Self-association of bovine immunoglobulin G1.

The self-association properties of bovine serum immunoglobulin G1 and colostral immunoglobulin G1 (IgG1) in 0.32 M-NaCl/0.01 M-Tris/HCl, pH 8.0, were investigated by analysing sedimentation data according to a monomer-dimer association model. The self-association was characterized by an equilibrium constant of 5.3 X 10(4) +/- 3.5 X 10(4) M-1 for serum IgG1 and 1.6 X 10(3) +/- 0.69 X 10(3) M-1 for colostral IgG1. The removal of the Fc portion of IgG1 by pepsin digestion abolished its property of self-aggregation. At high total protein concentrations of serum IgG1, low concentrations of the ostensible trimer species were observed. However, no self-aggregation was evident when 0.14 M-NaCl/0.01 M-sodium phosphate. pH 6.0, was used as a solvent, thus confirming results published previously [Tewari & Mukkur (1975) Immunochemistry 12, 925--930].     

Temperature and pH dependence of the self-association of human spectrin

The self-association of human spectrin between 21 and 35 degrees C and between pH 6.5 and 9.5 has been studied at sedimentation equilibrium. For a given set of solution conditions between pH 6.5 and 8.5, coincidence of omega function plots as a function of total spectrin concentration (0-2 g/L) indicated that equilibrium was attained and that no significant concentration of solute was incapable of participating in the self-association reaction. Above pH 8.5, however, irreversible aggregation occurred, inferred from a failure of overlap in the omega function and molecular weight distributions. The behavior of spectrin can best be described by a cooperative isodesmic model, in which the promoter for association is the heterodimer and for which K12 is between 10(6) and 10(7) M-1 (depending on pH and temperature) and all other K are approximately 10(6) M-1. The returned values of the second viral coefficient for this model fall within the range calculated from the charge and Stokes radius of spectrin. Association appears to be favored slightly by decreased temperature and by decreased pH. The pH dependence resides only in K12 and is consistent with the presence of a single group, possibly histidine, displaying a slightly higher pKa value in the tetramer than in the dimer. The association reaction appears to be driven by the loss of enthalpy associated with release of strain in the heterodimer. The association sites appear to be conserved in the association reactions, consistent with the images from electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of self-association and subsequent fibril formation in an alanine-rich helical polypeptide

Thermal unfolding, reversible self-association, and irreversible aggregation were investigated for an alanine-rich helical polypeptide, 17-H-6, with sequence [AAAQEAAAAQAAAQAEAAQAAQ] 6. Dynamic light scattering, transmission electron microscopy, and thermal unfolding measurements indicate that 17-H-6 spontaneously and reversibly self-associates at acidic pH and low temperature. The resulting multimers have a compact, globular morphology with an average hydrodynamic radius approximately 10-20 nm and reversibly dissociate to monomers upon an increase to pH 7.4. Both free monomer and 17-H-6 chains within the multimers are alpha-helical and folded at low temperature. Reversible unfolding of the monomer occurs upon heating of solutions at pH 7.4. At pH 2.3, heating first causes incomplete dissociation and unfolding of the constituent chains. Further incubation at elevated temperature induces additional structural and morphological changes and results in fibrils with a beta-sheet 2 degrees structure and a characteristic diameter of 5-10 nm (7 nm mean). The ability to modulate association and aggregation suggests opportunities for this class of polypeptides in nanotechnology and biomedical applications.     

Self-association of sperm whale metmyoglobin

The solution behavior of sperm whale metmyoglobin in 0.15 I phosphate-chloride buffer, pH 7.2, has been examined by sedimentation equilibrium, frontal gel chromatography, and sedimentation velocity. Results obtained from all three studies are shown to be consistent with a self-association model in which dimerization of the myoglobin is governed by an association equilibrium constant of 0.068 liter/g (580 M-1) at 20 degrees C.     

Comparison of the self-association properties of the 5'-triphosphates of inosine (ITP), guanosine (GTP), and adenosine (ATP). Further evidence for ionic interactions in the highly stable dimeric [H2(ATP)]2(4-) stack

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 degrees 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 greater than D(Ns)+/Ns in a 1:1 ratio greater than D(Ns)+. In contrast, a maximum is observed with ITP and GTP; the stacking tendency of GTP following the series: GTP4- less than or equal to D(GTP)3- (K approximately 0.7 M-1) less than D(GTP)3-/D2(GTP)2- in a 1:1 ratio (K approximately 2.9 M-1) greater than D2(GTP)2- greater than D3(GTP)- (K approximately 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 K-7 site is 50% protonated. Comparison of the association constant for the deprotonated species shows that here charge effects, i.e. repulsion between the negatively charged triphosphate chains, are important: Ino (K approximately 3.3 M-1) greater than ITP4- (K approximately 0.4 M-1) and Guo (K approximately 8 M-1) greater than GTP4- (K approximately 0.8 M-1). In addition the series holds: Ado (K approximately 15 M-1) greater than Guo greater than Ino. However, most important is the comparison of the ITP and GTP series with previous data for ATP: ATP4- (K approximately 1.3 M-1) less than D(ATP)3- (2.1 M-1) less than 1:1 ratio of D(ATP)3-/D2(ATP)2- (6 M-1) much less than D2(ATP)2- (approximately 200 M-1) much greater than D3(ATP)- (K less than or equal to 17 M-1).(ABSTRACT TRUNCATED AT 400 WORDS)     

Bovine cardiac myosin subfragment 1. Transient kinetics of ATP hydrolysis

The kinetics of binding and hydrolysis of ATP by bovine cardiac myosin subfragment 1 has been reinvestigated. More than 90% of the total fluorescence amplitude associated with ATP hydrolysis occurs with an apparent second-order rate constant of 8.1 X 10(5) M-1 S-1 and a limiting rate constant of approximately 140 S-1 (100 mM KCl, 50 mM 1,3-bis-[tris(hydroxymethyl)methylamino]-propane, 10 mM MgCl2, pH 7.0, 20 degrees C); the remaining 10% occurs more slowly (approximately 1 S-1). The observed rate constants are independent of subfragment 1 concentration under pseudo first-order conditions for ATP with respect to protein. The fraction of protein which hydrolyzes ATP rapidly is not a function of the nucleotide or protein concentration and appears to be constant irrespective of ionic strength or temperature within the range studied (50-100 mM KCl, pH 7.0, 15-20 degrees C). These data are compared to that obtained previously using subfragment 1 prepared by a different method which showed ATP-dependent aggregation of two protein species.     

Vincristine-induced self-association of calf brain tubulin

The vincristine-induced self-association of tubulin has been examined in a sedimentation velocity study as a function of free drug concentration in PG buffer (0.01 M NaPi and 10(-4) M GTP, pH 7.0) at 20 degrees C. Analysis of the weight-average sedimentation coefficient (S20,w) as a function of protein concentration showed a good fit with the model of an indefinite, isodesmic self-association mechanism. Analysis of the apparent association constants in terms of the Wyman linkage relations showed a good fit to mediation of the self-association by the binding of one ligand molecule. The intrinsic association constant for dimerization of the vincristine-liganded tubulin was found to be 3.8 X 10(5) M-1, and the intrinsic equilibrium constant for the binding of the self-association-linked vincristine molecule had a value of 3.5 X 10(4) M-1, consistent with that measured by fluorescence in our laboratory [Prakash, V., & Timasheff, S. N. (1983) J. Biol. Chem. 258, 1689-1697]. Both reactions are stronger in the presence of vincristine than of vinblastine, reflecting the oxidation of a -CH3 group to -CHO when going from the latter drug to the former one.     

Ligand-modulation of the stability of the glucose transporter GLUT 1

The glucose transporter GLUT 1 was isolated from human erythrocytes and reconstituted into endogenous membrane lipids. Results from thermal denaturation studies, using differential scanning calorimetry, indicate that the thermal denaturation temperature of GLUT 1 is significantly lower in the presence of ATP. The lowering of this transition temperature is very dependent on pH. At more acidic pH, ATP has a greater effect of lowering the thermal denaturation temperature of the protein. For example, with 4.8 mM ATP, the denaturation endotherm is lowered by over 10 degrees at pH 4.3, whereas at pH 7.4, ATP does not alter this transition temperature. However, a change in pH alone, in the absence of ATP, has very little effect on the denaturation temperature. Both glucose and salt partially reverse the lowering of the temperature of thermal denaturation caused by ATP. Studies of acrylamide quenching of the Trp residues of GLUT 1 indicate that at neutral pH, ATP increases the Stern-Volmer quenching constant, while glucose lowers it. The results indicate that ATP binds to GLUT 1 and destabilizes the native structure, leading to a lowering of the thermal denaturation temperature and an increase in acrylamide quenching. The effects of ATP are reversed in part by glucose and are also partly electrostatic in nature.     

Self-association of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) and promotion by metal ions

The concentration dependence of the chemical shifts of the protons H-2, H-8, H-10, H-11, and H-1' of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP4-) has been measured in D2O at 27 degrees C to elucidate the self-association. The results are consistent with the isodesmic 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 epsilon-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 Zn(ATP)2-, 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 epsilon-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(epsilon-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 epsilon-ATP systems are described. Care should be exercised in employing epsilon-ATP as a probe for ATP.     

Interaction of vinblastine with calf brain tubulin: multiple equilibria

The binding of the anticancer drug vinblastine to calf brain tubulin was measured by a batch gel filtration method in PG buffer (0.01 M NaPi, 10(-4) M GTP, pH 7.0) at three different protein concentrations. The Scatchard binding isotherms obtained were curvilinear. The binding of the first vinblastine molecule to each tubulin alpha-beta dimer (Mr 110,000) was enhanced by an increase in the protein concentration. Additional binding of vinblastine to the protein was independent of the protein concentration. Theoretical ligand binding isotherms were calculated for a ligand-induced macromolecule self-association involving various ligand stoichiometries and association schemes. Fitting of the experimental data to these isotherms showed that the system can be described best by a one-ligand-induced isodesmic indefinite self-association. The pathway giving the best fit consists of a ligand-mediated plus -facilitated self-association mechanism. The self-association-linked bound vinblastine binds specifically at a site with an intrinsic binding constant K1 = 4 X 10(4) M-1. Additional vinblastine molecules can bind less strongly to tubulin in probably nonspecific fashion, and the previous reports of two specific sites on alpha-beta tubulin for binding vinblastine are incorrect. The self-association constant K2 for liganded tubulin is 1.8 X 10(5) M-1. This analysis is fully consistent with the conclusions derived earlier from the linked function analysis of the vinblastine-induced tubulin self-association [Na, G. C., & Timasheff, S. N. (1980) Biochemistry 19, 1347-1354; Na, G. C., & Timasheff, S. N. (1980) Biochemistry 19, 1355-1365].     

The self-association of human spectrin at high concentration.

The self-association of purified human spectrin has been studied at sedimentation equilibrium over a wide range of concentration (0-20 g/L) at 30 degrees C and pH 7.5. Coincidence of apparent weight average molecular weight and omega (r) plots as a function of total spectrin concentration indicated that equilibrium was attained and that no significant concentration of solute was incapable of participating in the self-association reaction. Under these conditions, no significant dissociation of the heterodimer to component polypeptide chains could be detected. The behavior of spectrin between 0 and 20 g/L can be described reasonably well by a cooperative isodesmic model, in which the protomer for association is the alpha beta heterodimer. With this model, the equilibrium constant for the heterodimer-tetramer step, K24, is 2 x 10(6) M-1, and K(iso), the equilibrium constant describing all other steps, is approximately 0.2 x 10(6) M-1. The returned value of the second virial coefficient for this model, 1.0 x 10(-7) L mol g-2, is consistent with the lower limit of values calculated for the heterodimer from the charge and Stokes radius of spectrin. On the other hand, the attenuated indefinite association model fails to describe the self-association of spectrin adequately over the range 0-20 g/L. Systematic decreases in the estimates of the second virial coefficient and the equilibrium constants for association beyond the tetramer suggest that the assumption of a single value of the second virial coefficient may not be appropriate for spectrin, and that non-ideality would best be taken into account by consideration of the detailed solution composition.     

Polymerization pattern of insulin at pH 7.0.

Sedimentation equilibrium results, obtained with bovine zinc-free insulin (with and without a component of proinsulin) at pH 7.0, I o.2, 25 degrees C, and up to a total concentration of 0.8 g/l., are shown to be consistent with three different polymerization patterns, all involving an isodesmic indefinite self-association of specified oligomeric species. The analysis procedure, based on closed solutions formed by summing infinite series, yields for each pattern a set of equilibrium constants, It is shown that a distinction between the possible patterns can be made by analyzing sedimentation equilibrium results obtained in a higher total concentration range (up to 4 g/1.) with insulin freed of zinc and proinsulin, account being taken of the composition dependence of activity coefficients. The favored pattern, which differs from that previously reported in the literature, involves the dimerization of monomeric insulin (mol wt 5734), governed by a dimerization constant of 11 X 10(4) M-1 and the isodesmic indefinite self-association of the dimer, described by an association constant of 1.7 X 10(4) M-1. This polymerization pattern is also shown to be consistent with the reaction boundary observed in sedimentation velocity experiments.     

Self-association of myelin basic protein: enhancement by detergents and lipids

Self-association of basic protein has been proposed to be of functional significance in central nervous system myelin. In aqueous solution this protein self-associates, previous data being consistent with the formation of dimers, which then undergo an indefinite isodesmic self-association [Smith, R. (1980) Biochemistry 19, 1826-1831]. As this protein is membrane bound in vivo, we have now examined the effects of amphiphiles on the self-association equilibria. Contrary to the expected effects, at low molar ratios dodecyl sulfate, deoxycholate, Triton X-100, and lysophosphatidylcholine increased protein intermolecular attraction. The anionic detergents led to partial precipitation even at 1:1 protein:detergent molar ratios whereas the zwitterionic lipid and the nonionic detergent exerted less pronounced effects. Sedimentation velocity and equilibrium measurements have been used to define quantitatively the effects of lysophosphatidylcholine. The sedimentation coefficient increases up to a lipid:protein ratio of 4:1 and then remains constant up to a ratio of 12:1. The sedimentation equilibrium data suggest that the mode of protein-protein interaction is the same as in the absence of lipid but with substantially increased association constants. The dimerization constant is increased from 1.20 X 10(2) M-1 to 1.0 X 10(3) M-1 and the isodesmic association constant from 3.4 X 10(4) M-1 to 1.2 X 10(5) M-1. The effects of detergents on myelin basic protein are compared with the effects on other proteins, and the implications for the state of the protein with myelin are discussed.     

The self-association of ATP: thermodynamics and geometry.

The concentration and temperature dependence of the self-association of ademosin-5'-triphosphate (ATP) in aqueous solution was studied by means of ultraviolet absorption spectroscopy and circular dichroism (CD). Of several possible models, a model was indefinite linear self-association, in which each step has the same equilibrium constant, describes the data best. The two different methods lead within experimental error to the same thermodynamic parameters. At pH 8.7, IN 1 M Tris and 0.5 M 7gCl-2, we find deltaH-0 equals -5.1 kcal/mole and deltaS-0 equals -13.0 e.u. These values do not differ much from those found for the self-association of uncharged bases and nucleosides in aqueous solution. The CD spectrum that results from the self-association is conservative and quite similar in shape to that observed for some stacked dinucleotides: it is interpreted as a first approximation within the framework of the exciton model.     

Magnesium-induced self-association of calf brain tubulin. II. Thermodynamics.

The thermodynamic parameters of the magnesium ion induced self-association of calf brain tubulin in pH 7.0, 0.01 M phosphate buffer containing 10(-4) M GTP, were determined from sedimentation velocity experiments. This reaction proceeds by an isodesmic mechanism terminated by the highly favored formation of a closed ring shaped polymer of degree of association 26 +/- 4. Analysis of the variation of the apparent dimerization constant in the isodesmic mechanism s,ows that this self-association is characterized by positive enthalpy, entropy, heat capacity, and molar volume changes, as well as the binding of one additional magnesium ion, which is probably not involved as a bridge between the protein molecules. The addition of the last monomeric subunit has a free energy which is about three times that of dimer formation. Under the conditions of these experiments, tubulin binds 48 +/- 5 magnesium ions with a free energy of --2.8 kcal/mol.     

Nucleotide Binding to Na,K-ATPase: The Role of Electrostatic Interactions.

The contribution of electrostatic forces to the interaction of Na,K-ATPase with adenine nucleotides was investigated by studying the effect of ionic strength on nucleotide binding. At pH 7.0 and 20 degrees C, there was a qualitative correlation between the equilibrium dissociation constant (K(d)) values for ATP, ADP, and MgADP and their total charges. All K(d) values increased with increasing ionic strength. According to the Debye-Hückel theory, this suggests that the nucleotide binding site and its ligands have "effective" charges of opposite signs. However, quantitative analysis of the dependence on ionic strength shows that the product of the effective electrostatic charges on the ligand and the binding site is the same for all nucleotides, and is therefore independent of the total charge of the nucleotide. The data suggest that association of nucleotides with Na,K-ATPase is governed by a partial charge rather than the total charge of the nucleotide. This charge, interacting with positive charges on the protein, is probably the one corresponding to the alpha-phosphate of the nucleotide. Dissociation rate constants measured in complementary transient kinetic experiments were 13 s(-1) for ATP and 27 s(-1) for ADP, independent of the ionic strength in the range 0.1-0.5 M. This implies similar association rate constants for the two nucleotides (about 40 x 10(6) M(-1) s(-1) at I = 0.1 M). The results suggest that long-range Coulombic forces, affecting association rates, are not the main contributors to the observed differences in affinities, and that local interactions, affecting dissociation rates, may play an even greater role.     

Local anesthetic-phospholipid interactions. The pH dependence of the binding of dibucaine to dimyristoylphosphatidylcholine vesicles

The interaction of the local anesthetic dibucaine with unilamellar vesicles of dimyristoylphosphatidylcholine was studied by equilibrium dialysis. Saturating binding profiles (as a function of dibucaine) were found, with apparent association constant ranging from 1.26 X 10(3)M-1 to 2.57 X 10(3)M-1 as pH is increased from 5.0 to 7.5. The number of phospholipid molecules comprising a binding site was found to be about 5 at each pH. Analysis of the data was also achieved using the Stern model, which takes into account the electrostatic effect on binding of the cationic drug due to the build up of a surface potential.     

Influence of the protonation degree on the self-association properties of adenosine 5'-triphosphate (ATP)

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 degrees C under several degrees of protonation in the pD range from 1.5 to 8.4. The results at pD greater than 4.5 are consistent with the isodesmic model of indefinite noncooperative stacking, while those at pD less than 4.5 indicate a preference for the formation of dimeric stacks. The stacking tendency follows the series, ATP4- (K = 1.3 M-1) less than D(ATP)3- (2.1 M-1) less than 1:1 ratio of D(ATP)3-/D2(ATP)-2- (6.0 M-1) much less than D2(ATP)2- (approximately 200 M-1) much greater than D3(ATP)- (K approximately less than 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 = 15 M-1) greater than 1:1 ratio of Ado/D(Ado)+ (6.0 M-1) greater than D(Ado)+ (0.9 M-1)] and AMP [AMP2- (K = 2.1 M-1) less than D(AMP)- (3.4 M-1) less than 1:1 ratio of D(AMP)-/D2(AMP) +/- (5.6 M-1) greater than D2(AMP) +/- (approximately equal to 2 M-1) greater than D3(AMP)+ (K less than or equal to 1 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)]4-(2) dimer intermolecular ion pairs (and hydrogen bonds) are formed between the H+(N-1) site of one H2(ATP)2- and the gamma-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 (otherwise repulsion would occur as is evident from the adenosine systems). A detailed structure for the [H2(ATP)4-(2) dimer is proposed and some implications of the described stacking properties of ATP for biological systems are indicated.     

Binding strength of calcium ion to bovine alpha-lactalbumin

A Ca2+-sensitive electrode was used for determination of the binding strength of Ca2+ to bovine alpha-lactalbumin in 60 mM Tris buffer (pH 7.8-8.5) in the presence of various concentrations of NaCl. The dependence of the apparent binding constant on the concentration of NaCl was consistent with competitive binding of Ca2+ and Na+, and the binding constants of Ca2+ and Na+ were found to be 2.2 (+/- 0.5) X 10(7) M-1 and 99 (+/- 33) M-1, respectively, at 37 degrees C and pH 8.0. The temperature dependence of the binding constant of Ca2+ was examined between 30 and 45 degrees C; extrapolation of the dependence led to a binding constant of approximately 1 X 10(8) M-1 at pH 8.4 and 25 degrees C. The electrostatic contribution and conformational effect of the protein were also taken into consideration, and the intrinsic binding constant of Ca2+ to native alpha-lactalbumin was calculated to be (1.2-1.5) X 10(10) M-1 at 37 degrees C and pH 8.0.     

Self-association of adenosine 5'-monophosphate (5'-AMP) as a function of pH and in comparison with adenosine, 2'-AMP and 3'-AMP

The concentration dependence of the chemical shifts for protons H-2, H-8, and H-1' of adenosine (Ado), 2'-AMP, 3'-AMP and 5'-AMP was measured in D2O at 27 degrees C under several degrees of protonation. All results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for Ado decrease with increasing protonation: Ado (K = 15 M-1) greater than D(Ado)+/Ado (6.0 M-1) greater than D(Ado)+ (0.9 M-1). In contrast, a maximum is observed with 5'-AMP: 5'-AMP2- (K = 2.1 M-1) less than D(5'-AMP)- (3.4 M-1) less than D2(5'-AMP) +/- /D(5'-AMP)- (5.6 M-1) greater than D2(5'-AMP) +/- (approximately 2 M-1) greater than D3(5'-AMP)+ (less than or equal to 1 M-1). Self-stacking is most pronounced here if 50% of the adenine residues are protonated at N-1; complete base protonation reduces the stacking tendency drastically. Comparing the self-association of 2'-, 3'- and 5'-AMP shows that there is no influence of the phosphate-group position in the 2-fold negatively charged species, i.e., K congruent to 2 M-1 for all three AMP2- species. More importantly, there is also no significant influence observed if the stacking tendency of the three D2(AMP) +/- /D(AMP)-1:1 mixtures is compared (K congruent to 6-7 M-1); moreover, the measured association constants are within experimental error identical with the constant determined for D(Ado)+/Ado (K = 6.0 M-1). This indicates that any coulombic contribution between the -PO3(H)- group and the H+ (N-1) unit of the adenine residue to the stability of the mentioned stacks in D2O is small. However, experiments in 50% (v/v) dioxane-D8/D2O with the D2(5'-AMP) +/- /D(5'-AMP)- 1:1 system reveal, despite its low solubility, that coulombic interactions contribute to the self-association in an environment with a reduced polarity (compared to that of water). The implications of these observations for biological systems are briefly indicated.