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The occurrence of high concentrations of ATP in certain cell organelles prompted us to study the self-association of ATP via the concentration dependence of the ¹H-NMR chemical shifts for H2, H8 and H1′ in D2O at pD 8.4 (25°C) in the range 0.0025–0.4 M in the presence and absence of poly(α,ʟ-lysine), where [Lys units] was 0.4 M. The experiment in the presence of poly(Lys) was repeated at pD 12.1. At pD 8.4, the poly(amino acid) is protonated, i.e. poly(H·Lys)n4, whereas at pD 12.1 only approximately 10% of the ϵ-amino groups are positively charged. The results in all three systems are consistent with the isodesmic model of indefinite non-cooperative stacking. The stacking tendency follows the series: ATP4- (K= 1.3 M-1; pD 8.4)ATP4-/90% poly(Lys)/10% poly(H·Lys) n4 (K = 3.1 M-1; pD 12.1). It is evident that poly(H·Lys) n4 assists the association of ATP by a factor of approximately 10, and it is suggested that via its positively charged ϵ-ammonium groups, poly(H·Lys) n4 acts as a matrix by aligning ATP4- ions via ionic interactions with the negatively charged phosphate residues. The intragranular concentrations of various constituents of several storage or secretory cell organelles, as reported in the literature, are tabulated. The chromaffin granules of the adrenal medulla and the dense granules of blood platelets contain particularly high concentrations of nucleotides ([ATP] is approximately 0.14 M in the chromaffin granules and 0.5 M in the dense granules of rabbit blood platelets) and amines, such ,as epinephrine or 5-hydroxytryptamine. These granules, and probably also the storage vesicles of certain neurons (which seem to have a similar composition), appear, if the total concentrations of the various solutes are considered, to be osmotically unstable, which means that the intragranular solutes must be associated. This aggregation is discussed, especially with regard to the nucleotides. [ABSTRACT FROM AUTHOR]

The stability constants of the 1:1 complexes formed between Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+ and 2'AMP2-, 3'AMP2- or 5'AMP2- were determined by potentiometric pH titration in aqueous solution (I = 0.1 M, NaNO3; 25 °C). The experimental conditions were carefully selected such that selfassociation of the nucleotides and their complexes is negligibly small; i.e. it was made certain that the properties of the monomeric divalent-metal-ion-AMP [M(AMP)] complexes were studied. Based on recent measurements with simple phosphate monoesters, R-MP2- where R is a non-coordinating residue [Massoud, S. S. & Sigel, H. (1988) Inorg. Chem. 27, 1447-1453], it is shown that all the M(AMP) complexes of the alkaline earth ions, with the possible exception of Mg(5'AMP), have exactly the stability expected for a sole-phosphate coordination of the metal ion. The same property is revealed for the complexes with Mn2+, Co2+, Zn2+ or Cd2+ and 3'AMP2-; in case of Ni(3'AMP) and Cu(3'AMP) a slight stability increase just at the edge of the experimental-error limits is indicated. This slight stability increase is attributed to the formation of a macrochelate (possibly with N-3); in fact, additional information confirms macrochelation for Cu(3'AMP). About 45% of Cu(2'AMP) exists in aqueous solution as a macrochelate (probably involving N-3); the other M(2'AMP) complexes (M2+ = Mn2+ , Co2+, Ni2+, Zn2+, Cd2+) form (if at all) only traces of a base-backbound species. Most pronounced is macrochelate formation with 5'AMP2-: all mentioned 3d ions and Zn2+ or Cd2+ form to some extent macrochelates via N-7 (the structures of these closed species are indicated). In case of M(5'AMP) the base-binding site is certain: replacement of N-7 by a CH unit (tubercidin 5'-monophosphate) eliminates any increased complex stability, whereas formation of the l,N6-etheno bridge to form 1,N6-ethenoadenosine 5'-monophosphate results in the phenanthroline-like N-6,N-7 site which facilitates macrochelation significantly. [ABSTRACT FROM AUTHOR]

The concentration dependence of the chemical shifts for the protons H-2, H-8 and H-1' of ATP has been measured in D2O at 27°C under several degrees of protonation in the pD range from 1.5 to 8.4. The results at pD > 4.5 are consistent with the isodesmic model of indefinite noncooperative stacking, while those at pD < 4.5 indicate a preference for the formation of dimeric stacks. The stacking tendency follows the series, ATP4- (K = 1.3 M-1) < D(ATP)3- (2.1 M-1) < 1:1 ratio of D(ATP)³-/D2(ATP)2- (6.0 M-1) « D2(ATP)2- (≈ 200 M-1 » D3(ATP)- (K ... 17 M-1) (for reasons of comparison all constants are expressed in the isodesmic model). These results are compared with previous data for adenosine [Ado (K = I5 M-1) > 1:1 ratio of Ado/D(Ado)+ (6.0 M-1) > D(Ado)+ (0.9 M-1)] and AMP [AMP2- (K = 2.1 M-1) < D(AMP)- (3.4 M-1) < 1:1 ratio of D(AMP)-/D2(AMP)± (5.6 M-1) > D2(AMP)± (≈2 M-1) > D3(AMP)+ (K ≤11 M-1)] to facilitate the interpretation of the results for the ATP systems. Stack formation of H2(ATP)2- is clearly favored by additional ionic interactions; this is confirmed by measuring via potentiometric pH titrations the acidity constants of H2(ATP)2- in solutions containing different concentrations of ATP. It is suggested that in the [H2(ATP)]24- dimer intermolecular ion pairs (and hydrogen bonds) are formed between the H+(N-1) site of one H2(ATP)2- and the γP(OH)(O)2- group of the other; in this way (a) the stack is further stabilized, and (b) the positive charges at the adenine residues are compensated (otherwise repulsion would occur as is evident from the adenosine systems). A detailed structure for the [H2(ATP)]24- dimer is proposed and some implications of the described stacking properties of ATP for biological systems are indicated. [ABSTRACT FROM AUTHOR]

Solution structures of M(ATP)2- complexes are reviewed. First the self-stacking properties of ATP4- and M(ATP)2- are shortly described. It is emphasized that for an evaluation of solution structures of M(ATP)2- complexes only results from diluted solutions (below 1 mM) should be used. Next, a comprehensive set of stability data obtained under such conditions from potentiometric pH titrations is summarized for the complexes of Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+ with ATP, and for comparison also with pyrimidine nucleoside 5'-triphosphates (YTPs), i.e. CTP, UTP and TTP. The stabilities for the M(ATP)2- complexes are mostly larger than those for the corresponding M(YTP)2- species; this increased stability results from the metal ion backbinding to the base residue in M(ATP)2-, i.e. macrochelates are formed. Detailed analysis of the stability data allows calculation of the percentage of the closed form for the several M(ATP)2- complexes: back- binding is most pronounced in Cu(ATP)2- (67 ± 2%), remarkable in Zn(ATP)2- (28 ±7%), and not observable for Ca(ATP)2- (2 ± 6%). Comparison of these results with those from 1H-NMR and ultraviolet spectrophotometric studies allows the conclusion that two types of base back-bound macrochelates are formed: one with a direct, i.e. innersphere, M2+/N-7 coordination, and one with a water molecule between the metal ion and N-7, i.e. an outersphere interaction occurs [e.g. to about 10% in Mg(ATP)2-] through hydrogen bonding of a coordinated water to N-7. The formation degree of both forms of these closed isomers is quantified. The biological implications of these results are indicated and the versatility of ATP as a ligand is discussed by... [ABSTRACT FROM AUTHOR]

The concentration dependence of the chemical shifts of the protons H-2, H-8 and H-1′ for 2′-, 3′- and 5′-AMP²- and of the protons H-2, H-7, H-8 and H-1′ for tubercidin 5′-monophosphate (= 7-deaza-AMP²; TuMP²-) has been measured in D2O at 27°C to elucidate the self-association of the nucleoside monophosphates (NMPs). The results are consistent with the isodesmic model of indefinite non-cooperative stacking; the association constants for all four NMPs are very similar: K ≈ 2 M-1. These 1H-NMR measurements and those on the dependence of the chemical shifts on the pD of the solutions indicate that the NMP2 - species exist predominately in the anti conformation. Comparison of the shift data for 5′-TuMP and 5′-AMP shows that no hydrogen bonding between N-7 and -PO3H- occurs; hence, the previously observed and confirmed 'wrongway' chemical shift [Martin, R. B. (1985) Acc. Chem. Res 18, 32] connected with the deprotonation of the -PO3H- group most probably results from the anisotropic properties of the phosphate group which is in the anti conformation close to N-7. From the dependence between the chemical shift and the pD of the solutions the acidity constants were calculated for the four protonated NMPs, and for adenosine and D-ribose 5'-monophosphate. The measurements also allow an estimation of the first acidity constant of H3 (5&prim;e-AMP)+ ... The values for ... were also determined from potentiometric pH titrations in aqueous solution (I = 0.1 M, NaNO3; 25°C). The agreement of the results obtained by the two methods is excellent. The position of the phosphate group at the ribose moiety and the presence of N-7 in the base moiety influence somewhat the acid-base properties of the mentioned NMPs. Measurements with 5′-AMP in 50% (v/v) aqueous dioxane show that lowering of th... [ABSTRACT FROM AUTHOR]

The concentration dependence of the chemical shifts of the protons H-2, H-8, H-10, H-11, and H-1' of 1,N6ethenoadenosine 5'-triphosphate (ε-ATP4-) has been measured in D2O at 27°C to elucidate the self-association. The results are consistent with the isodesm&icaron;c model of indefinite noncooperative stacking; the association constant, K = 1.9 ± 0.2 M-1, is only slightly larger than the value for ATP4-, K = 1.3 ± 0.2 M -1. The self-stacking tendency of ε-ATP4- is promoted by a factor of about 4 by (1 :1) coordination of Mg2+ to the phosphate moieties, which probably links these together and also neutralizes part of the negative charge; Zn2+ is only about half as effective as Mg2+ in promoting the self-association. This result contrasts with the self-stacking properties of Mg(ATP)2- and, Zn2+ being considerably more effective in a 1:1 ATP system. It is assumed that due to the enhanced affinity of the N-6/N-7 site of the ε-adenine moiety towards Zn2+ repulsion of the bases occurs resulting thus in a lower stacking tendency; in addition, the simple isodesmic model is no longer applicable to the Zn(ε-ATP)2- system: to explain the experimental data, the formation of an intermolecular metal ion bridge in the dimeric stacks is proposed. The experimental conditions required for studies of the properties of monomeric ε-ATP systems are described. Care should be exercised in employing ε-ATP as a probe for ATP. [ABSTRACT FROM AUTHOR]

The stability constants of the 1:1 complexes between Cu2+ and Zn2+ with formate, acetate and several phenylalkanecarboxylates, i.e. C6H5-(CH2)n-COO¯ with n = 0 to 5, are summarized for water. 50% aqueous ethanol and 500 aqueous dioxane (I = 0.1 M; 25°C): Complex stability depends upon carboxylate group basicity. The influence of varying amounts of ethanol or dioxane (up to 90%) on the stability of the Cu2+ and Zn2+ (M2+) complexes with formate and acetate (CA) was measured by potentiometric pH titrations. The values for pKHH(CA) and log KMM(CA) increase, as expected, with increasing amounts of the organic solvents, i.e. with decreasing solvent polarity. The changes in the equilibrium constants are also evaluated with regard to the mole fractions of the organic solvents and the corresponding dielectric constants. These results may be used to estimate for low dielectric cavities in proteins the equivalent solution dielectric constant on the basis of enhanced carboxylate basicity or metal ion binding capability (method 1). Furthermore, the measured stability constants are used for comparisons of the coordination tendency of carboxylate ligands towards zinc(II)-metalloenzymes (method 2); in this way the equivalent solution dielectric constants in the active-site cavities of bovine carbonic anhydrase and carboxypeptidase A are estimated: the values are of the order of 35 and 70, respectively. This method seems to be generally applicable to metalloproteins. [ABSTRACT FROM AUTHOR]

The concentration dependence of the chemical shift of the protons H-2, H-10, H-11, and H-1 of 1,N6-ethenoadenosine 5-monophosphate (ε -AMP2-) has been measured. The results are consistent with the isodesmic model of indefinite noncooperative stacking; the association constant, K=2.5 ± 0.3 M1, is within experimental model identical to the value determined earlier for AMP2-, K=2.1 ± 0.4 M -1. The conditions for the potentiometric ph titration, used to determine the acidity constants of H2(ε-AMP), H2 AMP), and H(UMP)- and the stability constant of the metal ion (M2+) complexes of the corresponding nucleoside 5'-monophosphates (NMP), were similar; however, the stabilities of the Mn2+, Cu2+ and Zn2+ complex of (ε-AMP2- are much larger (in the case of Cu2+by a factor of 700) than those of AMP2-. This is due to the much larger metal ion affinity of the (ε-adenosine moiety compared to that of the parent adenosine residue. As the uridine moiety does not participate in complex formation, the stability constants of M(UMP) have been used to evaluated the extent of macrochelation (i.e. the simultaneous coordination of M2+ to the base moiety and the phosphate group) in the (ε-AMP) than for M(AMP). A comparison with previous result for the complexes with ADP3- and AFP4- indicates the order. M (AMP)..(ε-adenosine moiety towards Mn2+, Cu2+and Zn2+, the phosphate-monoprotonated complexes M(H-ε-AMP)+ also become important: the corresponding complexes play only a minor role in the M2+/AMP systems. Intramolecular aromatic-ring stacking occurs in the ternary Cu(2,2-bipyridy1)(NMP) complexes; about 80% of Cu(Bpy)(AMP) and Cu(&epsilon-amp)exist as the stacked isomer in aqueous solution ; for the former system it has been shown in a previous X-ray study that the intramolecular ligand-ligand interaction occurs also in the solid state[Aoki,K.(1978)J.Am Chem.Soc 100,7106]. Overall, the results emphasize that great care should be exercised in drawing conclusions based on studies of metal-ion-containing enzymic systems in which the natural adenine nucleotide cofactors have been replace by the corresponding 1,n6-etheno derivatives. [ABSTRACT FROM AUTHOR]

First-order rate constants (50 vC; I = 0.1 M, NaClO4) for the dephosphorylation of UTP and TTP (1 mM) in the pH range 2- 10 are compared with those of ATP and CTP; they all show the same properties indicating that the nucleic base has no influence on the rate. In the presence of Cu2 + or Zn2 + (NTP :M2 +' = 1 :1) this changes drastically: ATP-M2 + > UTP-M2 + &ap; TTP-M2 + &ap; CTP-M2 + > NTP, the Cu2 + systems being always more reactive than the Zn2 + systems, and these more than the Zn2 + systems. An Interaction between the nucleic base and metal ion is important for the Cu2 + -ATP and Zn2 + -ATP systems, but not for the pyrimidine nucleotide systems (these behave like methyl triphosphate). Accordingly. prevention of the Cu2 + -purine interaction by the addition of one equivalent of 2,2'-bipyridyl, leading to Cu(Bpy)(NTP)2 - , strongly reduces the activity and all four ternary Cu2 + systems now show the same dephosphorylation rate, Addition of a second equivalent of Cu2 + to the Cu2 + -UTP 1:1 system enhances the dephosphorylation rate significantly and Job's method provides evidence that a 2:1 complex is the most reactive intermediate. The relation between the initial rate, v0 = d[PO43-]/dt and the concentration of Cu2 + - UTP in 1:1 and 2:1 systems was determined. The results suggest that the reactive complex with pyrimidine nucleotides is a monomeric, dinuclear species of the type M2(NTP)(OH)- (its formation is inhibited byligands like tryptophanate), while with M2 +-ATP the reactive complex is a dimer. The connection between the indicated dephosphorylations in vitro, i.e. trans-phosphorytations to H2O, and related reactions in vivo are discussed. [ABSTRACT FROM AUTHOR]

The proton and charge stoichiometry of cytochrome c oxidase reconstituted into phospholipid vesicles has been analysed using a fast responding oxygen electrode and a system for the simultaneous measurement of H-, K+ or lipophilic cations, and O2. From initial rate measurements after addition of reductant (ascorbate,′′N,N,N′,N′-tetramethyl-p-phenylenediamine) the following stoichiometries could be extrapolated: K+/e-, between 1.85 and 2.05, H+/e-, between 0.58 and 0.75. Lipophilic cations can replace valinomycin potassium as the charge compensating system. Net H+ extrusion was observed for up to 11 turnovers of the enzyme (11 O2/cytochrome aa3). The variation of the internal pH buffering capacity of the vesicles has an influence on the number of turnovers during which net proton translocation can be observed, but has no influence on the determined stoichiometries. [ABSTRACT FROM AUTHOR]

The operation of cytochrome c oxidase with ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine as substrate in antimycin-A-inhibited rat liver mitochondria is coupled to proton ejection. Measurements of the initial rate of valinomycin-dependent K+ uptake have shown that nearly 4 K+ are taken up as 2 electrons are transferred from cytochrome c to oxygen. This proves directly that a charge separation of nearly 4 occurs across the inner mitochondrial membrane each time 2 electrons are transferred to oxygen. Measurements of the initial rate of proton movement after addition of the reductant show that about 1.6 protons are released by the mitochondria as 2 electrons are transferred from cytochrome c to oxygen. The data support the suggestion of a proton pump coupled to the operation of cytochrome c oxidase [Wikström, M. F. K. (1977) Nature (Lond.) 266, 271-273]. [ABSTRACT FROM AUTHOR]

The concentration dependence of the chemical shifts of the protons H-2, H-8 and H-1' of ATP4and of Mg(ATP)2-, of all non-labile protons of adenosine, of H-5, H-6 and H-1' of UTP, and of H-5, H-6, H-1', and H-2' of uridine have been measured. The results for the purine derivatives are consistent with the isodesmic model of indefinite non-cooperative stacking; for adenosine K = 15 ± 2 M-1, for ATP K = 1.3 ± 0.2 M-1 and for Mg(ATP)2- K = 3.6 ± 0.3 M-1. For the pyrimidines, uridine and UTP, stacking is much weaker and the stability constant could only be estimated; for uridine K ≤ 0.5 M-1, and for UTP K ≈ 0.3 M-1. [ABSTRACT FROM AUTHOR]

The energy-linked uptake of Ca2+ by heart mitochondria is inhibited by Mg2+ and this has been exploited to devise an inhibitor-stop method which has permitted direct measurements of the kinetics of Ca2+ uptake. The initial rate of Ca2+ influx has also been measured directly using a Ca2+--selective electrode. Half-maximal rate of uptake is attained at 12–15 μM Ca2+ and the maximum velocity is about 1.4 nmol of Ca2+/mg of protein per s at 25 C. The rate versus Ca2+-concentration curve is hyperbolic but becomes sigmoidal in the presence of Mg2+. Inorganic phosphate stimulates the initial rate of Ca2+ influx 2 to 4 times. [ABSTRACT FROM AUTHOR]

The dependence of the rate of dephosphorylation of ATP, ITP, GTP and CTP (= NTP), expressed as first-order rate constants (50°C; I = 0,1 M, NaClO4), on pH (2 to 10), in the absence and presence of Mn&sup2+;, Ni&sup2+; and Zn&sup2+;, was investigated. The reaction is accelerated by Zn&sup2+; and passes through a pH optimum at about 8 for the system Zn&sup2+;-ATP or 9 for Zn&sup2+;-ITP and Zn&sup2+;-GTP ; this is analogous to observations made earlier with the corresponding Cu&sup2+; systems. By computing the pH dependence of the distribution of the several species present in these systems it is shown that the highest rates are observed in the pH regions where the concentration of Zn(ATP)&sup2-;, Zn(ITP-H)&sup3-;, or Zn(GTP-H)3-dominates. By evaluating the pH dependence evidence is given that the attacking nucleophile is OH- or H20 for Zn(ATP)&sup2-; and H2O for Zn(ITP-H)&sup3-; or Zn(GTP-H)&sup3-;. For all these complexes metal-ion/nucleic-base interactions are known, leading to the formation of macrochelates. These metal-ion/nucleic-base interactions are crucial for the observation of a metal-ion-promoted dephosphorylation: in agreement with this, and the small tendency of the cytosine moiety to coordinate, the CTP systems are rather stable towards dephosphorylation. It should be noted that these experimental results do not necessarily mean that the macrochelates usually described are the reactive complexes, but only that the active complex must be closely related to them (e.g. isomers, etc.). Although for the Ni&sup2+; systems with ATP, ITP and GTP, and for the Mn&sup2+;-ATP system a metal-ion/nucleic-base interaction is also known, these systems are not very sensitive to hydrolytic cleavage of the terminal P-O-P bond. The only known significant structural difference between the Ni&sup2+;-NTP or the Mn&sup2+;-ATP complexes and those of Cu&sup2+; or Zn&sup2+; is that Ni&sup2+; and Mn&sup2+; coordinate to all three phosphate groups, whereas Cu&sup2+; and Zn&sup2+; involve only the β and γ ones. This structure-reactivity relationship is rationalized by the suggestion that in the active species the metal ion should be coordinated to the α,β-phosphate groups leaving the 9,-group open to nucleophilic attack. Obviously, an initial β,γ-coordination is suitable for a shift of the metal ion along the phosphate back-bone into the reactive α,β-position, while for an α,β,γ-coordination only the less favorable removal of the coordinated γ-group remains. The metal-ion/nucleic-base interaction is considered as being important for achieving this reactive structure. The connection between trans-phosphorylations in vitro and in vivo is discussed. It is also shown that the formation of mixed-ligand or ternary complexes inhibits the dephosphorylation process. This is on the one hand of interest with regard to the transport of hydrolysis-sensitive phosphates in nature, while on the other it casts doubts on conclusions based on experiments carried out in the presence of buffers, because these contain weak bases and hence potential ligands. [ABSTRACT FROM AUTHOR]

The chemical shift of the proton H(8) in the proton magnetic resonance spectrum of GTP was measured in aqueous solution in dependence on pH (2 to 12). Based hereon the acidity constants for protonation at N(7) and deprotonation at N(1) were determined. From the line broadening effects in the presence of Cu2+ it is concluded that this metal ion interacts with N(7) of the guanine moiety in Cu(GTP)2-. Hence, the structure of this latter complex corresponds to the one of Cu(ATP)2-, while in Cu(ITP)2- only a very minor interaction with N(7) occurs (cf. Eur. J. Biochem. 41, [1974] 209– 216). There is also evidence that in the complex Cu(GTP-1H)3- a 5-membered chelate involving N(7) and 0(6) is foxed, while in Cu(ITP-1H)3- the interaction with u2+ occurs only with the N(1), O(6)-site of the base moiety. The decrease in line broadening observed at higher pH indicates that the Cu2+-nucleic-base interaction is depressed in Cu(ATP)(OH)3-, Cu(GTP-IH)(OH)4- and Cu(ITP- 1H)(OH)4-, as well. In all the mixed-ligand systems which consist of Cu2+, 2,2′-bipyridyl (Bipy) and one of the three nucleotides (NTP) the ternary complexes, Cu(Bipy) (NTP)2-, are formed. Within these ternary complexes a charge-transfer interaction occurs between the purine part of the nucleotides and 2,2′-bipyridyl. [ABSTRACT FROM AUTHOR]

The acidity constant of protonated 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-l,3-propanediol (Bistris) has been measured. The influence of hydroxo groups on the basicity of Bistris and related bases is discussed. The interaction of Bistris with the metal ions (M2+) Mg2+, Ca2-, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 1.0 M, KNO3; 25 °C) and the stability constants of the M(Bistris)2+ complexes were determined. Unexpectedly Ca(Bistris)2+ is the most stable among the alkaline earth ion complexes (log KCa(Bistris)Ca = 2.25; the corresponding values for the Mg2+, Sr2+ and Ba2+ complexes are 0.34, 1.44 and 0.85, respectively). The ions of the 3d series follow the Irving-Williams sequence: log KMn(Bistris)Mn = 0.70, for Cu2+ 5.27 and Zn2+ 2.38. Ternary complexes containing ATP4- as a second ligand were also investigated: the values for Δlog KM (= log KM(ATP)(Bistris)M(ATP) -- log KM(Bistris)M) are in general negative (e.g. Δlog KCa = -0.40 or Δlog KCu = -1.65), thus indicating that the interaction of Bistris with M(ATP)2- is somewhat less pronounced than with M2+. However, even in mixed-ligand systems, complex formation may still be considerable, hence great reservations should be exercised in employing Bistris as a buffer in systems containing metal ions. Moreover, in several cases Δlog KM is relatively high [for Mg2+-ATP4- Bistris even positive], indicating some cooperativity between the coordinated ligands, possibly hydrogen-bond formation. Distributions of the complexes in dependence on pH are given, and the structures of the binary M(Bistris)2+ and the ternary M(ATP) (Bistris)2- complexes are discussed. The participation of Bistris hydroxo groups in complex formation is evident. [ABSTRACT FROM AUTHOR]

The interaction of 2-amino-2(hydroxymethyl)-l,3-propanediol (Tris) with the metal ions (M2+) Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I= 0.1 or 1.0 M, KNO3, 25°C). Stability constants of the M(Tris)2+ complexes were determined; those constants which were measured by both methods agreed well. Ternary complexes containing ATP4- as a second ligand were also investigated and it is shown that in the presence of Tris, mixed-ligand complexes of the type M(ATP)(Tris)2- are formed. The values for Δlog KM, whereΔ log kM = log M(ATP)M(ATP)Tris - log KMM(Tris), are all negative, thus indicating that the interaction of Tris with M(ATP)2- is somewhat less pronounced than with M2+. However, it should be noted that even in mixed-ligand systems complex formation with Tris may still be considerable, hence great reservations should be exercised in employing Tris as a buffer in systems which also contain metal ions. Distributions of the complex species in dependence on pH are shown for several systems, and the structures of the binary M(Tris)2- and the ternary M(ATP)(Tris)2- complexes are discussed. The participation of a Trishydroxo group in complex formation is, at least for the M(Tris)2- species, quite evident. [ABSTRACT FROM AUTHOR]

The chemical shift of the protons, H(2) and H(8), in the proton magnetic resonance spectra of ATP, ITP, and inosine was measured in aqueous solution in dependence on pR (2 to 12). Based on these data the sites of protonation or deprotonation are identified and the acidity constants determined. A comparison of tho constants duo to the nucleotides with the corresponding ones of the nucleosides evidences that the basicity of tho nucleic base increases somewhat under the influence of tho triphosphate chain. The possibility of an intramolecular hydrogen bound between N(7) and the proton of the γ-phosphate group is discussed.From the line broadening effects in the presence of Cu2+ it is concluded that this metal ion interacts mainly with N(7) of ATP, but with the N(1), O(6)-site of ITP. With inosine as ligand N(7) is the predominate binding site at pH < 6, while at pH > 7 N(1), O(6) is preferred. Experiments with ITP in the presence of [Cu2+] = [2,2'-bipyridyl] (under conditions where Cu(Bipy)2+ is nearly completely formed) reveal the surprising fact that in the pH range 2 to 6 the line broadening of the signal due to H(8), and H(2) as well, increases significantly compared with a system containing only ITP and Cu2+. This observation is easily explained by the assumption of a chargetransfer interaction within the ternary complex, Cu(Bipy) (ITP)2-, i.e. between the purine part of the nucleotide and 2,2'-bipyridyl. This interaction could indeed be confirmed by spectrophotometric measurements in the ultraviolet (λmax = 313 nm). Similar results were obtained for the mixed-ligand complex, Cu(Bipy) (ATP)2-. The distance between the planes of the aromatic rings in these systems is estimated and a structure for these metal-ion-bridged charge-transfer complexes is suggested. The biological implications of such ternary, metal, ion-bridged, charge-transfer complexes are briefly indicated. [ABSTRACT FROM AUTHOR]

The concentration dependence of the chemical shifts for the hydrogens H‐2, H‐8 and H‐1′ of ITP and for H‐8 and H‐1′ of GTP has been measured in D2O at 25°C under several degrees of protonation in the pD range 1.2–8.4. For reasons of comparison, inosine and guanosine have been included in the study. The results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for the nucleosides (Ns) inosine and guanosine decrease with increasing protonation: Ns > D(Ns)+/Ns in a 1:1 ratio > D(Ns)+. In contrast, a maximum is observed with ITP and GTP; the stacking tendency of GTP following the series: GTP4−≲D(GTP)3− (K≈0.7 M−1) < D(GTP)3−/D2(GTP)2− in a 1:1 ratio (K≈2.9 M−1) > D2(GTP)2− > D3(GTP)− (K≈1.5 M−1). The order of the series with ITP corresponds to that with GTP, but the association constants are slightly smaller. At the maximum of the self‐association tendency the triphosphate residue has only a minor influence; this follows from the fact that the association constants for the 1:1 ratios of Ino/D(Ino)+ and D(ITP)3−/D2(ITP)2− are identical within experimental error; this holds also for Guo/D(Guo)+ and D(GTP)3−/D2(GTP)2−; in all these pairs the N‐7 site is 50% protonated. Comparison of the association constants for the deprotonated species shows that here charge effects, i.e. repulsion between the negatively charged triphosphate chains, are important: Ino (K≈3.3 M−1) > ITP4− (K≈0.4 M−1) and Guo (K≈8 M−1) > GTP4− (K≈0.8 M−1). In addition the series holds: Ado (K≈15 M−1) > Guo > Ino. However, most important is the comparison of the ITP and GTP series with previous data for ATP: ATP4− (K≈1.3 M−1) < D(ATP)3− (2.1 M−1) < 1:1 ratio of D(ATP)3−/D2(ATP)2− (6 M−1) ≪ D2(ATP)2− (≈200 M−1) ≫ D3(ATP)− (K≲ 17 M−1). It is evident (a) that the self‐association tendency of the ATP species is larger and (b) that self‐association is most pronounced for D2(ATP)2− and not for the 1:1 ratio of D(ATP)3−/D2(ATP)2− [although the latter with a 50% protonation of the N‐1 site corresponds to that observed for Ado/D+ (Ado) (K= 6 M−1)]. The very large stability of the dimeric [H2(ATP)]4−2 stack has previously been attributed to the formation of intermolecular ion pairs (and hydrogen bonds) formed between the H+ (N‐1) site of one H2(ATP)2− and the γ‐P(OH)(O)−2 group of the other; in this way (a) the stack is further stabilized, and (b) the positive charges at the adenine residues are compensated. This interpretation is in perfect agreement with the properties observed now for H2(ITP)2− and H2(GTP)2−; both species show a low stacking tendency in accord with the fact that their neutral H(N‐1) site is not able to form the mentioned intermolecular ion pair with the (also present) γ‐P(OH)(O)−2 group, and the proton now located at the N‐7 site leads to repulsion of the purine moieties. The correctness of this interpretation is further confirmed by a study of the acid‐base properties of the purine residues in ITP, GTP and ATP in dependence on the concentration of the nucleoside 5′‐triphosphates (Corfù, N. A. & Sigel, H., unpublished results). The results of this study show that the acidity of the H+ (N‐7) site in H2(ITP)2− and H2(GTP)2− is enhanced upon stack formation (which leads to repulsion of the positively charged rings), while the acidity of the H+ (N‐1) site in H2(ATP)2− is reduced upon stack formation, because this site is also involved in ion pair and hydrogen bonding. Some consequences of these summarized results for biological systems are indicated. [ABSTRACT FROM AUTHOR]

Stability constants of mixed-ligand Cu(Arm)(AMP) complexes [where Arm = 2,2′-bipyridyl (Bpy) or 1,10-phenanthroline (Phen) and AMP2- = 2′AMP2- , 3′AMP2- or 5′AMP2-] were determined by potentiometric pH titrations in aqueous solution at I = 0.1 M (NaNO3) and 25 °C. The ternary Cu(Arm)(AMP) complexes are more stable than corresponding Cu(Arm)(R-MP) complexes, where R-MP2- represents a phosphate monoester with a group R that is unable to participate in any kind of interaction within the complexes as, for example. D-ribose 5′-monophosphate. This increased stability is attributed, in agreement with previous results, to intramolecular stack formation in the Cu(Arm)(AMP) complexes between the purine residue of the AMPs and the aromatic rings of Bpy or Phen. Based on correlation lines (previously obtained from log K versus pKa plots) for Cu(Arm)(R-MP) complexes without a ligand-ligand interaction, a quantitative evaluation was carried out. The degree of formation of the species with the intramolecular stacks increases for the Cu(Arm)(AMP) complexes in the series: 3′AMP2- < 5′AMP2- < 2′AMP2- ; e.g. in Cu(Bpy)(3′AMP) the stack reaches a formation degree of 45 ± 11% and in Cu(Bpy)(2′AMP) one of 96.1 ± 0.7% is obtained. It must be emphasized that these differences are due to the different steric orientations of the bridging metal ion, which result from the varying position of the phosphate group on the ribose ring. As shown by ¹H-NMR shift measurements, there is no significant effect of the position of the phosphate group on the stability of the binary (Phen)(AMP)2- adducts (K &assymp; 36 M-1 in D2O): such an effect is seen only if a metal-ion bridge is formed between the moieties forming the stack, i.e. metal-ion coordination imposes individual properties on the AMPs. By also taking into account some recent results on other nucleoside 5′-monophosphate complexes, the following trend for an increasing stacking tendency of the nucleic base moieties can be established: uracil [This symbol cannot be presented in ASCII format] cytosine [This symbol cannot be presented in ASCII format] thymine « adenine < 7-deazaadenine. Some additional conclusions of general importance are given and the relevance of the results with regard to bio-systems is indicated. [ABSTRACT FROM AUTHOR]

By means of UV difference spectra, the acidity constants KHLH of the N-1-proton in the nucleotides inonine, guanosine, uridine, and thymidine 5`-triphosphate were measured. The same deprotonation (KcuHLH) in the respective Cu2+ complexes was also determined. The measurement were carried out with and without (Table 1) addition of NaClO4 (0.1 M). It was found that the influence of the ionic strength is great, possibly due to the formation of Na+ complexes. The difference, ΔpKA = pKHLH-pK–H ≈ 2.0, for the nucleotide systems is of the same order as the respective value 2.2 of the Cu2+ guanosine system. This indicates a strong interaction between Cu2+ and the nucleic base in the Cu2+-nucleotide complexes. From experimental indices and the analogy to the Cu2+-ATP complex, it is concluded that macrochelates are formed. The acidity constants determined by means of UV difference spectra were confirmed by potentiometric titrations (Table 2). The Cu2+-nucleoside triphosphates are known to form stable complexes with 2,2`-bipyridyl. In such ternary complexes, the nucleic base can no longer coordinate to the Cu2+. The relatively small ΔpKBipy/A values (Table 2) are in agreement with this. The hydrolytic tendencies of Cu2+ bound to the different nucleotides were also estimated (Table 2). The pKCuL(H2O)xH values show that the hydrolytic tendency of the Cu2+ complexes decreases in the order CTP > ATP > TTP > UTP > ITP ≈ GTP. The influence of the coordination number of the different ligands and also other problems concerning nucleotide complexes were discussed. [ABSTRACT FROM AUTHOR]

The stability constants of the ternary Cu2+ complexes, ethylenediamine-Cu2+-pyrocatechol and histamine-Cu2+-pyrocatechol dianion, were determined. Both complexes are more stable than one would expect on pure statistical reasons. The imidazole-containing ternary Cu2+ complex is, however, especially stable. For the equilibrium, Cu(histamine)2-+-Cu(pyrocatechol)2 [This symbol cannot be presented in ASCII format]2[(histamine)Cu(pyrocatechol)], the constant is log X = 4.86 (I = 0.1; T = 25°), while the statistical value is 0.6. These results were compared with those taken (or calculated) from the literature; it is concluded that the π-system of the ligands is important for such a great increase in stability. Furthermore, the Cu2+-histamine 1:1 complex selectively binds to O-ligands rather than to N-ligands. Thus, the first coordinated ligand has an influence on the kind of the second to be coordinated, i.e. the Cu2+-histamine 1:1 complex shows discriminating qualities. The significance of these results is discussed with regard to Cu-proteins, sines it is suggested for several enzymes that the imidazole group of histidine is involved with the binding of metal ions. [ABSTRACT FROM AUTHOR]

By means of UV difference spectra, the acidity constants KHLH of the N-1-proton in the nucleotides inonine, guanosine, uridine, and thymidine 5`-triphosphate were measured. The same deprotonation (KcuHLH) in the respective Cu2+ complexes was also determined. The measurement were carried out with and without (Table 1) addition of NaClO4 (0.1 M). It was found that the influence of the ionic strength is great, possibly due to the formation of Na+ complexes. The difference, ΔpKA = pKHLH-pK–H ≈ 2.0, for the nucleotide systems is of the same order as the respective value 2.2 of the Cu2+ guanosine system. This indicates a strong interaction between Cu2+ and the nucleic base in the Cu2+-nucleotide complexes. From experimental indices and the analogy to the Cu2+-ATP complex, it is concluded that macrochelates are formed. The acidity constants determined by means of UV difference spectra were confirmed by potentiometric titrations (Table 2). The Cu2+-nucleoside triphosphates are known to form stable complexes with 2,2`-bipyridyl. In such ternary complexes, the nucleic base can no longer coordinate to the Cu2+. The relatively small ΔpKBipy/A values (Table 2) are in agreement with this. The hydrolytic tendencies of Cu2+ bound to the different nucleotides were also estimated (Table 2). The pKCuL(H2O)xH values show that the hydrolytic tendency of the Cu2+ complexes decreases in the order CTP > ATP > TTP > UTP > ITP ≈ GTP. The influence of the coordination number of the different ligands and also other problems concerning nucleotide complexes were discussed. [ABSTRACT FROM AUTHOR]