Jail. 5, 1954
STABILITY OF ZINCCOMPLEXES WITH GLUTATHIONE KHC-C=O I
I
H&
0
\ /
cu
/ \
0 NH:! I 1 O=C-CHR
and (b) an absorption maximum around 700 m p indicates a 1:l complex. Our conclusion may be compared to that of Klotz, et al.,5 who state that the absorption peak of copper(I1) ion in complexes shifts toward shorter wave lengths, from 700 to 600 mp, as coordination number is increased. Figure 6, curves 1, 2 and 3 are similar to Fig. 5 , except that here glycylglycinate is the ligand. Here all three curves have the same peak a t 630 mp. Therefore, from what has been said above, only one complex is present, namely, CuA2. It must be noted that in Figs. 4, 5 , and 6 the pH varies from mixture to mixture, depending on the ratio of potassium serinate or potassium glycylglycinate to copper(I1) nitrate in the mixture. This variation, however, has no effect on the conclusions
[CONTRIBUTIOS FROM THE
225
obtained on the formulas of the complexes. It is seen in Table I V that when an alkali is added to a given copper-serine mixture for the purpose of varying the pH, the values of Xmax fall in the region 635 to 617 mp over a pH range of 5.15 to 10.50. This wave length region of absorption maxima is, as noted above, an indication of the presence of a 1 :2 complex. In addition it is noted,in Table IV that over a pH range of 6.46 to 10.50 for the particular copper-serine mixture, the value of the molar extinction coefficient a t the wave length of maximum absorption is almost ionstant. For these reasons we are confident that the spectrophotometric results are valid in spite of the variation in pH. Summary on Number and Formulas of Complexes.-Tables I, 11, 111, and Fig. 4 show that the polarographic, potentiometric, conductometric and spectrophotometric methods are in agreement with respect to the existence of the 1 : 2 complex. The conductometric and spectrophotometric methods reveal an additional 1: 1 complex whenever the ligand is an a-amino acid ion, but fail to show this when the ligand is the anion of glycylglycine. PITTSBURGH 19, PESNA.
DEPARTMENT OF
CHEMISTRY,
DCQUESSEUSIVERSITY]
Stability of Zinc Complexes with Glutathione and Oxidized Glutathione] B Y NORMAN
c. LI, O S C h R G.4WRON
AND
GLORIABASCUAS
RECEIVED JULY 24, 1953 Several acid dissociation constants of glutathione and oxidized glutathione and the stability of zinc complexes with glutathione and with oxidized glutathione were determined a t 25'. Equations for calculation of the several acid dissociation constants by the Bjerrum method have been deduced and equilibrium formation constants for the 1: 1 complexes, i . e . , 1 mole of zinc ion to 1 mole of chelating agent, are reported. I t is suggested that in the zinc-glutathione complex, zinc is probably coordinated through the sulfur atom and the amino group, and that in the zinc-oxidized glutathione complex, the coordination is probably through the amino and a-carboxylate groups.
Because of current biochemical interest in gluta- the value, 9.62, reported by Pirie and pin he^.^ Bethione and oxidized glutathione, this paper reports cause of this large discrepancy we have redetermined the determination of the formation constants of zinc these values. The pK's of oxidized glutathione have complexes with glutathione and oxidized gluta- not been reported. thione, by an adaptation of Bjerrum's method.2 Calculation of Constants This method, which involves measurement of the Dissociation Constants of Oxidized Gluta(A) PH of solutions containing known amounts of the thione and Glutathione.-The dissociation conmetal ion, the peptide and a base, was carried out stants for oxidized glutathione which are necesa t a constant ionic strength of 0.15. This value of sary for calculating formation constants are dethe ionic strength was chosen so as to be the same as termined by the following equilibria present in the ionic strength used in zinc-albumin studies, inas- aqueous solutions much as the purpose of this study is to furnish backH4A = H3AH + ; K3 = (&A-)(H+)/(H4A) (1) ground information on metal-protein complexes. (2) I n order to obtain the concentration of the lig- H3A- = H Z A - ~ H + ; Kq = (H,A-')(H')/(H3A-) HA-3 H + ; Kj = (HA-a)(H+)/(H'A-z) (3) and, taken to be the anion of the peptide, it is nec- HzA-' (4) essary to determine the acid dissociation constants HA-3 = A-4 H'; K6 = (A-4)(H+)/(HA-3) of the peptides. Values for pK2 (COOH), 3.53, where ( ) represent molar concentration and pK3(NH3+), S.66, and @&(SH), 9.12, of glutathione H4=lis have been listed bv Cohn and Edsall.3 Their Pk, however, -OOC-(N~~+)-CH-(CH~)~-CONH-CH-(CH~S)--CONH-CH~COO~ is in serious disagreement with I
+
+
b
+ +
( l ) With the support of Grant No. -OOC-( NH3+)-CH-(CH2)2-CONH-CH-(CH~S)--CONH-CH2C00~ 1496 from t h e Penrose Fund of t h e American Philosophical Society. Removal of protons in the successive ionizations (1) J. Bjerrum, "hletal Ammine Formation in Aqueous Solutions." are assumed to take place in the order a,a,b,b for P . H a a s 8; Sans, Copenhagen, 1941. equations 2, 3) 4, respectively. f:i) E , J. C o h n a n d J. T. Edsall, "Proteins, Amino Acids and Peptides," Keinhold Publ. Curl,.. Ncw E'urk, S. Y., 1!143, p , 8:. (4) N. W. Pirie and K. C . Pinhey, J . Diol. C k e m , 84, 321 (ll12o).
--
XORMAN C. LI, OSCARGAWRON AND GLORIABASCTJAS
396
Equations relating concentration of peptide, Eoncentration of KOH arid PH to the dissociation constants can be derived as follows: for oxidized glutathione, let I' = total concn. of peptide in the solution T = +A-4 + FIAI--J+ H24-2 + H , , ~ I - ~ lf4dk ( 5 ) +
(XH) = total concn. of protons bound to peptide in the solution
+
+
+
(SEI) = H A - 3 2H,A-* m4A-' 4114'4 (SH) 4T - ( K O I i j - (H+) (O13-) ti = ( S H ) j T
+
((ji
n =
-1
11
oxidized glutathioiie, respectively. of moles of ligand baud per mole of illeta1 ioii
= A V , 110.
KsK4Ka(HX-+ ~ ~ : ~ ~ ~ ( H . !(H +A ~:-~ . ~ . ~ j ~ t:~ =~(AIA ( H T 2hfA2j/Thl ~ ~ ~ K:jK4K&6 f K3KdK,>(HT) K S K ~ ( H ' -f) ~IL(H+j3f ( H + j 4 (" For oxidized glutathione
(17)
+
=
PH
+ log
?I -
]
3
- ,z
log [ l
+
&(I/ -. 2'1 (H+)(n- 3 )
( IOi
ant1 P h 4
= total coiicii. uf metal ion iii soln. = total coiicii. of peptide iii soln. = total COI,C]1, of free cI1elati11g age11t i,l SO]Il, I;ro]Il \\.hat has l x e i i said, the chelating agents are A - 3 wheii t h e peptides are glutathioiie and miti 4-
l'\i
T
(20
In deriving equations for I
