Eur. J. Biochem. 199,659-669 (1991) 0 FEBS 1991

001429569100487X

Acid-base properties of nucleosides and nucleotides as a function of concentration Comparison of the proton affinity of the nucleic base residues in the monomeric and self-associated, oligomeric 5'-triphosphates of inosine (ITP), guanosine (GTP), and adenosine (ATP)

Nicolas A. CORFU and Helmut SIGEL Institute of Inorganic Chemistry, University of Basel, Switzerland

(Received December 21, 1990/March 8, 1991) - EJB 90 1525

The acid-base properties of the nucleic base residues of ITP, GTP, and ATP, and for comparison also as far as possible of the corresponding nucleosides, were studied in dependence on their concentration, i. e. on the effect of self-association. From the dependence between the 'H-NMR chemical shifts of H-2 (where applicable), H-8, and H-l', and the pD of the solutions, the acidity constants for the deprotonation of the D'(N-7) site in D2(ITP)2-, D2(GTP)2-, D(Ino)', and D(Guo)', and of the D'(N-1) site in D2(ATP)2- and D(Ado)' were calculated. Chemical shift/pD profiles for a whole series of varying concentrations of the nucleic base derivatives (= N) were constructed, including those for infinite dilution (&), which give the acidity constant for the monomeric N, and for infinitely concentrated solutions (h,,J, which give the acidity constant of an N in an infinitely long stack. The acidity constants determined from the 6,/pD plots are in excellent agreement with the pK, values measured by potentiometric pH titrations of highly diluted solutions of N. The effects of self-association are striking: e.g. the pKa value of the D'(N-7) site in D2(GTP)2- is lowered by about 1 (as calculated from the So/ pD and 6,/pD profiles), while the pKa value of the D'(N-1) site in D2(ATP)2- is increased by approximately 0.3; i.e. in the first case deprotonation is facilitated and in the second it is inhibited. The increasing inhibition of the H'(N-1) deprotonation with an increasing ATP concentration is due to the high stability of the dimeric [H2(ATP)]i- stack for which the intermolecular H+(N-l)/y-P(OH)(O); ion pairs between the two ATP molecules are crucial. In those cases where no other significant interaction but aromatic-ring stacking in the self-association process occurs, the release of protons from protonated nitrogen-ring sites is facilitated with increasing stacking; this holds not only for D2(GTP)'- as indicated above, but also for D2(ITP)2-, D(Ino)+, and D(Ado)'. The latter example especially suggests that the situation for the D2(ATP)2- system is exceptional. Some consequences of the considered acid-base properties for biological sytems are indicated.

Nucleotides and their metal ion complexes are among the most widely used substrates in cell metabolism [l], reaching rather high concentrations in certain cell organelles. For ex- ample, in the chromaffin granules of the (bovine) adrenal medulla ATP and GTP reach values of z 0.15 M and 0.02 M, respectively [2 - 41. Here evidently interactions and associ- ations must occur among the soluble constituents of the gran- ules to reduce the resultant osmolarity to isotonicity (about 0.3 M) [2], otherwise the granules would be osmotically un- stable. Indeed, in vitro studies suggest that self-association of nucleotides at the mentioned concentrations is significant and occurs via stacking of the nucleic base residues [5, 61.

However, increasing stack formation in a nucleoside or nucleotide system is expected to affect the acid-base properties of the nucleic base residues, because aromatic-ring stacking

Correspondence to H. Sigel, Institut fur Anorganische Chemie der Universitat Basel, Spitalstrasse 51, CH-4056 Basel, Switzerland

Abbreviations. N, nucleoside or nucleoside 5'-triphosphate, i. e. adenosine, inosine, guanosine, ATP, ITP, or GTP; NMP, nucleoside 5'-monophosphate; Ns, nucleoside; NTP, nucleoside 5'-triphosphate.

Definition. The expression 'protonation' is used throughout this study for the addition of H + or D + (= 'H') to a basic site, i.e. independent of the kind of hydrogen isotope. However, which isotope is considered in a given equilibrium is always clearly defined.

brings the sites of protonation, i.e. N-1 of adenine derivatives and N-7 of hypoxanthine or guanine derivatives (cf. Fig. l), close to each other and as a consequence repulsion between these sites should occur. In other words, provided there are no other binding interactions but self-stacking, then the release of protons should be facilitated and the pKa value of the protonated nucleic-base site should consequently be lowered. Obviously, this effect should be especially pronounced in species which are larger than the dimer, i.e. in oligomers, and under conditions which exceed a protonation degree of

To our knowledge, despite the obvious relevance for natural systems, no systematic studies on the acid-base prop- erties of nucleic base residues in dependence on self-associa- tion, i.e. on the concentration, have so far been carried out. The only indication of a self-association effect on acidity con- stants exists for ATP [7]: the acidity of the H'(N-1) site appears to decrease due to ion pair formation in the dimeric [H2(ATP)];- stack. Therefore, the present study was carried out with the aim of characterizing and comparing the acid- base properties of the nucleic base residues in ATP, ITP, and GTP (Fig. 1) [8 - 101 in dependence on the self-association degree; for reasons of comparison, the corresponding nucleosides were included in this study as far as possible.

50%.

660

Fig. 1. Chemicalstructures of the nucleoside 5'-triphosphates considered in this study. The nucleotides are shown in their dominating anti conformation [8 - 101

Our previous studies on the self-association of ATP [7], ITP and GTP [6], as well as of adenosine [ll], inosine and guanosine [6], were based on 'H-NMR shift experiments with varying concentrations of the nucleic base derivatives in DzO at a constant pD value. Hence, it was now possible to use the same data also for an evaluation of the acid-base properties of the nucleic-base residues in dependence on concentration. This procedure was especially appealing as it leads to compre- hensive results, because not only are constants provided, but also information about the protonation sites is obtained [lo]: protonation initiates downfield shifts which are especially pro- nounced for those signals of hydrogens that are close to the binding sites [12, 131.

EXPERIMENTAL PROCEDURES

The disodium salt of ATP (puriss.; research grade) was from Serva Feinbiochemica GmbH (Heidelberg, FRG) and the trisodium salts of ITP and GTP from Sigma Chemical Co. (USA). All the other reagents were the same as used earlier

The acidity constants for the release of a proton from the H+(N-1) or H+(N-7) sites of protonated adenine derivatives of hypoxanthine and guanine derivatives, respectively, were determined by exploiting the experimental data of our pre- vious 'H-NMR studies on the self-association of nucleosides and nucleotides. The corresponding 'H-NMR spectra for adenosine [l 11 and ATP [7] had been recorded with a Bruker WH-90 FT spectrometer (90.025 MHz) at 27°C in DzO as solvent; some of the spectra for inosine, guanosine, ITP, and GTP were measured on the same instrument [14] under the conditions described, but most of the spectra for these systems were obtained more recently with a Varian VXR 400 spec- trometer (399.96 MHz) at 25°C also in DzO solutions [6]. In all these experiments the center peak of the tetra- methylammonium ion triplet was used as internal reference; however, all measured chemical shifts were converted to the 3- (trimethylsily1)propane-1 -sulfonate reference [7,15] by adding

[6, 7, 101.

3.174 ppm (see also [16]). In addition, in all these experiments the ionic strength (4 was adjusted to 0.1 M with NaN03, where necessary; the ionic strengths valid for the various conditions are listed in the tables (vide infra).

From the examples of experimental data given in our previous figures [6, 7, 111, it is evident that the same or very similar concentrations of nucleic base derivatives, N, were used in different series at different pD values, now allowing the selection of series of chemical shifts which hold for a given concentration of N at different pD. As the concentrations used in the different series were not always exactly identical, we allowed a deviation of up to 10% from the anticipated concentration. When no experiment with the desired concen- tration within these limits was available, the chemical shift was taken from the curve calculated with the association con- stant, K,, (Table 1 in [7], and Tables 1 and 3 in [6]), for the corresponding chemical shift-concentration plot. About 20% of the chemical shift/pD data pairs used for the chemical shifts of H-2, H-8, and H-1' of the nucleic base derivatives (Fig. l), i.e. for the corresponding plots on pD, were obtained in this way. These data characterizing the variation of the chemical shift in dependence on the pD for DzO solutions with a con- stant concentration of N were evaluated with a curve-fit pro- cedure [lo]; such chemical shift/pD profiles are shown in Figs $ 6 and 8 (vide infra) in Results and Discussion. The pD of all solutions was measured as in [lo] and [ll].

From the preceding description, it follows that for each nucleoside or nucleotide the pD range available for evaluation is given by the experimental series of the self-association stud- ies; these are listed in Table 1 of [ l l ] (adenosine), Table 1 of [7] (ATP), and Tables 1 and 3 of [6] (inosine, guanosine, ITP, and GTP). For example, for inosine the pD range is between 1 .O - 6.9, and for the curve-fitting procedure at each concen- tration only four chemical shift/pD data pairs are available because self-association was investigated only at four different pD values (Tables 1 and 2 of [6]).

It is evident that the data listed in the tables of our previous work [6, 7, 111 for the chemical shifts at infinite dilution, 6,, and for the shifts of nucleic base derivatives in infinitely long stacks, 6,, may also be used for the determination of acidity constants valid for these conditions. Indeed, the 6,/pD data pairs have led throughout to reliable results; with the 6,/pD pairs the situation is more difficult, as the error inherent in 6, (e. g. Tables 2 and 4 in [6]), which is a consequence of the large extrapolation towards infinitely long stacks (see, e. g., Figs 4-6 in [6]), often prevents a reasonable curve-fit due to the scattering of the data.

THEORY For an evaluation and discussion of the results presented

below, some basic information on the acid-base properties and on the self-association tendencies of the nucleosides and nucleotides considered is necessary. This information is summarized here.

Acid-base equilibria of the nucleosides and nucleotides

In the monoprotonated nucleosides, H(Ns)', the proton is located at the N-1 [8 - 101 or the N-7 site [8,9] of adenosine or inosine and guanosine, respectively.

Deprotonation occurs according to equilibrium (1):

H(Ns)' +Ns + H +

K & N ~ ) = "sl [H+I/[H(Ns)+I. (1 a)

(1 b)

66 1

The neutral nucleosides, inosine and guanosine, may re- lease a further proton from their H(N-1) site [8,9] giving (Ns- H)- with pKE, = 8.76 and pK&, = 9.22 (Sigel, H., unpub- lished results) in diluted aqueous solution, but these de- protonation reactions are not of relevance in the present con- text and are therefore not discussed further.

The fully protonated nucleoside 5'-triphosphates, H5(NTP)+, liberate the first two protons in aqueous solution from the triphosphate chain (Fig. 1) at a very low pH. The resulting H3(NTP)- species release the next proton also from the still twofold protonated triphosphate residue (Eqn 2) [6, 71 :

H,(NTP)- HZ(NTP)'- + H + (2 a) G,(NTP) = [Hz(NTP)~-I [Htl/[H3(NTP)-l. (2b)

Deprotonation of H2(NTP)' - occurs now (mainly) from the monoprotonated nucleic base residue; i. e. from the H+(N-7) site of H2(ITP)2- or H2(GTP)'-, and from the Hf(N-1) site of H2(ATP)2- (Eqn 3):

H2(NTP)2- e H ( N T P ) 3 - + H + (3 a>

K!,(NTP) = [H(NTPl3-I [H+I/[H2(NTP)2-l . (3 b)

In the case of ITP the constants [6] PK;,(,~~) z 1.4 and pK:,(ITP) = 2.19 f 0.05 (Sigel, H., et al., unpublished results) are relatively close, i. e. the deprotonation steps according to equilibria (2a) and (3a) occur in an overlapping pH range, and consequently H2(ITP)2- exists in two isomeric forms: one form carries a proton each at N-7 and the y-phosphate group, H(N-7).ITP.H2-, and the other isomer has both pro- tons at the triphosphate chain, ITP.H:-; this problem is discussed further in Results. For H2(GTP)2- (see also third section of Results) and H2(ATP)2 - practically no isomerism occurs as the corresponding acidity constants are further apart. The remaining H(NTP)3- species carry the proton at the terminal y-phosphate group (Fig. 1); deprotonation occurs close to pH 6.5 according to equilibrium (4):

H(NTP)3-$NTP4- + H + (4 a)

(4 b) K&NTP) = [NTP4-] [H']/[H(NTP)3-].

Finally, ITP4- and GTP4- both can be deprotonated at their H(N-1) site (Fig. 1) with pKkp = 9.12 and pKgTp = 9.58 giving the (NTP-H)5- species (Sigel, H., et al., unpublished results), but these are not of interest in the present context.

For the discussions given under Results it is important to note that acidity constants valid for deprotonations in water may be transformed [17] with Eqn ( 5 ) into the corresponding constants which refer now to D 2 0 as solvent:

( 5 ) pKap20 = 1.015. pKa/H20 + 0.45.

This equation proved to give excellent results for the acid- base reactions of ATP [7, 181 and the adenosine monophos- phates [lo].

Indeed, we have transferred the acidity constants available in the literature for ATP [15, 19,201, ITP, and GTP (Sigel, H., et al., unpublished results) from determinations at low NTP concentrations in water into the corresponding constants for D 2 0 as solvent using Eqn (5) [6, 71. To facilitate the following discussion, these constants valid for diluted NTP solutions were used to calculate the distribution of species in D 2 0 in dependence on pD for the systems with ATP, ITP, and GTP (Fig. 2; from top to bottom).

0 1 2 3 4 5 6 7 8 9 1 0

PD

Fig . 2. Effect of p D on the concentration of the species present in a dilutedsolution of ATP ( top) , ITP (middle), or GTP (bottom part) in D 2 0 ( I = 0.1 M ; 2S°C). The results are plotted as the percentage of the total NTP present. The calculations for ATP are based on the constants pK&ATp) = 2.1, PK:~(AT~) = 4.52, and pK&TP) = 7.04 [7], those for ITP on PK;,(,~~) = 1.9, pK&ITP) = 2.67, P K $ , ~ ~ ) = 7.02, and pKEp = 9.71 [6], and those for GTP on pK&(GTP) = 1.9, pK;2(GTP) = 3.43, pK&Tp) = 7.04, and pKgTP = 10.17 [6]

Self-association tendency of the nucleosides and nucleotides

The 'H-NMR upfield shifts of the resonances of the nu- cleic-base hydrogens observed previously [6, 7, l l , 141 with increasing concentration of the nucleic base derivatives, N, confirm stack formation. They also allow a quantitative evalu- ation of the extent of stacking by employing the isodesmic model for an infinite noncooperative self-association [21- 241, which assumes for all equilibria (6 a) identical equilibrium constants (Eqn 6b):

(N>n + N*(N)fl + 1

K = "I + ll/"fll "I. (6a)

(6 b) This evaluation provides not only the association constant (Eqn 6b), but also the chemical shift, a,, at infinite dilution, i.e. for monomeric N, as well as the chemical shift, a,, for a molecule N in an infinitely long stack.

The results for the nucleosides (Ns) are easy to understand [6] ; the tendency for self-stacking decreases in the series: (a) Ado ( K = 15 M-I) > Guo (8 M-') > Ino (3.3 M-') [14], and (b) Ns > Ns/D(Ns)+ in a 1 : 1 ratio > D(Ns)+. This latter series indicates that progressing protonation of a nitrogen at a base residue inhibits stacking of the aromatic rings due to charge repulsion, as one would expect.

Some of the results for the self-association of the NTPs, which are pertinent for the present investigation, are summarized in Table 1. Fig. 3 gives an indication of the extent of stacking as a function of the concentration and the size of the association constant; the association constants selected

662

Table 1. Comparison of the association constants, K (Eqn 6 b ) , for sev- stacking (Eqn 6 a ) of several differently protonatedforms of ITP, GTP, and ATP us determined by ‘ H-NMR shift measurements in solutions of DzO at 25°C (ITP, GTP) or 27°C (ATP) and I = 0.1 - x 2 M

For comparison, all constants are expressed according to the isodesmic model (Eqn 6). The constants for ITP and GTP are from Tdbk 3 in [6], and those for ATP are from Table 1 in [7]. The values underlined are intra- or extrapolated from the association constant/ pD profiles shown in Fig. 8 of [6] by taking into account the distri- bution curves given in Fig. 2. The average error limits of the values are about

(NaNOd

50% ; for details see [6, 71

Form of NTP K for

ITP GTP ATP

M- ’ NTP4- 0.4 0.8 1.3

D(NTP)3 - / D(NTP)3 0.7 =o.tJ 2.1

D2(NTP)’- = 2 ~ N U z5 200 D3(NTP)- z0.J xi < @

D ~ ( N T P ) ~ - ( I : 1) 2 2.9 6.0

refer to the D(NTP)3-/Dz(NTP)z- 1 : 1 systems of ITP, GTP, and ATP (see Table 1). Further examples of such distribution curves are found in [6] and [7].

Several conclusions may be drawn from Table 1, most of which were discussed in detail previously [6]; only a few crucial points are emphasized now. (a) In contrast to the nucleosides, protonation of the NTPs at first facilitates self-stacking. (b) Consequently, association constant/pD profiles pass through a maximum [6, 71. (c) For ITP and GTP, self-association is most pronounced at the 1 : 1 ratio of D(NTP)3-/D2(NTP)2-. (d) For ATP, maximum self-association is observed for H 2(ATP)2 - .

Furthermore, it is evident from a comparison of the associ- ation constants listed in Table 1 that the situation for H2(ATP)’- must be special due to its very large association tendency. Indeed, the relatively small extent of the upfield shifts, i.e. A 6 = 6,-6,, observed for conditions where the Hz(ATP)2- species dominates (pD z 3.3; cf. Fig. 2) , suggests that mainly dimeric [H,(ATP)];- stacks are formed [7]. In

addition, there is evidence that intermolecular ion pair and hydrogen bonding is responsible for the large stability of the [H,(ATP)]:- stack and the tentative structure shown in Fig. 4 is proposed [7]. The crucial role of the Hf(N-l) / y-P(OH)(O); ion pair (see Fig. 4) is confirmed by the study [6] of the self-association of ITP and GTP; both NTPs carry a hydrogen at N-1 (Fig. l), but no positive charge. Indeed, Hz(ITP)’- and H2(GTP)’- do not show an especially en- hanced self-association, and the corresponding upfield shifts are much larger than expected for a single adjacent aromatic- ring system as in a dimer; hence, with the latter species clearly oligomers are formed.

RESULTS AND DISCUSSION

As indicated above, in the case of ‘pure’ self-stacking, i.e. without any other significant interactions, the formation of oligomers via aromatic-ring stacking should facilitate the re- lease of protons from protonated nucleic base residues. Based on the self-association properties summarized in the preceding section for adenosine, inosine, and guanosine, as well as for ITP and GTP, one expects for all these systems an acidification of the protonated base residues upon stack formation, i.e. with increasing concentration. A closer look at the structure of the dimer shown in Fig. 4 for H2(ATP)’- suggests just the opposite behavior: namely, an inhibition of the deprotonation of the H+(N-1) site due to the formation of an intermolecular H+(N-l)/y-P(OH)(O); ion pair in the dimeric [H,(ATP)]i- stack.

Before the acid-base properties of the NTP systems (Fig. 1) in dependence on the NTP concentration are evaluated, the simpler nucleoside systems will first be considered. In these latter cases no ambiguity in the interpretation of the results is likely, as there are no charge effects of phosphate groups possible and protonation can only occur at the nucleic base residues.

Acid-base properties of inosine, guanosine and adenosine: effect of the nucleoside concentration

The acidity constants determined from the ‘H-NMR shift measurements for the deprotonation of the D’(N-7) site in inosine at various concentrations of Ino are listed in Table 2. It is evident that the individual pK, values calculated for the chemical shifts of H-2, H-8, and H-1’ agree well with each

1:l ratio of h 1.1 ratio of

lOOh -I

5 40-

0 0.1 02 03 M 0 0 1 02 0.3 M 0 0.1 02 0.3 M

[I T PI [GT PI [ATPI Fig. 3. Variation of the proportions of ITP, GTP, or ATP (= NTP) present in the monomer ( I ) , dimer ( 2 ) , timer (3), etc. in D 2 0 solutions at 25°C for ITP and GTP. and 27”C.for ATP (1 = 0.1 to N 2 M , NaN03) as a function of the total NTP concentrations for the D(NTP13-/ D2(NTPI2- 1 : 1 ratios of ITP (K = 2.0 M - ’) [6] GTP (K = 2.9 M - ’ ) [6] , and ATP (K = 6.0 M - ’ ) (71. (See also Table 1)

663

0-P P,q r

Fig. 4. Simplified structure of the dimeric (HZ(ATP)]':- species which is held together by stacking, ionic interactions, and hydrogen bonds. Obviously, this structure could be modified in several ways: e. g. the insertion of a water molecule into the -OH . . . N-7 interaction would widen the 'bite' of the y-0-P-0 claw (see also [7]). The head-to-tail arrangement of the adenine residues with the amino groups on the same side corresponds to a previous crystal structure analysis [25]. The above structure is a modification of a version shown earlier [7]

Table 2. Influence of the concentration of inosine. i. e. of self-stacking, on the size of the acidity constant, K&,,,, (Eqn I b ) , for the deprotona- tion of the D+(N-7) site of D(Ino)' in D 2 0 solution at 25°C (I = 0.1 M ) [6] as determined from ' H - N M R shqt measurements The results were obtained by evaluating experiments such as those shown in Fig. 4 of [6] at a given concentration of inosine; i.e. the 6 versus pD profile for each concentration was evaluated by a curve- fitting procedure [lo] (see also Experimental Procedures). The values given in the first data row for a zero concentration of inosine refer to infinitely diluted solutions; they were calculated with the 6, values given in Table 2 of [6]. The range of error given with the values for pK, of the individual protons corresponds to the standard deviation (lo). The mean value is the weighted mean of the individual results; the range of error given here includes the internal and external errors and corresponds to twice the standard error (20). A general statement may be added: the acidity constants are always defined for indepen- dent (monomeric) sites (Eqns 1-4); this is also true for theevaluations at higher concentrations, as well as for the 6,/pD data pairs. As the curve fit was always excellent (see Figs 5 , 6 and 8), use of a more complicated cxpression applying overlapping pK, values for species with a different association degree would not be justified. These re- marks apply equally to Tables 3 and 4

Experi- pK, determined from the shift of PK&,,, mental (mean) [Ino] H-2 H-8 H-1'

0 1.55 f 0.06 1.52 f 0.02 1.56 +_ 0.05 1.53 f 0.04 0.005 1.58 f 0.06 1.54 f 0.02 1.56 f 0.05 1.55 k 0.04 0.010 1.54 f 0.06 1.48 +. 0.02 1.52 f 0.05 1.49 f 0.04 0.020 1.56 f 0.08 1.49 f 0.02 1.54 f 0.07 1.50 +_ 0.04 0.040 1.58 f 0.08 1.53 f 0.02 1.57 k 0.04 1.54 +_ 0.03 0.070 1.47 f 0.10 1.39 +_ 0.02 1.43 f 0.07 1.40 f 0.04 0.10 1.54 f 0.10 1.48 f 0.02 1.52 f 0.09 1.48 f 0.04

other for a given Ino concentration. That the error limits of the results obtained with the shifts of H-8 are smaller than those calculated from the shifts of H-2 and H-1' is in agree- ment with its larger chemical shift difference, which is downfield as expected; this difference decreases in the order H-8 > H-2 YH-1'. It is also expected that the downfield shift connected with the protonation of the N-7 site is most pronounced for H-8 as it neighbors N-7.

The mean values for pK&,,,) (Eqn 1 b) given in the column at the right in Table 2 indicate (especially when a graphical plot is made, see below) overall a slight but clearly not very pronounced decreasing trend of the acidity constants with

increasing concentrations of inosine. This result may at first sight appear disappointing, but actually it is not too surprising as stack formation is not very significant in the concentration range covered: even in 0.1 M inosine solution (which is the limit due to insolubility) [6, 141 the monomeric species still dominates with about 75%, next to the dimer with only about 20% (calculated with K = 1.9 M- ' [6]; cf. also the left part of Fig. 3). Indeed, the mean for all data in the concentration range of 0.005-0.10 M gives PK&~,,) = 1.49 f 0.03 (2a) which is identical within the error limits with pK&,,,) = 1.53 f 0.04 (first row in Table 2), calculated from the 6, values given in Table 2 of [6]. Transformation of these two results to aqueous solution (25°C; I = 0.1 M) using Eqn ( 5 ) gives pK&lno) = 1.02 0.03 and 1.06 f 0.04, respectively; it is very satisfying that these results agree excellently with the value determined in diluted aqueous inosine solutions by po- tentiometric pH titrations: pK~(,,,) = 1.06 k 0.15 (25°C; I = 0.1 M, NaNOJ [26]. Overall, the important result from these evaluations for inosine is that they prove that the 'H-NMR shift data provide reliable acidity constants.

The solubility of guanosine is too low [6, 141 to attempt an evaluation of the acidity constant for the D'(N-1) site in dependence on the concentration; hence, only the 6, values of Table 2 in [6], together with the corresponding pD values, were used for a calculation. The chemical shifts 6, for H-8 gave pKa = 2.59, those for H-1' 2.58, leading to the average pK&,,) = 2.59 -t 0.10 (Eqn 1 b). The error range given is an estimate because the curve-fit had to be carried out with the three available 6,/pD data pairs [6] only and therefore no error limit is obtained for the pKa values from the mathemat- ical treatment, as the fit also involves three variables (pK,, ~ D ( G , , , ) , 6~,,,) [lo]. Transformation using Eqn (5) of the acidity constant, valid for D 2 0 solutions, to aqueous guanosine solu- tions gives pK;,,,,, = 2.1 1 ; this result is identical with that obtained from potentiometric pH titrations of highly diluted aqueous guanosine solutions: PK&~,,,) = 2.11 f 0.04 (25°C; I = 0.1 M, NaN03) [26].

Evaluation of the available three 6,/pD data pairs (Table 2 of [l 11) for adenosine gives for the monomeric nucleoside PK&~,,) = 4.17 -t 0.05; this value agrees well with an earlier determination [lo] also by 'H-NMR in D20: PK&Ado) = 4.14 f 0.05 ([Ado] = 2.5 mM; I = 0.1 M, NaN03; 27°C). Trans- formation of these results with Eqn (5) for aqueous solutions gives PKE(Ado) = 3.67 and 3.64, respectively, in good agree- ment with the acidity constant obtained from potentiometric pH titrations in diluted aqueous solution: pK&Ad,) = 3.61 f 0.03 ( I = 0.1 M, NaN03; 25°C) [lo]. However, the main interest is the calculation based on the 6JpD pairs (Table 2 of [l 11) even though in this case only a limiting value can be obtained, i.e. pK&,d,) < 3.6, for the deprotonation of the D'(N-1) site in infinitely long stacks. Hence, acidification of the D'(N-1) site upon self-stacking is certain: ApK, =

Furthermore, the occurrence of some acidification upon stack formation with inosine is also indicated, despite the limited data set, by the negative slope of the least-squares regression line through the PK&,~,) versus [Ino] plot for the data of Table 2: y = -(0.93 f 0.50) . x + (1.54 f 0.02).

These results for adenosine and inosine (see also following section regarding the related ITP) prove that stack formation leads to an acidification of H(Ns)' independent of the lo- cation of the proton, which may be at N-1, as in adenosine, or at N-7, as in inosine. In accordance with this is the decreas- ing stacking tendency in the series, Ns > Ns/D(Ns)+ (1 : 1) > D(Ns)+ (cf. second section of Theory), because stack for-

D D PKD(Ado)/6,-PKD(Ado)/6, = (<3.6)-4.17 <

664

Table 3. Influence ofthe concentration of ITP, i.e. of self-stacking, on the size of the acidity constant, K&ITP) (Eqn 36 ) , for the deprotonation of the D'(N-7) site of DZ(ITPJ2- in D 2 0 solution at 25°C (I = 0.1 - 0 2 M ) [6] as determined from ' H - N M R shift measurements It should be emphasized that the values given in this table for pK&ITp) are actually micro acidity constants (see Results) for the H+(N-7) site. The remarks in Table 2 about the range of error given for the pK, values apply equally to the values in this table. The results were obtained by evaluating experiments such as those shown in Fig. 5 of [6] at a given concentration of ITP (see Fig. 5 and Experimental Procedures). The values given in the first data row for a zero concentration of ITP refer to infinitely diluted solutions; they were calculated with the 6, values of Table 4 in [6]

Experimental 1 [ITPI

pKa determined from the shift of

H-2 H-8

0 0.0028 0.0055 0.010 0.020 0.040 0.069 0.106 0.144 0.176 0.296 0.350

M 0.1 0.1 0.1 0.1 0.1

00.2 00 .3 00 .5 00 .7 00.8 01 .5 01.7

2.41 T 0.14 2.39 f 0.13 2.32 & 0.08 2.39 f 0.07 2.35 f 0.07 2.24 f 0.08 2.16 f 0.06 2.16 f 0.03 2.08 f 0.06 2.04 & 0.05 1.95 f 0.05 1.96 & 0.07

2.39 f 0.11 2.21 f 0.16 2.23 f 0.12 2.38 f 0.08 2.37 + 0.07 2.24 f 0.09 2.18 f 0.06 2.15 f 0.02 2.03 f 0.08 1.94 f 0.07 1.77 f 0.09 1.70 f 0.10

2.41 f 0.14 2.35 f 0.13 2.34 f 0.08 2.41 f 0.07 2.36 If: 0.07 2.25 f 0.08 2.16 f 0.07 2.15 f 0.04 2.09 f 0.05 2.06 f 0.04 2.01 f 0.03 2.02 f 0.06

2.40 f 0.15 2.33 f 0.16 2.31 f 0.10 2.39 f 0.08 2.36 f 0.08 2.24 f 0.10 2.17 f 0.07 2.15 f 0.03 2.08 f 0.07 2.03 f 0.06 1.98 f 0.09 1.94 f 0.16

mation has to inhibit protonation if ring protonation inhibits stack formation.

Self-association reduces the proton affinity of'the hypoxanthine residue in ITP

Evaluation of the available 'H-NMR shift data (Tables 3 and 4, and Fig. 5 of [6]) gave the results listed in Table 3 for the acidity constant due to the deprotonation of the D ' (N-7) site (Eqn 3b) in D2(ITP)2-. The shift difference, 6D(ITP) - is downfield and decreases in the series H-8 > H-2 % H-1' as expected (Fig. 5). Somewhat surprising is the fact that the protonation state of the phosphate group is not sig- nificantly reflected in the chemical shifts of the hydrogens mentioned (see Fig. 5); the same is seen with GTP (Fig. 6; vide infra), but not with ATP (Figs 7 and 8, and [7]). This property of the chemical shifts means that the micro acidity constant for the deprotonation of the H t (N-7) site in H2(ITP)'- at different ITP concentrations is actually deter- mined.

Comparisons in Table 3 show that the individual pKa values calculated for the chemical shift of H-2, H-8, and H-1' at a given ITP concentration agree within one standard deviation; only at ITP concentrations of 0.296 M and 0.35 M are the errors larger, but the values still agree within two standard deviations. Hence, it may definitely be concluded from the acidity constants listed in the column at the right in Table 3 that, with increasing stack formation, an increasing acidification of the D+(N-7) site occurs. For example, the acidification due to stacking between an 0.35 M ITP solution (pK, = 1.94 0.16), which contains the ITP in the form of about 50% monomers, 30% dimers, 12% trimers and 5% tetramers (calculated with K = 2 M-'; see the left part of Fig. 3), and an infinitely diluted solution, which contains only monomeric ITP (pK, = 2.40 k 0.15), is quite remarkable, i.e. A pKa = -0.5 k 0.2, and far beyond any experimental error. This result confirms those of the preceding section and proves further that oligomer formation of nucleic base derivatives via stacking decreases the basicity of the neutral site (in this case

63

61

5 . 9 5 0 1 2 3 L 5 6 7 8 9

PD

Fig. 5. Variation ofthe chemical shut for H-2, H-8 and H-1' of ITP in dependence on p D f o r various concentrations of ITP. [ITP]: (1) 0 M (= 6, values from Table 4 of [6]) (e); (2) 0.040 M (0); (3) 0.144 M (0); (4) 0.350 M (9). The spectra were measured in D 2 0 at 25°C (for I see Table 3) on a Varian VXR 400 spectrometer at 399.96 MHz, relative to internal (CH3)4N'/NO; and converted to values relative to sodium 3-(trimethylsilyl)propane-l -sulfonate by adding 3.174 ppm (Experimental Procedures) [6]. The solid curves are the computer- calculated best fits [lo] of the experimental data which result in the individual pK, values given in Table 3

N-7) because, with increasing protonation, charge repulsion within the stack occurs.

It should be emphasized that the described acidification is not an ionic strength effect. This is evident from the following pK!2(ATP) values which hold for the deprotonation of the H t (N-1) site in H2(ATP)'- and which were determined by potentiometric pH titrations in strongly diluted aqueous ATP solutions (25" C) of different ionic strength (adjusted with

665

NaN03) [7]: pKEZ(ATp) = 3.99 f 0.02 ( I = 0.1 M; [ATP] = 0.00025 M), 4.01 f 0.01 (I = 0.1 M; [ATP] = 0.0005 M), and 3.99 f 0.03 (I = 1.0 M ; [ATP] = 0.0005 M). These acidity constants are identical within their error limits; hence, there is clearly no ionic strength effect in the range of 0.1 - 1.0 M for the deprotonation of H’(N-1) and the same may safely be surmised for the H ‘(N-7) site; in addition, organic solvents like 1,4-dioxane also affect the basicity of the N-1 site relatively little [7, 10, 151. This is very different for the acid- base properties of phosphate groups; these are strongly affec- ted by changes in ionic strength [7] or the addition of organic solvents [7, 10, 15, 271.

There is one further important aspect: the acidity constant determined by potentiometric pH titrations in aqueous,

2.19 & 0.05); (Sigel, H., et al., unpublished results) if trans- formed with Eqn (5) to D 2 0 solutions (pKE2(ITp) = 2.67 f 0.05) apparently agrees poorly with the corresponding value in Table 3 for an infinitely diluted ITP solution in D 2 0 (pK&lTP) = 2.40 f 0.15). However, this apparent discrepancy is easily resolved : the value determined by potentiometric pH titrations in H 2 0 is a macro acidity constant for the species H2(ITP)’-, ignoring the location of the protons. Indeed, there are two isomers present, one with both protons at the phos- phate chain, ITP.Hi-, and another one with one proton at N-7 and the other at the triphosphate residue, H(N-7) . ITP . H’- (see first section of Theory). As stated above, the value determined now by the ‘H-NMR shift experiments is actually a micro acidity constant, i.e. p,kg2!;)’?&DD = 2.40 f 0.15 (= pK&(lTP) in Table 3) which describes the release of the proton from the Dt(N-7) site in D(N-7).ITP.DZ-.

Indeed, the micro acidity constant mentioned fits excel- lently into the general picture as is evident from the following reasoning. In inosine and guanosine no ambivalency exists as the proton can be located only at N-7; on the other hand, the basicity of N-7 in Hz(ITP)’- and H2(GTP)’- should be influenced in exactly the same manner by the presence of a monoprotonated triphosphate chain. Hence, if the micro acidity constant is used for H(N-7).ITP.HZ - and the macro acidity constant for H2(GTP)2-, as in this latter species practi- cally no ambivalency exists (see second section of Theory and next section), the differences between corresponding acidity constants should be equal. These dpKa values for D 2 0 as solvent are PK&~) -pK&lno) = (2.59 f 0.10) -(1.53 f 0.04) (see preceding section and Table 2) = 1.06 f 0.11 and pKEZ(GTp) - pk$&)’.;F,DD = (3.52 f 0.07) -(2.40 f 0.15) (see Tables 3 and 4 and next section) = 1.12 0.17; it is evident that both differences agree excellently within their error limits. Now that the micro acidity constant, pkH(N-7). ITP. H , is known, the complete micro constant scheme [28, 291 for the equilibrium H(N-7).ITP.H, $ (N-7).ITP . H3- + 2 H + can be resolved; as this scheme is of no interest in the present connection, it will be presented elsewhere.

strongly diluted ITP solutions for H2(ITP)’- - -

(N-7) ITP H

Effect of self-association on the protonation degree of the guanosine residue in GTP and comparison with the situation in ITP

The influence of self-stacking on the Hf(N-7) site in Hz(GTP)’- is expected to correspond to the observations made with ITP; this is indeed largely the case but, for GTP, a pK, value of a H ‘(N-7) site in an infinitely long stack could also be calculated. The chemical shifts of H-8 and H-1’ of GTP are plotted in dependence on pD for several concentrations of

90

88 GTP

60

58

--._.

PD

Fig. 6. Variation of the chemical shifi for H-8 and H-I’ of GTP in dependence on p D for various concentrations of GTP. [GTP]: (1) 0 M (6, values from Table 4 of [6]) (0 ) ; (2) 0.0307 M (0); (3) 0.106 M (0); (4) 0.243 M (0); ( 5 ) 0.351 M (0); (6) co (6, values from Table 4 of [6]) (0). The measurements were made in D 2 0 at 25°C (for I see Table 4; see also legend to Fig. 5 and Experimental Pro- cedures). The solid curves are the computer-calculated best fits [lo] of the experimental data which result in the individual p K , values given in Table 4. The broken-line curve is the computer-calculated best fit through only five 6,/pD data pairs, i.e. the poorly fitting point at pD 3.44 is deleted; the resulting two individual pKa values are given in the third section of Results

GTP in Fig. 6; even without any mathematical treatment, it can be clearly seen that the buffer region of the h/pD profiles shifts to lower pD values with increasing GTP concentration, indicating that the D ’ (N-7) site is increasingly acidified. The solid curves shown in Fig. 6 are the computer-calculated best fits of the experimental data; the resulting acidity constants for the D ‘(N-7) site in D,(GTP)’- are summarized in Table 4.

The acidity constant measured in D 2 0 for infinitely diluted GTP solutions ( P K & ~ ~ ) = 3.52 f 0.07) may be transformed with Eqn ( 5 ) into the corresponding constant for an aqueous solution: pK!2(GTp) = 3.02 f 0.07. This value is in fair agree- ment with the acidity constant determined by potentiometric pH titrations in diluted aqueous GTP solutions: 2.94 f 0.02 (25°C; I = 0.1 M, NaN03; Sigel, H., et al., unpublished results). It may be added that the least-squares regression line through the first seven pK,/[GTP] data pairs, i.e. for [GTP] = 0.0028-00.153M,ofTable4~ = -(1.30 f 0.12).x + (3.41 f O.Ol)] (cf. also Fig. 9, vide infra) gives an intercept with the y-axis that is identical with the weighted mean of the pKa

3.41 f 0.06 (2a); this value, and the one derived from it for water as solvent (pKi2(GTP) = 2.92 f 0.06), also agree well with the corresponding constants mentioned. Hence, these NMR results confirm further that the proton in Hz(GTP)’- is overwhelmingly bound at the H ‘(N-7) site (see Theory).

The acidification that the H+(N-7) site experiences upon self-association in GTP is comparable to that in ITP, as a comparison of the acidity constants for 0.35 M NTP solutions with those of 0.0028 M solutions demonstrates (cf. Tables 3 and 4): ApK, z -0.4 for both NTP systems. Hence, this

values for the two lowest GTP concentrations: pK&(Gm - -

666

Table 4. Influence of the concentration of GTP, i.e. of self-stacking, on the size of the acidity constant, K&GTP) (Eqn 3 b ) , for the deprotona- tionofthe D+(N-7) si teofD,(GTP)=- in D20solutionat2S0C (I = 0.1 - z 2 M ) [6] as determined from ‘ H - N M R sh$t measurements The results were obtained by evaluating experiments such as those shown in Fig. 6 of [6] at a given concentration of GTP (see Fig. 6 and Experimental Procedures). The values given in the first data row for a zero concentration of GTP refer to infinitely diluted solutions and those given in the last row to infinitely concentrated solutions; i.e. to conditions of complete stack formation; the results were calculated with the values given in Table 4 of [6] for 6, and a,, respectively. The remarks in Table 2 about the range of error given for the pK, values apply equally to the values in this table

Experi- I pK, determined from the shift of pK&(GTP) mental (mean) [GTPI H-8 H-I’

0 0.0028 0.0055 0.0102 0.0307 0.065 0.106 0.153 0.196 0.243 0.297 0.351 co

M 0.1 3.51 f 0.05 3.53 f 0.05 3.52 f 0.07 0.1 3.41 f 0.09 3.40 f 0.05 3.40 f 0.09 0.1 3.43 f 0.08 3.40 f 0.05 3.41 f 0.08 0.1 3.41 f 0.05 3.36 f 0.02 3.37 f 0.04

-0.2 3.42 f 0.03 3.35 f 0.03 3.39 i 0.07 -0.3 3.35 f 0.02 3.27 f 0.06 3.34 f 0.05 -0.5 3.27 & 0.02 3.17 f 0.07 3.26 f 0.05 -0.7 3.22 & 0.02 3.04 f 0.08 3.21 -t 0.08 -0.9 3.16 f 0.03 2.93 f 0.07 3.12 f 0.17 -1.2 3.12 f 0.03 2.85 f 0.08 3.09 f 0.18 -1.5 3.08 & 0.04 2.79 f 0.06 2.99 -t 0.27 -1.7 3.06 f 0.05 2.74 0.08 2.97 f 0.29

2.39 f 0.11 3.45 f 0.28 2.5 f 0.7

H-8

H-2 - H-1’ A

- 8

Fig. 7. Variation of the chemical shift, ti,, for H-2, H-8 and H-1‘ at infinite dilution of ATP in D 2 0 in dependence on pD at 27°C (I = 0.1 M , N a N 0 3 ) . (See also Experimental Procedures.) The plotted So/ pD data pairs are from Table 2 in [7]; for H-8, no more data are available (for details see [7]). The solid curves shown are the computer- calculated best fits of the 6JpD data pairs by employing pK&(ATP) = 2.1, pK&(ATp) = 4.52, and pK&ATP) = 7.04 (these acidity constants are from [7]). The vertical dotted lines indicate pD = pK,, where the values for pK, correspond to the given acidity constants

result confirms again (see the two preceding sections) the expectation mentioned in the introduction that oligomer for- mation leads to an acidification of the Hf(N-7) site. A more appropriate comparison may even be the one between the pK&TP) values valid for an infinitely concentrated GTP solu- tion, which describes the properties of GTP in an infinitely long stack, and an infinitely diluted solution, that quantifies the properties of monomeric GTP: ApK, = ~K&(GTP),s, - pK;2(GTP),60 = (2.5 0.7) - (3.52 f 0.07) = -1.0 f 0.7 (Table 4).

This acidification of A pK z 1 is quite impressive despite its relatively large error range (20). This error mainly originates in the extrapolation inherent in 6, (see e. g. Figs 4 - 6 and Table 4 in [6]). Indeed, from the 6,/pD profiles shown in Fig. 6 it is evident, especially from that for H-l’, that the 6, value at pD 3.44 fits poorly on the calculated curve. Deletion of this S,/ pD pair leaves only five data pairs for the curve-fitting pro- cedure giving the results pK, = 2.38 * 0.05 (lo) and 2.88 & 0.04 for the shifts of H-8 and H-l’, respectively, and for the weighted mean (including the external error) pK&GTP) = 2.7 f 0.5 (2o), which again describes the acid-base properties of the N-7 site in a GTP molecule in an infinitely long stack. Comparison of these results with those in the bottom row of Table 4 shows that the pK, value obtained from the shifts of H-I’ in particular has changed and that the error limits have become smaller, although the final results are still close, i.e. pKE2(GTPl = 2.5 f 0.7 and 2.7 f 0.5. Moreover, the extent of acidification as described in the preceding paragraph has not significantly changed; the new result is ApK, = -0.8 f 0.5. Hence, these calculations and comparisons prove that, despite the relatively large error limits, the trends are un- equivocal and the conclusions are based on solid grounds.

Self-association enhances the proton affinity of the adenine residue in ATP

The chemical shift data for a quantitative evaluation of the values for PK;,(,,~~) in dependence on the ATP concen- tration are available from our previous study of the self- association of ATP [7]. Fig. 7 shows plots for the chemical shifts, So, of H-2, H-8, and H-1’ of ATP in infinitely diluted D 2 0 solutions in dependence on pD. In contrast to the experi- ence with ITP (Fig. 5) and GTP (Fig. 6), the chemical shifts of these ATP hydrogens are also somewhat (more) sensitive to acid-base reactions at the triphosphate chain and not only to those at the nucleic base residue. A fit of the chemical shifts by varying all three acidity constants (pK;,(ATp), pK&(ATP), pK&ATp)) of D,(ATP)- is not possible however, because, due to the small shift differences, not enough data points are available for a resolution (cf. the situation for AMP in [lo]). Therefore, the known pK, values [7] were used to calculate, together with the 6,/pD pairs, the shift/pD profiles, i.e. the solid curves seen in Fig. 7.

Despite this shortcoming, these shift/pD profiles clearly show (cf. with the top part in Fig. 2) that between pD 3 - 5.8 the chemical shift is largely determined by the acid-base reaction at N-1, i.e. by equilibrium (3a). Hence, we used the chemical shifts available [7] for an evaluation in this pD range. Unfortunately, only the chemical shift of H-2 is large enough at all ATP concentrations for a suitable curve-fitting pro- cedure and therefore only these results are listed in Table 5. However, using these pK;,(ATp) values, a perfect fit of all the other accessible data for H-8 and H-1’ is also obtained, as demonstrated in Fig. 8 for the extremes, i.e. for the 6,/pD and 6,/pD data pairs.

Indeed, the following comparison shows that the calcu- lated acidity constants are reliable. Using Eqn (9, the acidity constant determined from the ‘H-NMR shift experiments for

667

Table 5. Influence ofthe concentration of ATP, i. e. of self-stacking, on the size of the acidity constant, K&(aTPl (Eqn 3 b ) ,for the deprotonation o f the D' (N-1) site of D2(ATP)2- in D 2 0 solution at 27°C (I = 0.1 - N I M ) 171 as determinedfrom ' H - N M R shqt measurements The results were obtained by evaluating experiments such as those shown in Fig. 3 of [7] at a given concentration of ATP (see also Fig. 8 and final section of Results); in the present case, only the chemical shifts due to H-2 (see Fig. 1 ) were suitable for the curve-fitting pro- cedure (Fig. 8). The results given in the top and bottom data row were calculated with the values for 6, and A,, respectively, listed in Table 2 of 171

Experimental [ATP] I PK&ATP)

0 0.0025 0.0055 0.0101 0.0202 0.051 0.079 0.100 0.153 0.200 X

M 0.1 0.1 0.1 0.1 0.1

x 0.2 - 0.3 -0.4 -0.6 -0.8

4.53 f 0.17 4.64 f 0.09 4.57 f 0.11 4.68 f 0.05 4.66 f 0.02 4.62 & 0.10 4.54 0.04 4.56 f 0.04 4.56 f 0.04 4.60 f 0.03 4.85 f 0.06

infinitely diluted D2(ATP)2- in D 2 0 solution (pK;,(ATp) - - 4.53 f 0.17) may be transformed into the corresponding value in aqueous solution: pKE,(ATP) = 4.02 f 0.17. This result is in perfect agreement with constants determined by potentio- metric pH titrations (25°C; I = 0.1 M) in strongly diluted aqueous ATP solutions: ~K&(A,,, = 4.01 f 0.01 [15] and 4.00 f 0.01 [19].

A detailed interpretation of the acidity constants listed in Table 5 is made easier by graphical presentation. Therefore the available pKE2(ATp) values are plotted in Fig. 9 in depen- dence on their corresponding ATP concentration, together with results obtained for D2(ITP)'- and D2(GTP)2- (Tables 3 and 4). We are not sure that in the ATP systems the displace- ment towards larger pK, values is real for the five data points due to the low ATP concentrations between 0.0025 - 0.051 M, as the deviations are mostly within the error limits. It could however be that at these low ATP concentrations dimers are formed preferably, e.g. use of the (rather low) association constant K = 10 M- ' (see Table 1, and especially Table 1 in [7]) indicates that in 0.02 M solutions 20% dimers are already present (cf. also Fig. 3), and that these strongly inhibit the release of the proton from the D+(N-1) site, in agreement with the hypothesis given above (introductory paragraph to Results) and that this effect is partially offset with the enforced formation of longer associates at higher ATP concentrations. It should be recalled that the transfer from oligomers (dominating at pD > 4.5) to dimers (dominating at pD < 4.5) and vice versa occurs in the pD range of the pKE2(ATP) value [7]. A further observation, supporting the indicated idea, was made with potentiometric pH titrations of aqueous solutions containing different concentrations of ATP: pK:2(ATP) = 4.24 & 0.02 for [ATP] = 0.030 M and pKi,(ATP) = 3.99 f 0.02 for [ATP] = 0.0025 M gives the large positive ApK, = 0.25 f 0.03 ( I = 0.1 M, NaN03; 25"C), whereas pKE,(ATp) = 4.10 & 0.02 for [ATP] = 0.20 M and pKi2(ATP) = 3.99 f 0.03 for [ATP] = 0.005 M gives only ApK, = 0.11 0.04 ( I = 1.0 M, NaCIO,; 25°C) [7]. It is amazing how close these differences are to the corresponding

Fig. 8. Variation of the chemical sh$ at 27°C for H-2, H-8 and H-I' at (0) infinite dilution (tio; I = 0.1 M , N a N 0 3 ) of ATP in D 2 0 and (a) in an infinitely long stack (6,; I F=Z I M ) in dependence on pD. (See also Experimental Procedures.) The plotted A,/pD and 6JpD data pairs are from Table 2 in [7]; for H-8 no more data are available (for details see [7]). The solid curves are the computer-calculated best fits [lo] of the five AJpD and 6,/pD data pairs (see final section of Results) available for H-2 which result in the individual pK, values given in Table 5. The broken-line curves are the computer-calculated best fits of the data pairs for H-8 and H-I' by using the pK, values (see Table 5 ) obtained from the chemical shifts for H-2

4.81

I . . I , . , .$+

0 0.1 0.2 0.3 M 00

"TPI Fig. 9. Dependence of the negative logarithm of the acidity constant, pK&NTp,, f o r the D ' (N- I ) deprotonation of DZ(ATP)'- and for the D'(N-7) deprotonation of Dz (GTP) ' - or D2(ITP) ' - on the N T P concentration. The plotted data are from Tables 3 to 5 ; it should be noted that for ITP the micro acidity constant for the D+(N-7) deprotonation of the D(N-7) . ITP . D2- species is plotted (see second section of Results)

668

ones in Fig. 9. That an ionic strength effect is not observed here was discussed in the second section of Results and is also evident from the two pK, values given which are valid for low concentrations of ATP at different ionic strengths.

Whether the indicated effect at the lower ATP concen- trations is real or not, the overall trend is definitely clear: the release of the proton from the D’(N-1) site is inhibited with an increasing ATP concentration, i.e. with the formation of stacks (Fig. 9; Table 5). Indeed, comparison of the pKa value for infinitely concentrated solutions ( h a ; Fig. 8) with that of infinitely diluted solutions (do; Fig. 8) gives an unequivo- cally positive A pKa difference: A pK, = pK&(ATP),S, -PK;~(ATP),S, = (4.85 k 0.06) -(4.53 & 0.17) = 0.32 f 0.18 (Table 5).

CONCLUSIONS In the second and third sections of Results, it was seen

that oligomer formation leads to an acidification of the H+(N- 7) site of ITP and GTP. In the case of ATP, the most basic site at the purine residue is N-1 [7, 8, 261 and there is strong evidence [6, 71 (see second section in Theory) that this site is involved in the stabilization of dimeric [H,(ATP)]‘:- stacks by forming an ionic bridge (hydrogen bond) between the H+(N- 1) of one H2(ATP)’- and the y-P(OH)(O); group of the other as is indicated in Fig. 4. Clearly, if this hypothesis about the structure of the [H,(ATP)]$- dimer is correct then the release of the proton from the H+(N-1) site should be inhibited upon formation of these stacks, as pointed out in the introductory paragraph of Results; that this is indeed the case is described in the last section of Results. This means that the dependence of the acid-base behavior on the concentration of the purine residue in ATP is the opposite to that found for ITP and GTP, as is very evident from the pK,/[NTP] profiles shown in Fig. 9. Hence, these results correspond to expectation and confirm the special role of the H+(N-1) site in the stabilization of the dimeric [H,(ATP)]‘:- stacks; in other words, the ionic interac- tions contribute significantly to the large stability of these dimers (Fig. 4).

That the acid-base properties of the H(ATP)3-/ H2(ATP), - system in their dependence on concentration are ‘unique’ (i.e. ApK, = pKg2(ATP),d, = +0.3) is also confirmed by a tentative evaluation for the H(AMP)-/ H2(AMP)’ system. With the chemical shift data for H-2, H-8, and H-1’ given in [ l l ] for 6, and h,, we calculated the corresponding pK&AMp) values: despite shortcomings in the available data set which is incomplete and partially impaired by solubility problems, the trend is clearly opposite to that observed for ATP; i.e. the estimate for AMP gives ApK, z -0.1. Indeed, here also the evaluation for the Ado/ D(Ado)+ system presented in the first section of Results should be recalled; although only an upper limit for the differ- ence, A pK, = pK&Ado),d, - pKD(Ad,),a,, D could be obtained, this difference is definitely negative, i. e. A pKa < - 0.6. Hence, in the AMP and adenosine systems H+(N-l), like H+(N-7) in the ITP and GTP systems, is becoming more acidic upon stack formation. These latter results with AMP and adenosine are not only interesting as they confirm the ‘uniqueness’ of the situation in the dimeric H,(ATP)]i- stack (Fig. 4), but also because they show that a phosphate residue is compulsory and even that a monophosphate group is still too small. Clearly, under this aspect, a study of the self-association of ADP in dependence on pD is quite desirable.

Finally, it should be pointed out that the observed change of the acid-base properties of nitrogen sites at nucleic base

residues upon oligomer formation via self-association is evi- dently a very subtle tool for nature as changes can occur in both directions: in ITP and GTP oligomer formation con- siderably lowers the pK, of the H+(N-7) site thus facilitating the release of the proton, while stack formation with ATP increases the pKa of the HC(N-1) site, inhibiting the release of the proton (Fig. 9). A combination of these effects with those created by a reduced solvent polarity [30], i.e. by a reduced so-called ‘effective’ or ‘equivalent solution’ dielectric constant compared to that of bulk water as it occurs in proteins [31, 321 and in active-site cavities of enzymes ([33] and page 258 in [34]), may well mean that the described alterations of the acid- base properties of nucleic base residues are also important in nature. This appears especially likely for the adenine residue, considering that in certain cell compartments the pH may be rather low, and hence relatively close to the pKa value for H,(ATP),-, e. g. for neurosecretory granules [35] and chromaffin granules of the adrenal medulla [2, 361, which also contain large concentrations of nucleotides (about 0.2 M) [2- 41, a pH range of 5.2 - 5.8 is given.

The recording of the N M R spectra during the past years by the technicians of the NMR-service laboratory of the Institute for Organic Chemistry, the support towards the costs of these NMR measure- ments by the Ciba-Stiftung Basel, and a research grant from the Swiss National Science Foundation are gratefully acknowledged.

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