R.M. SMITH AND R. A. ALBERTY
2376
[CONTRIBUTION FROM
THE
VOl. 78
DEPARTMEST O F CHEMISTRY, UNIVERSITY O F WISCONSIN]
The Apparent Stability Constants of Ionic Complexes of Various Adenosine Phosphates with Divalent Cations's2 BY R E X
hf. SMITH3 AND ROBERT -4. ;iLBERTY RECEIVED OCTOBER 10, 1955
Equations have been derived for the calculation from pH measurements of apparent stability constants of complexes for cases in which more than one complex may be formed. The formation of complexes of adenosine-5-mon0, di and triphosphate, orthophosphate and creatine phosphate with Ca-+, &lg+-, S r - + and &In++ has been studied at 0.2 ionic strength and 25'. The stability constants of the complexes were calculated on the basis of the assumption that (CHzjdN;, ( C 2 F s j 4 N + and (n-C3H7)+J+d o not form complexes with the phosphate anions. Sincc the highest pK,' values were obtained in solutions containing (n-C3Ht)4N+, it is considered that the most accurate values of the stability constants are obtained on the basis of the assumption t h a t (n-C3H7)4N+ ions do not form complexes. The l o ~ vsolubility of the (n-C4Hg)4Nf halides did not permit the extension of the data to larger tetraalkylammonium ions. Only a single complex is detected in the case of orthophosphate, adenosine m o n o p h o s p l ~ a t eand ~ creatine phosphate while in the case of adenosine di- and triphosphate two complexes are important, and there is indication of a third. The relative values of the stability constants are in the order expected from electrostatic theory; > A D P + > P-2, and .lTP-3 > A%DP-2 andMg++;> e a + + > Sr-+. Creatine phosphate forms weaker complexes than any of t h e other phosphate compounds, and M n + + forms complexes which are considerably more stable than those containing the alkaline earth cations. The ionization constants of the complexes of ATP and B D P with C a + + , M g + + , S r + + and b.In+T have bccn calculated.
Introduction Many reactions of biochemical importance involve organic phosphate compounds, and i t is characteristic of these reactions that their equilibria and kinetics are markedly affected by the pH and metal ion concentration. In order to understand such effects, it is necessary to have knowledge of the ionization constants of the phosphate compounds and of the stability constants of complexes which are formed. The variation with pH of the free energy of hydrolysis of some organic phosphate compounds has been calculated by Dixon,j and Hill and Morales6 have discussed the magnitude of the free energy of hydrolysis of a number of compounds and the effect of pH. The adenosine phosphates AMP, ADP and ATP4 have 1, 2 and 3 strongly ionized groups, respectively. The apparent ionization constants in 0.15 M sodium chloride for the amino and the last phosphate ionizations of these compounds were determined by Albertji, Smith and Bocki a t 25 and 38'. These apparent ionization constants were used to calculate the pH variation of the apparent free energy and the number of equivalents of acid produced P, or consumed in the reactions XTP = XDP ADP = AMP P and AMP = X P. NelchiorS showed that lower values of the apparent ionization constants are obtained in solutions containing tetraalkylaminonium halides rather than alkali halides. He showed that this indicated the formation of complexes of N a + and E(+ with ATP and ADP and determined the stability coiistnnts
+
+
+
(1) Presented a t t h e American Chemical Society Meeting i n N e v York, Sept., 1954. (2) This paper is based u p o n a dissertation presented by R e x 32. Smith t o the Graduate School of the University of Wisconsin in partial fulfillment of t h e requirements for t h e degree of Doctor of Philosophy, June, 1955, (3) Pure Oil Fellow, 1053-1055. (4) Adenosine mono-, di- and triphosphate are represented by A I f P , A D P and A T P , respectively, orthophosphate by P and creatine phosphate by C r P . ( 3 ) AI. Dixon, "Alulti-enzyme Systems," Cambriiljie 1;niversity Press, 1949. (6) T. L. Hill and 11.F. Lforales, THISJ O U R I ~ ' A L , 73, 165G (1951). (7) R . A, Alherty, R . hf. Smith and R . M.Bock, J . Biol. C k e i i i . , 193, 42;1 (1951). ( X ) N,C . llclcliii,r ;hi,/ , 2 0 8 , f i l i ( l ! I . i . l j ,
for these coniplexes. This work has been extended in the preceding article in this seriesg The exects of N g + + and Ca+f on many biocheniical reactions have been investigated. I t is found that the number of equivalents of acid produced, the apparent equilibrium constant for the reaction and the kinetics may be markedly changed. These various types of effects are illustrated by the studies of myosin ATPase by Green and Xommaerts'O and of ATP-creatine transphosphorylase by Kubq-, Soda and Lardy.llml? The stability of the complex formed between M g + + and HP04" has been studied by a number of workers whose results will be summarized in the Discussion section. Evidence for the formation of a complex with ATP, other than that given by the effect of Ca++ and Mg++ on the equilibria and kinetics of chemical reactions, has been given by Hersla who has reported that the titration curve for ATP is displaced t o lower pH values by the addition of niagnesium chloride and by SpicerI4 who has nieasured the quantity of acid produced when calcium or magnesium salts were added to solutions of 9TP. Burton and Krebs'j have made preliminary measureinelits of the stability constants for the magnesium complexes of ATP and ADP and used these values to estimate the el'fect of magnesium concentration on the free energy of hydrolysis of
.vw.
Treatment of Data.--The acid HA is defined by PII = pk':,
pk'; value of a weak
+ log l Y , , - l r € I a l + log [ a / ( l - u)l PKi -I-log [a/(l -
=
(1)
where a is the fraction in the conjugate basic form, and ? A - and ~ 1 z . are 4 the activity coefficients for the conjugate basic and acidic forms, respectively. The apparent ionization constant KL may be considered to be equal to the product of the thermody(9) I
