J. Phys. Chem. 1981, 85,3523-3528
tention for recoil atoms with energies below some threshold of displacement energy, (2) halide-halide substitution reactions (billiard-ball reactions), (3) reactions in which two or more halide ligands are displaced with subsequent filling up of the vacancies by halides from the surroundings, and (4)formation of interstitials which form free halide during the dissolution of the irradiated substances. No ligand vacancy transfer processes as proposed by RosslerlO are needed for the discussion of the experimental results. The only qualitatively slightly different results can be understood by assuming some influence of the recoil energy which increases in the order 82Br< 38Cl < 36 C1. With increasing recoil energy the primary retention and
3523
the direct displacement reaction yields decrease and the yield of the products resulting from processes of larger disorder increases. The free-halide yield increases in the mixed crystals because of the less effective momentum transfer in C1-Br impacts.
Acknowledgment. The investigations were supported financially by Fonds der Chemie, Deutsche Forschungsgemeinschaft, and Freiburger Wissenschaftliche Gesellschaft. The numerical calculations were performed with the UNIVAC 1100/81 computer a t the University of Freiburg. We thank the Kernforschungszentrum Karlsruhe-Isotopenstelle for the irradiations.
Interactions of Ni2+ with Inosine and Inosine 5’-Monophosphate. Equilibria and Dynamics A. Nagasawa’ and H. Diebler” Max-Planck-InstM fur Biophysikalische Chemie, 34 ~ttingen-Nikolausberg,West Germany (Received: Februaty 20, 198 1; In Flnal Form: JuW 7, 1981)
Spectrophotometric techniques have been used to evaluate the strength of binding of Ni2+to inosine (In) and to inosine 5’-monophosphate (IMP). At 15 “C the following stability constants were obtained: NiZt-In, K = 14 M-l (ionic strength, p, = 1.0 M); Ni2+-IMP2-,K = 920 M-* (p = 0.2 M); and Ni2+-IMPH-, K = 75 M-’ ( p = 0.2 M). These values are considerably higher than those for the corresponding adenosine compounds. The rates of the complex formation reactions have been determined by the temperature-jump relaxation method. For Ni2++ In a formation rate constant k f = 790 M-’ 8 ( 1 5 “C, p = 1.0 M) is obtained, a value which is consistent with those of similar ligands. The kinetic data for complex formation between Ni2+and IMP2-are interpreted in terms of a stepwise binding process. The rate of the first step is about typical for substitution at Ni(H20)62t, whereas that of the second step is substantially lower, due to steric effects. All the experimental evidence (spectral changes, complex stability, and kinetic data) indicates that in the IMP complex the nucleotide is bound inner sphere via the phosphate group and the base moiety to the metal ion. Introduction Most reactions in biological systems involving nucleotides require the presence of divalent metal ions. Magnesium ions are of particular importance in this respect. In vitro they can often be replaced by other metal ions like Co2+, Mn2+,or Zn2+. There are indications that interactions of nucleotides with transition metal ions and Zn2+ are of relevance also in biological systems, but not much is known with certainty in this area.2 Nucleotide ligands offer a variety of potential binding sites for metal ions. In addition, these ligand molecules can adopt various conformations and thus exhibit complex steric properties. The whole area of metal ion-nucleotide interactions is therefore of interest also from the point of general coordination chemistry. Main-group metal ions apparently bind only to the phosphate groups of nucleotides, whereas transition metal ions and Zn2+bind to the organic base as ell,^^^ forming a chelate complex. Base binding is most easily recognized by changes in the UV (1) Department of Chemistry, Faculty of Science,Tohoku University, Sendai 980, Japan. (2) (a) L. G. Marzilli, T. J. Kistenmacher, and G . L. Eichhorn in “Nucleic Acid-Metal Ion Interactions”, T. G. Spiro, Ed., Wiley, New York,1980, p 179; (b) G. L.Eichhorn in “Inorganic Biochemistry”, Vol. 2, G. L. Eichhorn, Ed., Elsevier, New York, 1973, Chapters 33 and 34. (3) R. M. Izatt, J. J. Christensen, and J. H. Rvttinn. - -. Chem. Reu... 71.. 439 (1971). (4) A. T. Tu and M. J. Heller in “Metal Ions in Biological Systems”, Vol. 1, H.Sigel, Ed., Marcel Dekker, New York, 1974, p 2.
absorption of the base moiety. Also, the chelate structure leads to an enhanced stability of the metal-nucleotide complex, as compared to binding alone by the phosphate group or the base g r o ~ p . ~The ? ~ formation of a chelate structure is reflected furthermore by the kinetics of the complexation reaction. Relaxation studies of the complex formation reactions of 5’-AMP with Ni2+and Co2+revealed a nonlinear relationship between the reciprocal relaxation time and the reactant concentrations6-8which could be rationalized only by assuming a stepwise binding process in which the monodentate species is not a steady-state intermediate. A detailed analysis of the kinetic data enabled the evaluation of all the rate constants involved and thus also of the stepwise e q ~ i l i b r i a . ~ ? ~ Of particular interest in transition metal ion-nucleotide interactions is the effect of the respective nucleotide base. In the present paper studies are reported on the interaction of Ni2+with inosine and inosine 5’-monophosphate (5’IMP). Like adenosine, inosine (= hypoxanthine riboside) is a nucleoside with a purine-type base. Formally it is obtained by substituting the adenosine’s amino group (at the c6 position) by an OH group, but in solution it is predominantly present in the isomeric keto formeQ The (5) C. M. Frey and J. E. Stuehr, J.Am. Chem. SOC.,94,8898 (1972). (6)R. S. Taylor and H. Diebler, Bioinorg. Chem., 6,247 (1976).
(7) A. Peguy and H. Diebler, J. Phys. Chem., 81, 1355 (1977). (8) J. C.Thomas, C. M. Frey, and J. E. Stuehr, h o g . Chem., 19,505 (1980).
0022-3654/8112085-3523$01.25/0 0 1981 American Chemical Society
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The Journal of Physical Chemistry, Vol. 85,
No. 23,
Nagasawa and Diebler
1981
2-amino derivative of inosine is the biologically important nucleoside guanosine. Inosine itself also occurs in living systems, e.g., in small amounts in some tRNA’s.’O Experimental Section Materials. All chemicals were of the best quality commercially available and were used without further purification: inosine (Serva, p.a.), disodium 5’-IMP (Boehringer, cryst.), nickel perchlorate (Fluka, purum p.a.), sodium perchlorate (Merck, p.a.) NaH2P04(Merck, p.a.), cacodylic acid (Fluka). The concentrations of stock solutions of nickel perchlorate were determined by complexometric titrations with EDTA, using murexide as indicator.’l Stock solutions of inosine and 5’-IMP were prepared by weight and standardized by pH titration and spectrophotometry,using literature values of the extinction coefficient^.^^^^^ Methods. Since the kinetic reaction effects showed somewhat larger amplitudes at lower temperatures, all measurements were carried out at 15 “C (f0.l “C). Except were stated otherwise, the ionic strength was 1.0 M in studies involving inosine and 0.2 M in studies involving 5’-IMP, adjusted with sodium perchlorate. The constants for the protolytic equilibria of inosine and 5’-IMP were determined by potentiometric titrations of M solutions of the substances against 0.05 about 3 X M solutions of NaOH or HC104. pH measurements were carried out by means of a digital pH meter (Radiometer, PHM 52) with a Metrohm EA 125 combined electrode in which the 3 M KC1 solution had been replaced by a 3 M NaCl solution, The pH meter was standardized with Merck “Titrisol” standard buffer solutions of pH 4.0-10.0. Nitrogen was bubbled through the solutions during the pH measurement. The equilibrium constants for the binding of Ni2+to inosine and to 5’-IMP were determined by UV spectrophotometry with a Cary 118 recording spectrophotometer. Since the spectral changes due to binding of Ni2+are rather small, difference absorbance measurements were carried out: (metal ligand) vs. ligand at high sensitivity of the instrument (0.05-0.2 OD units/full scale). A small contribution by free Ni2+to the absorbance was either corrected for or directly compensated by using two-compartment cells in each path: (metal + ligand)(H20)vs. (metal)(ligand), with a light path of 1 cm of each compartment. Studies of the kinetics were carried out by means of the temperature-jump relaxation technique.14 The cell with the solution to be studied was thermostated at 9.7 “C before raising the temperature to 15 “C by discharging a 0.05-pF capacitor of 40 kV. the time constant for the increase in temperature of a solution of ionic strength 0.2 M is about 3 p s at 15 “C. The chemical relaxation process was observed spectrophotometrically at 280 or 265 nm. This is actually off the absorption maximum which is close to 250 nm, but here the relative change in absorption associated with complex formation is large (see Figures 1
+
(9)A. Psoda and D. Shugar, Biochirn. Biophys. Acta, 247,507(1971).
(IO) (a) T. V. Venkstern, “The Primary Structure of Transfer RNA”, Plenum Press, New York, 1973,p 234; (b) R. P. Singhal and P. A. M.
Fallis, Prog. Nucl. Acid Res. Mol. Biol., 23, 227 (1979). (11) G. Schwarzenbach and H. Flaschka, “Die komplexometrische Titration”, F. Enke, Stuttgart, 1965,p 197. (12)G. B. Beaven, E. R. Holiday, and E. A. Johnson in “The Nucleic Acids”, Vol. 1, E. Chargaff and J. N. Davidson, Ed., Academic Press, New York, 1965,p 493. (13) P. L.Biochemicals, Biochemicals Reference Guide and Price List 106, 1979. (14)M. Eigen, and L. De Maeyer in “Technique of Organic Chemistry”, 2nd ed, Vol. VIII/2, A. Weissberger, Ed., Interscience, New York, 1963.
0.61
0.1
n
1
‘\ ‘\ ‘._
A
00 220
2LO
260
280
300
hlnrni
Figure 1. Spectrum of 4.9 X IO4 M IMP in absence of Ni2+(solid line) and in presence of 0.010 M Ni2+(broken line): A = base line (15 “C, p = 0.2 M, pH 7.0).
and 2) and reasonable relaxation amplitudes are obtained. The measured signal was stored in a transient recorder (Datalab DL 905) and displayed on a two-channel oscilloscope. Simultaneously,an exponential signal of variable time constant and amplitude, produced by an electronic device which was constructed by C. R. Fbbl, was displayed on the oscilloscope and fitted to the measured curve. This procedure allows a quick and convenient evaluation of the signals. The relaxation times given in this paper are averages of 5-10 individual determinations. The deviations of the individual values from the mean were usually within &lo%. The pH of the reactant solutions in the spectrophotometric and kinetic studies was stabilized by adding 5 X M of cacodylic acid buffer. A few of the or 1 X kinetic experiments were carried out also with phosphate buffer instead of cacodylate, with no change in the results. At these low concentrations the interaction of the buffers with Ni2+is negligible. Results Equilibrium Studies. Protolytic Equilibria. (a)Inosine. The neutral inosine molecule (HIn) adds a proton to the base (at N7) around pH 1.2-1.5; it loses a proton from the base (N, or C60) around pH 8.9 and another one from the ribose moiety between pH 12 and 13.3 In the present studies only the pK -8.9 might be of relevance. The value of this equilibrium constant was therefore redetermined under the experimental conditions of this study by potentiometric titration of a solution of inosine with a solution of NaOH. The evaluation of 14 points of the titration curve between pH 8.47 and 9.32 (not too far away from pH = pK) gave KI = [HIn]/[H+][In-] = 6.94 (fO.lO) X lo8 M-l (15 “C, p = 0.2 M) or log KI = 8.84 (f0.01). In the determination of KI the activity coefficient y of the H+ ion was taken as yH = 0.80 under the conditions of this study16and the ionic concentration product of water M2. The latter value as Kw = [H+][OH-] = 0.79 x results from the thermodynamic ionization constant of water at 15 “C, Kw’ = 0.45 X M2,16and YH = 0.80, ?OH = 0.71.l‘ (b)5’-IMP. The protolytic equilibria of interest in this study are the addition of a first proton to the dinegative phosphate group around pH 6 and the dissociation of a (15) See, e.g., R. G. Bates, “Determination of p H , Wiley, New York, 1964,pp 52-3. (16) H. S. Harned and B. B. Owen, “The Physical Chemistry of Electrolytic Solutions”, 3rd ed, Reinhold, New York, 1958 p 638.
Ni2+ Binding with In and IMP
0.OL
0.0 2
The Journal of Physical Chemistry, Vol. 85, No. 23, 198 1 3525
1
0.L
j
-
-
0.3 -
E
0.2
-
5
-0.06-
IO
20
15
~ O ~ ( N ~ , +( M L I~ I
Figure 3. 2.
Plots of [Ni],/lA - A,I vs. ([Nil,
+ [L],)
at pH 7.0, see eq
TABLE I: Apparent Stability Constants for Complex Formation of Ni2+ with Inosine Monophosphate (16 "C, = 0.2 M )
5.00 (t0.02) 6.00 (t0.02) 7.00(k0.02) 7.50 (k0.02)
148 (t20) 519 (t45) 847 (+.40) 1143 (t65)
which is valid if (C[NiL])2
