6526 infer that orotidine is in a n unstrained syn conformation with a C(2’)-endo ribose. Acknowledgment. We are indebted to Professor F. Cramer for his interest and support of our work. We thank Miss U. Wittenberg for her skillful technical
assistance and Dr. Ph. C. Manor for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft. The computations were done with the UNIVAC 1108 of the Gesellschaft fur wissenschaftliche Datenverarbeitung, Gottingen.
Interaction of Metal Ions with Polynucleotides and Related Compounds. XXI. Metal Ions as Agents for the Stacking of Nucleotides. A Specific Interaction of Zinc( 11) and Adenosine Monophosphate Joseph M. Rifkind and Gunther L. Eichhorn” Contribution from the Gerontology Research Center, National Institutes of Health, National Institute of Child Health and Human Development, Baltimore City Hospitals, Baltimore, Maryland 21224. Receiced January 17, 1972 Mconcentration in the presence of a polycation to produce Abstract: Zinc(I1) ions react with 5‘-AMP at 1 x a highly rotatory complex with conservative circular dichroism bands. This complex is characterized in the uv by hypochromicity and shoulders at 280 and 290 nm, and it exhibits a titration curve in which the phosphate pK is lowered and the pK for zinc hydrolysis is raised. Conservative CD bands are also produced by Zn(I1) and 3’-AMP. These observations indicate that the zinc promotes parallel stacking of 3’- and 5‘-AMP. The effect is remarkably specific. Other AMP isomers (2’, 2’,3‘ cyclic, and 3’,5’ cyclic) and other nucleotides (5’-GMP, 3’-GMP, 5’-CMP, 5’-UMP, and 5’-IMP) do not produce a highly rotatory complex. Metal ions other than Zn(II), e.g., Mn(II), Co(II), Ni(II), Cu(II), Cd(II), Hg(II), Ag(I), Fe(III), Al(III), and Ce(III), also do not produce such a complex. The failure to produce the effects that have been noted does not indicate a lack of complex formation, but only the inability of the metal to induce parallel stacking of the nucleotides. A metal that does not induce parallel stacking at low concentration may do so at high concentration, e.g., Cu(II), but some metals apparently do not induce such stacking at any concentration. Highly rotatory complexes that do not display the conservative CD effect are formed between Pb(I1) and 5’-AMP and Zn(I1) and 3’- and 5‘-dAMP. The absence of the 2’-OH group produces a much greater change in the CD effect of the 3’-AMP complex than of the 5’ complex, suggesting that the 2‘-OH group plays a more significant role in the former than in the latter.
T
he participation of metal ions in the biochemical reactions of nucleotides and nucleic acids has provided a great deal of interest in the determination of the structures of the metal-nucleotide complexes. Recently a nuclear magnetic resonance study has suggested that the reaction of Cu(I1) with 3‘-AMP and 5’-AMP results in a complex containing two atoms of Cu(I1) and two molecules of AMP, with the AMP molecules located in such a way as to permit T interaction between the p ~ r i n e s . ~ If metal ions are able to induce such stacking of nucleotide bases, this stacking should result in the drastic alterations of their O R D and C D spectra. The present study reveals that metal ions can indeed produce large increases in the rotatory strength of nucleotides which are consistent with an interaction between the bases. Perhaps the most striking aspect of the production of these Cotton effects is that, far from constituting a general phenomenon, they are produced only in certain very restricted cases. Copper ions unexpectedly d o not produce the effects. Only zinc(I1) and lead(II), of all common metal ions, (1) G. L. Eichhorn, N. Berger, J. Butzow, P. Clark, J. Rlfkind, Y . Shin, and E. Tarirn, Adian. Chem. Ser., N o . 100, 135 (1971). ( 2 ) U. Weser, Sfruct. Bonding (Berlin), 5 , 41 (1968). (3) N. A . Berger and G. L. Eichhorn, Biochemistry, 10, 1847 (1971).
Journal of the American Chemical Society
and only AMP (either 3’ or 5 ’ ) , of all the common nucleotides, will produce Cotton effects, and only the optical characteristics of zinc, and not lead, are indicative of parallel stacking. Such specificity in the reaction of metal and ligand is quite remarkable. This paper describes the studies that demonstrate this specificity and provides some clues for its occurrence. The study of the optical properties of metal-nucleotide complexes is complicated by the fact that many of these complexes precipitate at neutral pH. This problem has been overcome by carrying out the optical studies in the presence of poly-r-lysine and certain other polycations. It is then possible to probe the effect of metal ions on the uv, ORD, and CD spectra of various nucleotides and thus to determine whether metal ions can induce the stacking of nucleotide bases.
Experimental Section The nucleosides and nucleotides were obtained from Sigma except for 3’-GMP which was obtained from P. L. Biochemicals. The basic polypeptides were obtained from Pilot Chemicals, polyethyleneimine from Pfaltz and Bauer, and DEAE-dextran from Pharmacia. All other chemicals were reagent grade. The concentrations of nucleotides were determined from their uv spectra. The concentrations of basic polypeptides were deter-
/ 94:18 / September 6 , I972
6527 mined by the method of Lowry which was checked by nitrogen analysis. The concentrations of metal ions were determined with a Zeiss atomic absorption spectrometer. Titrations were performed using a Radiometer TTA3 titration assembly with an ABUl autoburet. Titrations were performed at 25” under nitrogen using nitrate salts. All titrations were made at 5 min/pH unit and at a slower rate of 20-200 min/pH unit. The faster and slower results were always qualitatively similar and often identical. Uv spectra were measured using a Cary 14 spectrophotometer, and O R D and C D spectra were obtained with a Cary 60 spectropolarimeter with the 6001 circular dichroism attachment. Cylindrical cells of 1 mm path length at an ambient temperature of 24 =k 1 were used unless specified otherwise. In the preparation of samples for uv, CD, and ORD spectroscopy the acetate salts were frequently used because of the greater solubility of zinc acetate. However, the same results are obtained with the chloride or nitrate salts, and when the acetate was not available other salts were used. The solutions were usually prepared by adding the nucleotide t o the polycation, then adding the metal, and finally adjusting the pH with a Radiometer Model 25 pH meter to the desired pH. Variations in the protocol were investigated, and particularly with 3’AMP some of the details of the spectra are sensitive to the manner in which the solutions are prepared. The spectra of 5’-AMP in the presence of poly-L-lysine and Zn(I1) are insensitive to the order in which the solutions are added, but some of the qualitative features of the spectra d o depend on pH, particularly in the region of pH 6. The position of the peaks and the relative intensity of the peaks are very sensitive to the relative concentrations of polypeptide, nucleotide and metal. For this reason all reported spectra are at p H 7.0 with a 2: 1 :1 polypeptide: nucleotide :metal concentration. In all cases the effect of time on the observed spectra was investigated. Particularly in those cases in which no significant effect on the C D spectra was observed, the solutions were reexamined after standing for at least 1 day. When the elapse of time produced a significant enhancement in CD, as was the case for the solutions of Zn(I1) 2’-AMP in Figure 9, and the solutions used in Figure 11, the later spectrum is reported.
Results and Discussion The Reaction of Zn(1I) with 5’-AMP. The addition of a twofold excess of poly-L-lysine to 5’-AMP produces a 9 % decrease in the extinction coefficient at 259 nm at neutral p H 4 and no significant effect on the O R D or C D spectrum of 5’-AMP. The further addition of zinc(I1) has a dramatic effect on all three types of spectra (Figure 1). The uv spectrum exhibits a hypochromicity of 37% at 259 nm relative to that of the already hypochromic 5‘-AMP after the addition of polyL-lysine, or a total hypochromicity of 42%. In addition, two shoulders appear, at 280 and 290 nm. The O R D and C D spectra indicate a very large increase in rotatory strength to a value that is somewhat greater than that obtained for the ordered conformations of polyadenylic acid. The C D spectrum consists of two sets of conservative C D bands and at least one nonconservative band indicated by the high-wavelength minimum at 282 nm. The near-uv conservative bands consist of a minimum at -277 nm (corresponding to a shoulder) and a maximum at 251 nm, while the far-uv conservative bands consist of a minimum at 222 nm and a maximum at 212 nm. The presence of the conservative C D bands constitutes a clear indication that the Zn(I1) holds the nucleotide bases in a parallel stacked configuration which splits the T- T* bands into two bands whose rotatory strengths are approximately equal but opposite in sign5s6 Such a conclusion is (4) K. G . Wagner and R. Arav, Biochemistry, 7, 1771 (1968). (5) I. Tinoco, Jr., J. Amer. Chem. SOC.,86, 297 (1964). (6) C. A. Bush and I. Tinoco, Jr., J . Mol. Biol., 23, 601 (1967).
0
I
2 X
u,
6 -
t
B A 2
41
-
1
\A\-“-]i 1 /+- _------ I--._____-
2t
0
A
CD
-2 -41
................... f .................
7 210
230
250
i
W
270
290
310
WAVELENGTH, nm
Figure 1 . Uv, CD, and ORD spectra of a solution of 1 X M 5’-AMP at p H 7: (. . .) without further addition; (- -) in the in the presence of presence of 2 X M poly-L-lysine; (-) 2x Mpoly-L-lysine and 1 X Mzinc(I1).
-
strengthened by the fact that the inversion of all of the bands of Figure 1 produces a spectrum that is strikingly similar to the C D spectrum of poly A at neutral P H , ~or of an adenosine din~cleotide,~ in both of which the bases are considered to be in a parallel stacked helical configuration. The inversion of the bands in the Zn(11) 5’-AMP spectrum can readily be explained by a change in the relative orientation of the bases and their transition moments which determine the signs and magnitudes of the C D bands.6 Such an inversion has been observed in a dinucleotide in which the D-ribose is replaced by L-ribose,* as well as in a dinucleotide in which the 5’ positions are separated by three or four phosphate^.^ The poly-L-lysine is not required for the production of these optical effects; other polypeptides, e.g., polyD-lysine, poly-L-ornithine, and poly-L-arginine, may be substituted. Indeed, other unrelated polycations, e.g., polyethylenimine, a globular polyamine containing primary, secondary, and tertiary amines, and DEAEdextran, a polysaccharide with positive diethylamine ethyl ether substituents, can be used instead of polylysine. The range of structural differences that can be tolerated in the polycation suggests that it brings the zinc(I1)-AMP complex into solution by a process that leaves the complex intact, though some interaction with the polypeptide probably takes place. M 5’-AMP in The addition of zinc(I1) to 1 X the absence of polycation immediately precipitates most of the nucleotide at neutral pH. The uv spectrum of the 5’-AMP remaining in solution when zinc(I1) is (7) C. A. Bush and H. A. Scheraga, Biopolymers, 7, 395 (1969). (8) I. Tazawa, S. Tazawa, L. M. Stempel, and P. 0. P. Ts’o, Biochemistry, 9, 3499 (1970). (9) J. F. Scott and P. C. Zamecnik, Proc. Nat. Acad. Sci. U.S., 64, 1308 (1969).
(10) A very large excess (e.g., 40-fold) of polycation, however, will disrupt the complex.
Rifkind, Eichhorn
Specific Interaction of Zinc(II> and AMP
6528
-ii -
0.0
.-c \
-3
0
E
1.0
s! X
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$-
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%
0 Y K 0-
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3.0
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2z
4.0
i
0
u
0.0 0.1 0 2
0 3 0 . 4 0.5 0 6 0 7
08 0 9
10
MOLE FRACTION OF 5 ’ A M P
Figure 2. Continuous variation curve t o determine stoichiometry of the Zn(II)-5’-AMP complex. Stock solutions of 5‘-AMP and Zn(I1) acetate, both 1 X M , were mixed so as t o produce solutions with the same total concentration of Zn(I1) and 5’-AMP, but varying ratios, as indicated in the abscissa: analytical measurements performed on supernatant; precipitate concentration determined by difference; (0) concentration of 5’-AMP, from absorbance remaining after precipitation; ( 0 ) concentration of Zn(II), by atomic absorption spectrophotometry.
0
2
4
6
8
1 0 1 2
moles OH-/mole AMP (or Z n )
Figure 3. Titration curves of the zinc(I1) complexes of the isomers of AMP compared with the titration curves of the components. M in nucleotide and/or Zn(NO&. Enough Solutions 1 x M in HNOJ HNOBwas added to each solution to make it 5 X before titration with 5 X 10-2 M NaOH under nitrogen at 25”: (-)Zn(NO&; ( - - - ) A M P ; (-.-)2’-AMPandZn(N03)2; (- --) 3’-AMPandZn(NO&; (--)5’-AMPandZn(N03)r.
added to the nucleotide is virtually identical with that of 5’-AMP in the absence of zinc, and it appears therefore that the zinc(I1)-AMP complex is insoluble. The continuous variation curve (Figure 2 ) measuring the absorbance and zinc concentration of solutions of zinc(l1) and 5‘-AMP, from which precipitate has been removed, exhibits a minimum at a 1 :1 ratio of the constituents, indicating an equimolar ratio of zinc and 5’-AMP in the complex. The zinc(II)-5’-AMP complex dissolves at high and low pH. It is possible to study the formation of an insoluble complex by a titration that is carried out slowly Journal of the American Chemical Sociely
/
94:18
4
5
7
6
8
PH
Figure 4. Spectropolarimetric titration of solutions containing (A) 5 X M 5’-AMP and zinc(I1) acetate and 1 X M poly-L-lysine, and (B) 1 X M 3’-AMP and zinc(I1) acetate M poly-L-lysine. The solutions were adjusted with and 2 X concentrated NaOH.
enough to attain equilibrium” at all points of the titration curve. In Figure 3 the titration curve of a 1 :1 mixture of Zn(I1) and 5’-AMP is compared with titration curves of the components. Two features of the titration of the complex may be noted. First, complex formation lowers the pK of the secondary phosphate. Second, the hydrolysis of zinc(I1) occurs at higher pH in the presence of the nucleotide.12 These results indicate that the complex formed involves the phosphate of the nucleotide and that the complexed zinc is protected from the action of hydroxide ion. The requirement of doubly negative phosphate prevents formation of the complex at low pH and the hydrolysis above pH 8 destabilizes the complex at high pH. Such a stability range is demonstrated by the spectropolarimetric titration in the presence of poly-L-lysine shown in Figure 4A. The pH titration and the spectropolarimetric titration lead to the same conclusion. The fact that the stability range of the complex as determined by pH titration in the absence of poly-L-lysine and by spectropolarimetric titration in the presence of polylysine is so similar again indicates that a similar complex is formed in the presence or absence of poly-L-lysine. When the concentrations of all components of a 2: 1 :I mixture of poly-L-lysine, 5’-AMP, and zinc(I1) are gradually decreased, the rotatory strength of the SOM lution decreases in such a way that at a 1 X concentration of zinc no more complex formation is (11) G. L. Eichhorn and P. Clark, J . Amer. Chern. Soc., 85, 4020 (1963). (12) A titration was also carried out in the presence of poly-r-lysine; the p K of the secondary phosphate of 5’-AMP is lowered as in Figure 3 . However, the etTect on the Zn(I1) hydrolysis is masked by titration i n that region of the polypeptide e-amino group, whose titration could itself be altered by the presence of the Zn(I1)-AMP complex.
1 September 6, I972
6529
t
10 l1
gt 210
230
250
270
290
310
WAVELENGTH, nm
Figure 5. Comparison of CD spectra at pH 7 of solutions of 1 X M 5'-AMP and 2 X M poly-L-lysine: (- - -) in the abin the presence of 1 X 10-3 M sence of divalent metal ions; (-) zinc acetate; (- - -) in the presence of 1 X Mlead nitrate; (. . .) in the presence of 1 X M copper(I1) chloride. Other metal ions that, like Cu(II), produced no appreciable enhancement are Ca(II), Mg(II), Mn(II), Co(II), Ni(II), Cd(II), Hg(II), Ag(I), Fe(III), Cd(III), and Al(II1).
indicated. This concentration is only slightly higher than the concentration of unprecipitated Zn(I1) at a 1 :1 ratio of 5'-AMP and Zn(I1) in the absence of poly-Llysine (Figure 2). These results indicate that the insoluble complex in the absence of polycation and the polycation-solubilized complex are both disrupted at similar concentrations. The rotatory strength of the complex is not affected by heating to 75" ( 2 X M poly-L-lysine, 1 X M ZII(NO~)~, and 1 X M 5'-AMP at pH 8.0), and is diminished by only 25 at 98". Are Conservative CD Bands Found When Zn Is Replaced by Other Metal Ions? Figure 5 shows the effect on the C D spectrum of replacing Zn(I1) by other metal ions. In the presence of Cu(II), Ca(II), Mg(II), Mn(II), Co(II), Ni(II), Cd(II), Hg(II), Ag(I), Fe(III), Ce(III), and AI(II1) the effect on the circular dichroism of the nucleotide is insignificant. Pb(I1) is the only other metal ion besides Zn(I1) which was found to produce a large effect on the C D spectrum of the nucleotide. Some of these metals have minor effects on the uv spectrum. However, no metal other than Zn(I1) produces the shoulders at 280 and 290 nm, and no other metal produces a conservative C D effect. The near-uv absorption peak of 5'-AMP is shifted in the presence of Pb(I1) to 262 nm with a small decrease in extinction coefficient. The titration curve of a 1:l mixture of 5 ' A M P and Pb(I1) reveals that lead has an even greater effect on the phosphate pK (-5) and the metal hydrolysis (>9) than Zn(I1). On the other hand, lead produces no significant effects on the uv and C D spectra of 3'-AMP and deoxy-5'-AMP, in contrast to the behavior with Zn(I1). Thus, lead exhibits an even greater specificity than zinc in its reaction with the ribosephosphate portion of the nucleotide. These results imply that the effect of lead on 5 ' AMP may be produced by a different type of complex than the effect of zinc. A rotatory strength much greater than that of 5'-AMP has been observed with some adenosine derivatives in which the ribose is rigidly attached to the base.13,14 If the lead causes a (13) D. W. Miles, R. K. Robins, and H. Eyring, Proc. Nar. Acad. Sci. U.S . , 57, 1138 (1967).
1 0
2
4
6
2 4 moles OH-/mole AMP (or Cu) 6
1 0 1 2 0
6
8
1 0 1 2
Figure 6. Titration curves of the copper(I1) complexes of the isomers of AMP, compared with the titration curves of the components: (A) solutions 1 x 10-3 M in nucleotide and (or) Cu(N03)2; M enough H N 0 3 was added to each solution to make it 5 X in H N 0 3 before titration with 5 X 10-2 M NaOH under nitrogen at 25"; (B) solutions 5 X lo-* M in nucleotide and (or) Cu(N03)~; enough HN03was added to each solution to make it 2.5 X 10-I M in HNOI before titration with 2.57 M NaOH under nitrogen at 25"; (-) Cu(NO&; (---)AMP; (---) 3'-AMPandCu(N03)2; (- -) 5'-AMP and Cu(N03)2.
similar rigidity in the ribose-base attachment, the large rotatory strength observed with Pb(I1) can be explained. Alternatively, the C D spectrum can be interpreted as resulting from a complex similar to that found for zinc, with the difference that lead produces a nonparallel interaction between bases. Such an interaction would result in nonconservative C D bands. 15,16 A comparison of the titration results indicates a similarity between the zinc and lead complexes. The failure of Cu(1I) to produce any effect on the C D spectrum is surprising in view of the nmr evidence indicating a stacked structure for the Cu(l1)-AMP complex. The apparent discrepancy between these nmr results, which were obtained at a high concentration of AMP, and the uv and C D results, obtained at a much lower concentration, has perhaps been resolved by titration of Cu(II)-5 '-AMP mixtures at two different concentrations. Figure 6A contains titration curves at 1 X M concentration. At this concentration the pK of the phosphate is lowered in a manner analogous to that observed with zinc. However, one OH- reacts with CU(11) in the presence of 5'-AMP at a lower pH than in its absence; the second OH- requires higher pH. As Figure 3 has shown, both OH- ions in Zn(I1) 5'-AMP are titrated at a higher pH, and this phenomenon has been attributed to a structure in which the zinc ions are relatively protected from the action of OH- ions. Apparently copper ions react with one OH- ion under (14) D. W. Miles, M. T. Robins, R. I
