, AMINO ACIDS, POLYAMINES
431
above results. Either hypothesis will also account for ,themuch smaller effect observed in the case of the polymer involving the smaller base, uracil. Whatever the cause of the hypochromic effect in poly A, the results shown in Fig. I indicate once again that there is a strong interaction between divalent ions :and polynucleotides, and that the sites on the polynucleotide are saturated when one equivalent of divalent ion has been added/mole of phosphate. The results of SHACK,JENKINS AND THOMPSETT8 bear an interesting resemblance to those reported here for poly A. These investigators added salts of both monovalent and divalent ions to solutions of DNA in distilled water, and observed the O.D. drop. It was subsequently realized9 that DNA in distilled water has a large portion of its hydrogen bonded system disrupted. In view of our results, it is not necessary to postulate the partial reconstitution of the DNA hydrogen bonded structure to account for the increased hyperchromicity. The disrupted DNA structure may behave somewhat like two single strands over considerable lengths. The effect of divalent ion upon the formation of poly (A + U) is also characteristic of strong interaction. At the highest polynucleotide concentrations one equivalent of divalent ion/mole of phosphorus is required to complete the reaction. The excess concentration of divalent ion required to complete the reaction at lower polynucleotide concentrations, and the existence of a limiting curve, suggest a partial dissociation of the poly (A + U) - Mg++ complex at these low concentrations. It is not possible to account quantitatively for the results of Fig. 3 on the basis of any simple reaction scheme of the kind which was used 8 in the study of threestranded poly (A + 2U). The widely-varying conditions of ionic strength in the experiments described here are sufficient to explain this difficulty. On the other hand, any one of a number of reaction schemes involving partial dissociation of the poly (A + U) - magnesium complex at low concentrations will qualitatively account for the results. Despite the partial dissociation of the complex at low concentrations the interaction is a "strong" one (i.e., the reaction goes to completion with a very small excess of Mg++) at phosphate concentrations of IO-4 M and above. It should be pointed out that partial dissociation of the poly (A + U) - metal complex is not necessarily inconsistent with a stronger affinity of single polynucleotide strands for divalent ion, since the unit which combines with divalent ion in the two-stranded case may be an uncombined, but neighboring, pair on a partly-formed two-stranded structure. This structure could be somewhat less flexible than the single-stranded polymers, with a consequent decrease in affinity for metal ion x°. Unlike divalent ions, the effect of NaC1 upon the polynucleotide combination is characteristic of a weak interaction. Both the large excess of ion required and the absence of polynucleotide concentration dependence in Fig. 4 are consistent with this interpretation. Amino acids and polyamines The contrast between the effects of "strong" and "weak" cation interaction on formation of two-stranded poly (A + U) provide a useful method for studying cation binding to polynucleotides. This is particularly true in the case of strong binding, which can be studied at low ionic strength, thus providing complementary information to that obtained by means of dialysis equilibrium at high ionic strength. At high ionic strength, there is competition for the polynucleotide sites from the sodium Biochim. Biophys. Avta, 37 (I96o) 4z5-433
432
G. FELSENFELD, S. HUANG
ion or other cation used to raise the ionic strength. At low ionic strength the interaction between the cation of interest and the polynucleotide is enhanced by the absence of this competition. The application of this technique to amino acid binding shows conclusively that even under optimal conditions, charged amino acids are not bound in a selective manner to the polynucleotides studied. Earlier studies n-is of the binding of amino acids to DNA and RNA at high ionic strength have resulted in the same conclusion. It is particularly interesting that the large base of the histidine molecule, which might be expected to stabilize the binding through "stacking", does not give histidine any advantage. The diamines on the other hand are obviously bound strongly to the polynucleotides. The fact that slightly higher concentrations of the diamines than of Mg++ are required to cause the poly A - poly U interaction suggests that the diamines are a little less strongly bound than the metal ion, but the affinities are certainly of the same order of magnitude, and it is likely that diamines would compete strongly with divalent ions for sites on polynucleotide chains under conditions of equal concentration, even at higher ionic strength. Naturally, the presence of monovalent ions such as Na+ under physiological conditions tends to reduce the absolute amount of diamine bound, but in systems which involve diamine concentrations of the order of divalent ion concentration, as much diamine as divalent ion will be bound. Preliminary studies of binding of putrescine to poly U by means of conductometric titration show that there is also strong interaction between single-stranded polynucleotides and diamines. Otheg titrations involving the tetra-amine spermine indicate that in distilled water spermine tetrahydrochloride is capable of displacing divalent barium stoichiometrically from poly U, precipitating a spermine-poly U complex. Thus, it appears that in distilled water the affinity of single-stranded polynucleotides for polyamines is strong, and it is probable that the affinity increases as the number of amine groups per molecule increases. In a physiological situation such as that encountered by the DNA of certain bacteriophages 14, which involves high polyamine concentrations, it is likely that nucleic acids will carry significant amounts of polyamine. The quantity which is carried will of course depend upon the particular polyamines present, their concentration, and the concentration of small ions which can compete for sites on the nucleic acid. In addition, the presence of strongly bound protein chains may block the combination with polyamines. Our preliminary experiments with spermine indicate a behavior strongly resembling that of polylysine*. If the more highly charged polyamines do approach polylysine in their affinity for polynucleotide, they may exert a marked influence upon the physical properties of the polynucleotide to which they are attached. This possibility must be considered whenever a nucleic acid has been isolated from an environment containing polyamines. ACKNOWLEDGEMENTS
We are indebted to Dr. B. AMES for helpful discussions. This work was supported by Grant C-3883, National Cancer Institute, Public Health Service.
Biochim. Biophys. Acta, 37 (196o) 425-433
POLYNUCLEOTIDES AND METAL IONS, AMINO ACIDS, POLYAMINES
43~
REFERENCES I R. C. WARNER, J. Biol. Chem., 229 (I957) 711. 2 G. FELSENF~LD AND A. RICH, Biochim. Biophys. Acta, 26 (1957) 457. 8 G. FELSESFELD, D. R. DAVmS AND A. RICH, ]. Am. Chem. Soc., 79 (1957) 2023. 4 G. FELSENFELD AND S. HUANG, Biochim. Biophys. Acta, 34 (1959) 234. 8 j . R. FRESCO AND P. DorY, J. Am. Chem. Soc., 79 (1957) 3928. 6 R. F. BEERS AND R. F. SrEINER, Nature, 179 (1957) lO76. 7 A. M. MICH~LSON, Nature, 182 (1958) 15o2. s j . SHACK, D. J. JENKINS AND J. M. THOMPSETT, ]. Biol. Chem., 203 (I953) 373. 9 R. THOMAS, Biochim. Biophys. Acta, 14 (1954) 231. t0 F. E. HARRIS AND S. A. RICE, J. Phys. Chem., 61 (1957) 136o. 11 C. D. JARDErZKY, J. Am. Chem. Soc., 80 (1958) 1125. 18 j . S. WIBERG AND W. F. NEUMAN, Arch. Biochem. Biophys., 72 (1957) 66. 18 G. ZUBAYAND P. DoTe, Biochim. Biophys. Acta, 29 (1958) 47. It B. N. AMES, D. T. DUBIN AND S. M. ROSENXHAL, Science, 127 (1958) 814.
Biochim. Biophys. Aaa, 37 (196o) 425-433
STUDIES ON T H E ENZYMIC OXIDATION OF AMINOPURINES F E L I X BERGMANN, HANNA KWIETNY*, GERSHON LEVIN* AND HANNA E N G E L B E R G
Department o[ Pharmacology, The Hebrew University, Hadassah Medical School, Jerusalem (Israel) (Received April 24th, I959)
SUMMARY
I. 8-Aminopurine is oxidised by mammalian xanthine oxidase along the following pathway: 8-Aminopurine ---> 6-hydroxy-8-aminopurine ~
2,6-dihydroxy-8-aminopurine
It is thus evident that oxidation of all three aminopurines takes a course different from the corresponding hydroxypurines. 2. Adenine and 6-methylaminopurine are oxidised along the same pathway, but all other methyl derivatives of adenine are refractory. 3-2-Aminopurine is converted by a bacterial enzyme system into 2-amino8-hydroxypurine. These results are discussed in terms of specific enzyme--substrate complexes.
INTRODUCTION WYNGAA~DEN AND DUNN I h a v e r e c e n t l y e s t a b l i s h e d t h a t a d e n i n e is a t t a c k e d b y m a m m a l i a n x a n t h i n e o x i d a s e (XO) first a t p o s i t i o n 8 a n d t h e r e a f t e r a t c a r b o n a t o m ~. T h e o r d e r of s t e p s is t h u s a t v a r i a n c e w i t h t h e s e q u e n c e , a p p l y i n g t o t h e e n z y m i c o x i d a t i o n of h y p o x a n t l f i n e : Hypoxanthine --~ xanthine ~ uric acid Adenine ~ 8-hydroxyadenine ~ 2,8-dihydroxyadenine. * The results of this investigation are parts of Ph.D. theses, submitted to the Faculty of Science, The Hebrew University, Jerusalem.
Biochim. Biophys. Aaa, 37 (196o) 433-441