Nov. 20, 1953
REACTIONS BETWEEN MERCURY (II), CYSTEINE AND GLUTATHIONE [CONTRIBUTION FROM THE
SCHOOL OF
5673
CHEMISTRY O F THE UNIVERSITY O F MINNESOTA]
Reactions between Mercuric Mercury and Cysteine and Glutathione. Apparent Dissociation Constants, Heats and Entropies of Formation of Various Forms of Mercuric Mercapto-Cysteine and -Glutathione BY W. STRICKS AND I. M. KOLTHOFF RECEIVEDJUNE 5, 1953 Reactions of cysteine and of reduced glutathione with mercuric mercury in media of various pH have been studied polarographically. In the absence of chloride, and in the pH range between 3 and 9, cysteine and glutathione can form at least three compounds with mercury: Hg(RS)z, Hg,(RS)t and Hgg(RS),, in which mercury is bound firmly as mercaptide. In the presence of much chlonde the formation of the compounds Hg2(RS)*and Hgg(RS)2is suppressed by the complex HgCt--. The formation of compounds of the type RSHgCl could not be demonstrated polarographically. The apparent dissociation constants of three charge types of the mercaptides of cysteine and glutathione were calculated from e.m.f. measurements of mercury in equilibrium with solutions of the mercaptides in the entire pH range in the presence of a large excess of either cysteine or glutathione. The constants of the mercaptide species with two charged amino groups are found to be of the same order of magnitude for both cysteine and glutathione, while the constants of other species differ to a greater extent. From the values of the dissociation constants a t 12 and 25” the heats and entropies of formation were calculated.
The stable bond between mercury and sulfur of sulfhydryl groups has been made wide use of in biology. Thus mercury compounds have long been used as specific reagents for sulfhydryl groups in biological materials. l-6 Mercury has also found application in the isolation of proteins like mercaptoalbumin.6 More recently analytical use of mercury compounds was made in the quantitative mercurimetric determination of sulfhydryl and disulfide groups in proteins, peptides and amino acid^.^.^ Considering the wide biochemical applicability of mercury as a sulfhydryl reagent, a more complete knowledge of the reactions between mercury and sulfhydryl containing amino acids, peptides and proteins is desirable. This paper deals with a quantitative study of the mercuric-cysteine (RSH) and the mercuricglutathione (GSH) systems a t various pH and temperatures. It is shown polarographically that mercury can form a t least three compounds with RSH and GSH, respectively. The mercury bound to the sulfur is very slight1.y dissociated. Apparent dissociation constants, heat and entropy of formation of the mercaptides of cysteine and glutathione have been calculated from e.m.f. measurements. The results obtained with the amino acid (RSH) are compared with the data of the peptide (GSH). Materials.-Cysteine used as free base was a product from Mann, glutathione was a Pfanstiehl product. The purity of these products was 99% as determined by titration with cupric copper.0 Stock solutions of these compounds (0.01 and 0.2 M ) were prepared in air-free water. Only freshly prepared stock solutions of the amino acid and peptide were used. The stock solution of mercuric acetate (a C.P. product from Powers-Weightman-Rosengarten Co.) was 0.05 M in mercury and 0.08 M in acetic acid. The concentration of the stock solution of mercuric chloride (a (1) L. Hellerman, Cold Spring Harbor Symposia Quant. B W . , ‘7, 166 (1939). (2) H. Neurath, J. P. Greenstein, F. W. Putnam and J. 0. Erickson, Chcm. Revs., 84, 157 (1944). 361 (1945). (3) M. L. Anson, Advances Prokin Chcm., I, (4) H. S. Olcott and H. Fraenkel-Conrat, Chcm. Revs., 41, 151 (1947). (5) G. E. S. Barron, Aduancss Enrymol., 11, 201 (1961). 69, 1836 (1947); Cold S9rin.s (6) W. L. Hughes, Jr., Tma JOUENAL, Harbor Symposia Quant. Biol.. 14, 79 (1949). (7) W. L. Hughes, Jr., “Proceedings, First Conference on Cancer Diagnostic Tests,” p. 26, 1950. ( 8 ) W. Stricks and I. M. Kolthoff, Anal. Cham., 96, 1060 (1963). (9) I. M. Kolthoff and W. Stricka, ibid., It, 768 (1951).
Mallinckrodt product, better than 99.5% pure) was 0.05 M in Hg(I1). All the other chemicals used were C.P. reagent grade products. The stock solutions for the preparation of the buffers were: M hydrochloric acid, M acetic acid, M sodium acetate, 0.2 M disodium phosphate, 0.2 M monosodium phosphate, 0.1 M borax, 0.4 M boric acid, 2.6 M sodium hydroxide, M ammonia, M ammonium nitrate. The ionic strength was adjusted by addition of appropriate volumes of 2 M potassium nitrate or 4 M potassium chloride solutions. Experimental Methods.-Current-voltage curves were determined at 25.0 f 0.1’ with the manual apparatus and circuit described by Lingane and Kolthofflo and automatically with a Heyrovsky self-recording polarograph. The characteristics of the capillary were: m = 1.56 mg. set.-', 6 = 4.82 sec. (open circuit); m’/$/*= 1.748 mg.’/l set.-'/*; h = 80 cm. A layer of chloroform at the bottom of the cell was used to prevent the mercury metal from reacting with free mercury in solution. As maximum suppressor to 2 X 10-8% gelatin was used. Dissociation constants of the mercuric mercaptides of cysteine and glutathione were calculated from e.m .f. measurements of mercury metal in equilibrium with mercuric mercaptides in the presence of a large excess of uncomplexed thiol. Under these conditions all the mercury in the solution is present in the mercuric form. Mercurous cysteinate and glutathionate are unstable and readily decompose to mercury metal and the mercuric compounds.11 The mercuric ion concentration was calculated from the measured potential. All potentials were measured with a Leeds and Northrup student potentiometer against the saturated calomel electrode (S.C.E.) which was kept at the same temperature as the test solution (25.0 f 0.1’ and 12 f 0.1’) using a salt bridge with saturated potassium chloride solution. The potential of the S.C.E. against the hydrogen electrode was calculated by the expressionl* EE.c.E.= -0.2420 0.00076 ( t 25) (1)
+
-
where t is the temperature in OC. Oxygen was removed from the solution in the cell with a stream of oxygen-free nitrogen which was purified by bubbling through vanadous sulfates.1* An atmosphere of nitrogen was maintained over the solution during the measurements. The pH was measured with a Beckman pH meter, Laboratory Model G, using the Beckman “General Purpose” glass electrode for the entire PH range. pH measurements a t above 10 were corrected for the sodium ion concentration. The cell for the e.m.f. measurements was provided with a rubber stopper with holes for a nitrogen inlet tube, for a tube with sintered glass bottom which contained the salt bridge, and with a hole which served for introducing solutions into the cell by means of a pipet. Ten to 20 ml . of pure mercury was placed into the cell and a given volume of buffer solution (IO) J. J. Lingane and I. M. Kolthoff, THISJOURNAL, 61, 825 (1939). (11) W. Stricks and I. M. Kolthoff, ibid., ‘74, 4646 (1952). (12) S. Glasstone, ‘‘Textbook of Physical Chemistry,” D. Van Nostrand and Co., Inc., New York, N. y., 1940, p. 922. (13) L. Meites and T. Meites, And. Chrm., 30, 984 (1948).
5674
W. STRICKS
c
AND
r. M. KOLTHOFF
:/
VOl. 75
to the line of the mercaptide wave. The slope of these lines is considerably smaller than that of the blank C L E ~ R line, obtained by adding mercuric ion PRECIFITATE to an acetate buffer in the absence of GSH. The location of the various 4 SOLY/TION I a* waves is made clear in Fig. 2 which illustrates polarograms obtained during a titration of GSH at pH 5.6 with mercury. All observations indicate that most of the mercury added after 3 the first end-point is not present as free mercuric ion but is bound to the mercaptide. When mercury has been added in the mole ratio GSH:Hg = 1:1 the two cathodic waves are of the 2 same height. Apparently a compound Hgg(GS)* is formed in which one mercury is bound !irmly as mercaptide and one much more loosely, probably by way of carboxyl groups. g 1 The addition of more mercury after the second end-point gives rise to the formation of a white predpitate which results in a reduction in height of the two cathodic waves to the same ex0 tent. Thus the change in height of the two waves on the addition of increasing amounts of mercury can now be represented by the same line as is illustrated in Fig. 1. Since no new wave appears in the polarogram it is -1 concluded that the mercury added after the second end-point, combines with Hgs(GS)p to form a slightly soluble compound Hga(GS)z. When the amount of mercury added is in the -2 vicinity of the mole ratio CSH :Hg = 2 : 3 (third end-point) the cathodic wave a t the more positive potential increases in height again but starts 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1 2 1.3 now a t +0.28 v. instead of +0.20 v. It is seen from Fig. 1 that this ‘‘ex0.05 M Hg( 11) solution added, ml. cess reagent” line is curved at first, Fig. 1.-Titration of 30 ml. of 10-8 M G S H (acetate buffer, pH 5.6, 1.7 X l O - 3 % becomes steeper after the third endgelatin) with 0.05 M mercuric acetate solution. Plot of diffusion currents versus ml. point and finally attains a slope which is practically equal to that of the of Hg(1I) solution added: 0 , O ; ia measured a t -0.10 v.. 0 , id measured a t -0.65 blank line. The mercaptide wave, volt. however, decreases continuously along a curved l i e and faally disadded. Nitrogen was bubbled through and appropriate appears in the presence of an excess of mercury. Apparently volumes of air-free stock solutions of cysteine or glutathione precipitation of the compound Hgg(GS)2is not complete a t and of mercuric acetate were added to the air-free buffer, the third end-point and therefore a mercaptide wave is still the total volume being either 10 or 20 ml. The sintered observed at this point. Addition of more mercury decreases glass tube with the salt bridge was lowered into the solution the solubility of Hg,(GS)n and gives rise t o a further decrease and the e.m.f. measured. The pH of each solution was de- and finally disappearance of the mercaptide wave. The termined a t the temperature specified in Tables I to IV. slow precipitation of Hg,( GS)z also explains why the excess of mercury line is not parallel to the free mercury line until I. Polarographic Measurements a considerable excess of reagent is present. The white precipitate formed during the titration was cenT o air-free 10-8 M RSH or GSH solutions in the absence and presence of chloride ion and a t various pH were added trifuged, washed with air-free acetate buffer (PH 5.6) and known volumes of 0.05 or 0.025 M mercuric solutions and suspended in an air-free acetate buffer of the same pH. complete polarograms were taken after each addition of mer- Part of the precipitate dissolved and the resulting solution cury. From the known polarographic characteristics of waa polarographed. The polarograms showed two cathodic waves starting at +0.22 and -0.3 v. with diflusion cysteine, glutathione and their mercuric mercaptide+J* the di&sion currents (corrected for residual current and currents of 0.613 pamp. (measured at -0.1 v.) and of 0.232 change in volume) of the various compounds present in the ramp. (measured a t -0.65 v.) corresponding to the recourse of a titration could be plotted. As sn utam le such duction of loosely bound and mercaptide mercury, respeca plot is presented in Fig. 1, which ives the various $iffusion tively. The ratio of the heights of the first and the second currents in a titration of a IO-* M ~ Wsolution in an acetate waves was found to be 2.6 indicating an appreciable disbuffer a t +H5.8 in the absence of chloride ion. From Fig. 1 sociation of Hg,(GS)Z into H g + + and Hgz(GS)2, the wave it is seen that the anodic diffusion current of GSH decreases of free mercuric ions overlapping with the first wave of on the addition of mercuric ions and disappears when GSH Hg,(GS)z. Figure 3 presents the results of a titration of a 10-3 M and mercury are present in a mole ratio 2: 1 (first end-point). At this point only one cathodic wave is observed, starting GSH solution in an acetate buffer (pH 6.6) which was 2 M at a potential of -0.3 v., corresponding to the reduction of in potassium chloride. In the presence of chloride ion no mercury in the slightly dissociated merca tide Hg(GSX. precipitate is formed and only one end-point corresponding On further addition of mercury the height o?the mercaptide t o the formation of the mercaptide can be detected. After wave remains constant and another cathodic wave starts this end-point the excess mercury line is slightly less steep at a more positive potential of about +0.2 v. This wave than the blank line but on further addition of mercury beincreases in height along a straight line which is parallel comes parallel to the blank. Evidently the complex HgCld-- in the presence of 2 M chloride is stable enough to (13a) I. M. Kolthoff and C. Barnum, THISJOURNAL,69,308 (1940). suppress the reaction of excess mercury with the mercaptide. CI
I
f
s
Nov. 20, 1953
If
WAVE OF FREE
REACTIONS BETWEEN MERCURY (11), CYSTEINE AND GLUTATHIONE Hslnb
5675
6
E
5
4
3
e;
x$
2
LI 0
2 1 -0.05 -0.25 -0.45 -0.65 E d e . , volt vs. S.C.E. Fig. 2.-Polarograms taken during a titration of 30 ml. of 10-3 M glutathione (acetate buffer, PH 5.6, 1.7 X l O - 3 % gelatin) with a 0.05 M mercuric acetate solution. A, before addition of mercury. After addition of (B) 0.15 ml., (C) 0.3 ml., ( D ) 0.6 ml. 0.05 M Hg(I1) solution. (E) Blank: l o v s M Hg(I1) in acetate buffer in the absence of GSH. $0.35
+0.15
Since the dissociation constant of HgC14-- is 8.3 X 10-16,14 the constants for mercury bound by carboxyl groups of glutathione must be considerably larger. Titrations of glutathione carried out a t pH 9 and PH 3 in the absence and presence of chloride ion gave results which were similar to those presented in Figs. 1 and 3. At pH markedly lower than 3 no precipitate is formed during the titration in a chloride-free solution and chloride ion has no M effect on the titration lines. Thus a titration of GSH in 0.1 M perchloric acid, 0.1 M sodium perchlorate gives only the mercaptide end-point and the diagram of the titration is practically identical with that in Fig. 3. Mercurimetric titrations of cysteine indicate that the amino acid qualitatively behaves in the same way as the peptide in its reactions with mercury. A titration of M RSH in a borax buffer (pH 9.06) in the absence of chloride is presented in Fig.4. The results indicate that cysteine can also form three compounds with mercury: Hg(RS)a, Hgg(RS), and Hg,(RS)Z in which one mercury atom is bound firmly to sulfur. In the presence of chloride a diagram is obtained which is practically identical with that in Fig. 3.
T
.1" -1
-2
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 0.025 M Hg(I1) solution added, ml. Fig. 3.-Titration of 20 ml. of lo-* MGSH (acetate buffer, PH 5.57, 2 M KCI, 2.5 X lo-*% gelatin) with 0.025 M mercuric chloride solution. Plot of diffusion currents versus ml. of Hg(1I) solution added. 0 , O ; i d measured a t -0.16 v.; 0 , id measured a t -0.70 volt.
found to contain one cysteine residue and one mercury, thus corresponding to the formula Hg2(RS)2. When he mixed cysteine and mercuric chloride in the mole ratio 2 : 1 the amorphous white precipitate first formed disappeared on stirring, but on stwding of the clear solution a white precipitate separated which microscopically was found Polarographic experiments thus indicate that to consist of transparent needle shaped crystals. cysteine as well as glutathione can form a t least Analysis showed this compound to be Hg(RS)1. three compounds with mercury in which all (in When an excess of mercuric chloride was added to Hg(RS)2) or a part (in Hg2(RS)2 and Hg,(RS)2) cysteine a white amorphous precipitate was formed of the mercury is bound firmly as a mercaptide. the composition of which corresponded to the No polarographic evidence can be obtained for formula Hg(RS)2*2HgC12. The formation of the the formation of compounds of the type RSHgCl compounds Hg(RS)t and Hg2(RS)2 was also demonin which one mercury is bound by sulfur as well as strated by potentiometric titrations with the dropping mercury electrode as indicator electrode. In a by chloride or any other anion. The polarographic findings on the system potentiometric titration of 20 ml. of 6 X M cysteine-mercury substantiate and extend M RSH in a buffer of PH 4.48 with 2 X Barnum'sls results on the reactions between mercuric chloride Barnum obtained two endcysteine and mercuric chloride. Barnum mixed points corresponding to the compounds Hg(RS)s equimolar solutions of cysteine and mercuric and Hg2(RS),. chloride and observed the precipitation of a white II. Determination of the Apparent Dissociation amorphous compound which was analyzed and Constants. Heats and Entropies of Formation of (14) A. Johnson, I . Quartfort and L. Sillen, Acto Ckem. Scand., I. 46, the Mercuric Mercaptides of RSH and GSH 473 (1947). From the mercuric ion concentration determined ( 1 5 ) C. P. Barnurn, Jr., Ph.D. Thesis, University of Minnesota in a given mixture of mercury(I1) and an excess of (1040).
Vol. 75
W. STRICKS AND I. M. KOLTHOFP
5676 4
.
I
,
Ip
t
P R E CI P I T A T E 3
2
g
1
a
$
.-a
0
E
-1
-2 A
!?
I
-3 01
0
03
0,4
0
0,s
0,7
1,0
I2
1,l
1,3
I,.&
7
Fig 4.-Titration of 40 ml. of lo-* M RSH (0.05 M borax, 0.1 M KNO,, 2.5 X lo-*% gelatin, QH 9.06) with 0.05 M mercuric acetate solution. Plot of d ~ u s i o ncurrents wcrsus ml. of Hg(I1) solution added: O,O, id measured at -0.3 v.; 0 , & measured at -0.80 volt.
RSH or GSH the apparent dissociation constant of the mercaptide can be calculated. All measurements were carried out a t an ionic strength of 1 0.1 and concentrations instead of activities of the various ionized forms of RSH and GSH were used in the calculations. The values of the apparent dissociation constants K have been calculated by introducing the proper concentration values in equation 2 K e p p . = [a&+] [RS-I */IHdRSM (2) The mercuric ion activity was calculated from e.m.f. measurements vs. S.C.E. using the equation RT c.m.f. = Eo + 2~ In [a&*] - 0.2420 +
*
0.00076 ( t
- 25)
(3)
effect on the dissociation constants of the mercaptides and therefore the constants of the carboxyl groups need not be considered. Constants of cysteine a t 12 and 25’ are given in equations 4,5, 6 and 7. The concentrations of the various species are denoted by letters above the symbols. C
RSH
I
-
f
NH,+
d
RS-
I
NHJ
d
-
4-H+, pKA
= 8.66at 12and 25’ (4)
+
a
where Eo = 0.854 volt,Is the standard potential (vs. N.H.E.) of the mercury-mercuric system. b U The charge forms of cysteine and glutathione as RSH RSwell as of the mercaptides vary with the PH. I f- I H+, p K f , = 10.51 at 12 and 25’ (7) The acid-base constants of the various groups of 1“ “a cysteine are known and their variation with tem2 Cysteine = a + b + c d = perature was estimated previously. l7 The experimental data in this paper indicate that the charge of the carboxyl groups in RSH and GSH has no Equation 8 was used to calculate the concentration (IS) W. M. Latimer, “The Oxidation States of the Elements and of each of the species of cysteine a t a given pH. theu Potentials in Aqueous Solutions,” Prentice-Hall, Inc., New The individual constants of glutathione are not York, N. Y.,1962, p. 179. known. However, approximate values of the con(17) W. Stricks and I. M. Kolthoff’,THISJOURNAL., IS, 4569 (1951).
+
+
REACTIONS BETWEEN MERCURY(II), CYSTEINE AND GLUTATHIONE
Nov. 20, 1933
centrations of the various charge species can be calculated, assuming that in analogy to cysteine and homocysteine, the values for glutathione of PKA and pKb are practically identical a t 25'. This assumption is reasonable considering the fact that the measured dissociation constants of the peptide A' - 2.2 X (NH3+) and K f 4= 7.6 X lo-'" (SH)19 differ to a much smaller extent than the corresponding constants of cysteine (K'z = 4.7 X K'3 = 1.7 X The constants K13and K'4 as determined directly by pH titration are related as follows to the individual constants1g-20
5677
Combining equations 13 with 14 and 14 with 15 and substituting from equation 6 gives
and Kp = KII -IC&
KIII
From equations 18 and 19 it is seen that the dissociation constants of the amino groups in the mercaptide can be expressed in terms of the constants KI, KII, KIII and of the individual constant K'a = [H+l(d b , = Kta + KIB K b of the amino acid or peptide. c (9) The constant KIII which involves the species with uncharged amino groups is found directly from experiments with solutions of sufficiently high K'aK'4 K'AK'c = K'BK'D (11) pH. An example of the calculation of KIII for The concentration of the total glutathione is given cysteine follows: original mixture: 4 X M RSH, 5 X M Hg(II), PH 13.3, ,u = 1, t = by 1 2 O , e.m.f. = -0.6968~. vs. S.C.E. [H+l + Zglutathione = a b c d =a 1 From equation 3: [Hg++] = 1.2 X K'4 Z uncombined cysteine = total cysteine added (12) -2 X total mercury added = 4 X = K'aK'p 3.9 x 10-2. where a[H+]/K'd = b d. The small letters From equation 8: a = 3.88 X d = 1.28 refer to the same charge species as indicated for X It is seen that d is negligibly small as cysteine. Equality of K'A and K'B means that b compared to a, while a is practically equal to the and d are also equal. The values of b and d can concentration of total uncombined cysteine. Therebe calculated from equation 12. Using equations fore e and g can be neglected as compared to f, 9, 10 and 11 and the known values of K t 3and K f 4 , while f is equal to the concentration of the total the approximate pK values of the individual con- mercury added (5 X M ) , the mercuric ion stants of glutathione are found to be ~ K ' A= concentration being negligibly small. ~ K ' B= 8.96 and pK'c = ~ K ' D= 8.82 a t 25'. From equation 15: KIII = 3.6 X This At 12' ~ K ' A = 8.96, ~ K ' B = 9.33, PK'c = 9.21 simplified calculation of KIII can be applied for and ~ K ' D= 8.82. It is now possible to calculate the dissociation constants of the mercaptides with cysteine a t PH higher than 11.5 a t 12 and 25' reference to the various species of cysteine and and for glutathione a t PH higher than 10.9 and 9.5 a t temperatures of 12 and 25O, respectively. glutathione, respectively. It is seen from TabIes I to IV that the values of KIII e d as calculated in the above way are practically RS-Hg-SR RS[Hg++]d' constant within these PH ranges. Below is given I s 2 1 + H g + + ; Kr =I e an example of the calculation of KI: original NH: NH: KH: mixture: 4 X M RSH, 5 X M Hg(II), (13) P d a pH 2.97, p = 1, t = 1 2 O , e.m.f. = -0.2737 volt vs. S.C.E., [Hg++] = 1.1 X Z Uncombined cysteine: 4 X - 2 X 5 X lom4F 3.9 X a = 1.073 X d = 7.95 X e =
+
+
+ + +
1
+
1
+
f
a
From equation 13: KI = 1.4 X The concentration of the species with uncharged amino groups can be neglected over a PH range which e s extends for RSH from pH 0 to PH 7 a t 12' and RS-Hg-SR RS-Hg-SR I + I IT + H + ; K E = -M+Ig from pH 0 to pH 5.5 a t 25', and for GSH from pH I IiHC hH2 KH: NH: 0 to PH 7.5 both at 12 and 25'. Beyond these PH (16) ranges the species with uncharged amino groups * must be considered. The constant KII can now be calculated from measurements in solutions in the higher pH range and from KI and KIII. An example for the calculation of KII follows: original mixture: 5 X M RSH, 7.5 X M Hg(II), (18) L. R. Ryklan and C. L. A. Schmidt, Arch. Biochcm., 5, 89 (1944). pH 9.10, p = 1, t = 2 5 O , e.m.f. = -0.6338 v. (19) E. J. Cohn and J. T. Edsall, "Proteins, Amino Acids and Pepvs. S.C.E., [Hg++] = 7.4 X Z Uncombined 1943,p. 85. tides," ReinhoId Publ. Corp., New York, N.Y., = 5 X 1.5 X = 4.85 X cysteine (20) J. T. Edsall and M. H. Blanchard, THISJOURNAL, 55, 2337 M. a = 8.48 X d = 1.90 X (1933). f
I
5678
ii? STRICKS . AND
I. M. KOLTHOFF
Vol. SEI
TABLE I APPARENTDISSOCIATION CONSTANTS OF THE THREE CHARGE TYPES OF MERCURIC MERCAPTO CYSTEINATE (Hg(RS)2)AT 25" RSH added,
Hg(I1) added,
M
2 3 4
x x x
M
5 5 5
10-2 10-2
10-2
x x x
10-2 10-2 10-2 10-2
2 x 2 x 2 x 3 x 10-2 4 x 10-2 6 X 6 X 4.44 x 10-2 2 x 10-2
10-4 10-4 10-4 10-8 10-3
10-8 10-3 10-8
10-3
10-3 2 X 2 . 2 x 10-8 10-3
2
x
10-2
10-3
2
x
10-2
10-3
2
x
10-2
10-3
2
x
10-2
10-3
5
x
10-2
2
x
10-2
7.5
x 10-4 10-3
3
x
10-2
2
x
10-2
2
x
10-2
10-3
2
x
10-2
10-3
2
x
10-2
10-3
3
x
10-2
10-3
4
x
10-2
10-3
5
3 x 10-2 2 x 10-2 6 X
4
x
5
x
x
10-4
10 -3 10-4 10-3
2 X 10-3 10-3
10-2
Electrolyte
0.2 M NaOH, 0 . 8 1cf KN03 . 2 M NaOH, .8 M KNOa . 5 M NaOH, .5 M KNO: . 2 M NaOH, .8 M KNOI . 2 M NaOH, .8 M KN03 . 3 M NaOH, .8 M KN03 .3 M NaOH, .8 M KCl . 3 M NaOH, .8 M KCI .3 M NaOH, . 8 M KN03 . 3 M NaOH, .7 M KN03 , 3 M NaOH, . 7 M KNOa . 3 M NaOH, . 7 M KN03 .05 M NaBPO, .078 M NaOH, . 8 M KN03 .08 it4 Na2HPO4 .078 M NaOH, .7 -1J KN03 .08 M NaZHPO, ,039 M NaOH, . 7 M KN08 . 1 M NHiNOs . 2 M "2, .9 KNOa .05 M Borax ,026 I1 XaOH, ,F,i JI KCl .05 ,2.I Borax ,052 M NaOH, .8 it4 KC1 ,0175 M Borax, .13 M boric acid ,026 M NaOH, . 9 M KC1 ,015 M Borax, .14 Jl boric acid, M KCl .08 M NazHPO4, .75 M K N 0 3 . O l M NaH2P04 .08 M NazHPOa, .75 Jf KXO3 .01M PiaHpPOl .05 M Na2HP04 .05 M NaHZPOa, .76 M KNOI .18 M CHgCOONa .02 M CHsCOOH, . 8 M KNO3 . 1 M CHsCOOSa . 1 M CHsCOOH, . 9 M KNOa . 2 iM CHsCOOH, ;If KNOi . 2 M CHaCOOH, M KNOi .2 CHsCOOH, M KNOa . 2 31 HC1, , 8 M KC1 . 2 Jf HC1, .S M KNO3
From equations 13 and 15 and the known values of K I and KIII, given in Table I e = [HgC+ld2 = 4.79 x 10-6,f =: E = KIII
KI
1.95 x 10-6 hence g = ZHg(I1) - e - f = i o - - 6 - 1.95 x 1 0 - 6 = 7.26 x 10-4
+ + g;
f ZHg(I1) = e 7.5 x 10-4 - 4.79
x
ExperiFrom equation 14: K ~ =I 1.6 X ments were carried out over the entire +H range with mixtures of varying mercury and RSH(GSH) concentrations, respectively. The experimental data and the constants K I , KII and KIII are listed in Tables I, 11, 111 and IV. The variation of the mercuric ion concentration with PH for a mixture of the same original concentration in mercury -11) is pre( l o + M ) and RSH(GSH) ( 2 X
fiH
fi[HgC+l
>13 >13 > 13 >13 >13 > 13 >13 >13 >13 >13 >13 > 13 11.61
43.36 43.81 44.04 42.25 43.03 43.02 43.03 43.47 43.81 44.27 43.91 43.41 43.02
11.24
x
KI
Krr
KIII
X 10"
X 104'
... ...
...
...
...
3.1 2.6 2.8 3.6 3.0 3.1 3.0 2.7 2.2 1.8 2.0 2.8
1041
...
...
...
...
...
...
... t
.
.
.,.
... ...
...
...
...
...
...
...
...
. . I
...
1.2
...
42.92
...
1.6
...
10.23
42.31
...
1.7
...
9.40
41.28
...
2.8
...
9.15
40.81
...
4.3
...
9.10
42.13
...
1.6
...
8.22
39.45
...
3.1
, . .
7.59 7.27
38.57 37.51
... ...
1.9 4.9
...
7.16
36.99
...
2.1
...
6.40
35.31
...
2.8
...
5.52
33.50
5.4
...
...
4.62
32.07
5.6
...
3.07 3.00 2.90 1.00 0.88 Av. K:
29.21 29.17 28.35 25.11 24.80
5.9 5.5 4.4 5.8 6.3 5.6
...
...
... ... ...
...
...
...
...
...
...
2.5
2.7
sented graphically in Fig. 5. It is seen that the p[Hg+f] values of the particular mixtures are identical for RSH and GSH in the lower PH range but differ by about two units for mixtures in the higher p H range a t which the p[Hg++J becomes independent of the hydrogen ion concentration. The relation between hydrogen ion and mercuric ion concentration can be given by the equation
where A is the original concentration of cysteine (glutathione) and B the concentration of mercury added. K', and K'S are the titration dissociation constants of IiSH (the corresponding constants of
REACTIONS BETWEEN MERCURY (11), CYSTEINE AND GLUTATHIONE
Nov. 20, 1953
5679
TABLEI1 TABLEIV APPARENT DISSOCIATION CONSTANTS OF THE THREECHARGE APPARENTDISSOCIATION CONSTANTS OF THE THREE CHARGE TYPESOF MERCURIC MERCAPTO CYSTEINATE AT 12" TYPES OF MERCURIC MERCAPTO GLUTATHIONATE AT 12' RSH added, M
2 x 10-2 4 X 2 x 10-2 5 x 10-2 6 X 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 4 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 5 x 10-2 4 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 5 x 10-2 6 X lo-*
?%M:
pH
[Hi;+]
5X 5 X lo-' lo-'
13.20 13.32 13.20 13.20 2 X 13.26 12.64 10-3 11.37 10.42 10-3 9.52 9.32 8.32 10-3 7.19 6.40 5.49 4.57 3.60 3.00 5X 2.97 2.86 1.55 0.71 loy3 0.83 2 X 0.87
45.18 45.93 44.85 45.86 45.63 44.79 44.67 44.10 42.90 43.58 41.12 38.47 36.77 34.90 33.09 31.25 30.94 30.97 29.76 27.13 25.56 26.45 26.37 Av. K:
KI
KII
KIII
IO44
lOM
IOU
x
x
.. ..... ..... ...
4.7
.....
3.6
...
..
...
..
4.6 3.2 3.7 5.0 3.4
..
...
... ,
3.8 7.8 4.04 5.7
..
... ... 1.1 1.6 1.9 1.7 1.4 1.3 1.4 1.4 1.4 1.1 1.8 1.8 1.5
.. ,.
.. , . ..
.. ..
GSH added,
X
... ,
,
.
...
... ...
... ... ... ... ...
..,
., .. .. .. , .
...
5.3
4.0
... ...
... , . .
M
M
2 4 3 2 4 4 4 2 2 4 2 2 2 2 2 2 2 2 2 2 2 2 3 4 2 4 2
X lou2 5 X X 10-2 5 X IO-' X 10-2 7 . 5 X x 10-2 lod3
x
10-2
X
x x x x
2 X 10-2 10-2
lo-' lo-'
lo-*
X
x x x x x x x x x x
10-3
5 X lo-'
1010-2 10-2
10-3
10-
10-8 10-8 10-3
10-2
10-2
10-2 10-2 10-2 10-2 X 10-2
10-8 10-8
10-3
10-8 10-8 X IOd2 2 X 10-8 x 10-2 5 x 10-4 x 10-2 10-8 X 10-2 5 X 10-4 x 10-2 10-8
pH
13.2 13.0 13.1 13.2 13.0 13.2 12.8 12.7 11.90 11.28 10.87 10.86 9.70 9.30 8.48 7.64 6.53 6.00 5.13 4.32 3.37 2.73 2.74
43.24 44.27 43.61 42.84 43.93 43.50 43.77 42.78 42.77 43.89 43.23 42.82 42.73 42.49 41.24 39.01 36.85 35.76 34.09 32.62 30.62 29.46 29.51 1.80 28.58 1.60 27.06 0.88 26.60 0.88 25.56 Av.K:
KI
KII
KIII
104a
10"
10"
x
[H:;+]
x
. . . . . .
x
......
4.1 1.7 2.7 4.6 1.7 2.0 2.5 5.4 5.4 1.8 4.1 4.6
3.9 2.7 2.2
... ...
.
.
,
.,
,
, , ,
,
, ,
. . . . . .
. . . . . . . . . . . .
. . . . .
. . . . .
...
... ... 4.2 6.3 6.7 5.8 4.1 5.2 3.9 3.9 4.1 5.5 5.3 6.1 5.1
. . . . .
. . . . .
. . . . .
. . . . .
...
...... . . . . . . . . . . . .
...... . . . . . . . . . . . ,.. . . . ,., . . . ,
. . . . . . , ,
,
. . . , ,
.
. . . . , ,
. . . TABLEI11 2.9 3.4 APPARENT DISSOCIATION CONSTANTS OF THE THREE CHARGE TYPESOF MERCURIC MERCAPTO GLUTATHIONATE, Hg(GS)2 glutathione are denoted as K l g and K ' J . The full AT 25" lines in Fig. 5 were constructed from PHg values GSH Hg(II) KI KII KIII calculated from equation 20 for A = 2 X added, M
1.5 x 10-2 2 x 10-2 2 x 10-2 4 x 10-2 2 x 10-2 2 x 10-2 4 x lo-" 2 x 10-2 4 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x 10-2 2 x lo-' 2 x 10-2 2 x 10-2 8 x 10-2 2 x 10-2 4 x 10-2 3
2
x x
10-2
10-2
added, M
5 5 5 5
x x x x
10-4 10-4 10-4 10-4 10-3 10-3 10-3
2 2
x x
10-3
10-3 10-3 10-3 10-a 10-3 10-3 10-3 10-3 10-3 10-3
10-3 10-3
2
x
10-8 10-3 10-3 10-3
10- 8
5
x 10-4 10-3
2%'
pH
>I3 >13 >13 >13 >13 >13 >13 >13 >13 12.80 11.37 9.50 9.18 8.32 7.49 6.53 5.19 4.43 4.41 3.41 2.80 2.47 2.30 1.87 0.87 0.83 0.83
[Hg++]
41.07 41.41 41.48 42.35 41.00 41.11 42.08 40.55 41.50 41.03 40.98 40.90 40.72 39.46 37.51 35.54 32.82 31.36 31.28 29.39 28.17 27.49 28.10 26.26 24.96 24.96 24.22 Av. K:
x
x
x
IO41
IO42
IO42
...... ...... ......
3.4 2.8 2.4 . . . . . . 1.4 . . . . . . 3.2 . . . . . . 2.5 . . . . . . 1.2 . . . . . . 3.6 . . . . . . 2.1 . . . . . . 3.0 . . . . . . 3.3 . . . . . . 1.9
... ...
... ... ......
1.3 1.0
1.0 1.3 . . . . . . 1.4 . . . . . . 1.2 . . . . . . 1.4 . . . . . . 1.1 . . . . . . 1.0 . . . . . . 1.1 . . . . . . 1.1 . . . . . . 1.2 . . . . . . 1.0 . . . . . . 1.0 . . . . . . 1.1 . . . . . . 1.1 1 . 2 2.6
and B = a t varying [H+]. The values of KI, KII and KIII were taken from Tables I and 11. The experimental points fit the calculated lines satisfactorily over the entire pH range. It is readily seen that a t low pH the first two terms a t the left side of equation 20 are negligibly small as 46 44
42 40 38 36 834t 32 30
+ 0 1 2 3 4 5 6 7 8 9
m.
11
13
Fig. 5.-pHg versus pH of mixtures of 10-8 M Hg(I1) with 2 X lo'% M RSH ( 0 )and 2 X lo-* M GSH (0),respectively, at 12' (- - - -) and 25" (-).
W. STRICKS AND I. M. KOLTHOFF
5 680
Vol. 75
compared to the third term and on the right side the last two terms in the bracket can be neglected as compared to the first term. Thus for low pH values, equation 20 can be written in the simplified form
heats and entropies of formation of the various species of the mercaptides are summarized in Table VI. It is seen that the mercaptides of the amino acid and the peptide have values of AH and A S of comparable magnitude.
or
TABLE VI APPARENT DISSOCIATION CONSTANTS (pK VALUES),FREE ENERGIES, HEATSAND ENTROPIES OF FORMATION OF MERCURIC MERCAPTO CYSTEINATE AND GLUTATHIONATE
At low pH the relation between fiH and PHg is thus presented by a straight line of slope 2. This relation is actually observed in Fig. 5 a t 12 and 25'. The values of the three constants for cysteine and glutathione are not affected by the composition of the buffers used. K I I I and K I I are practically equal and smaller than K I for both GSH and RSH, the difference being more pronounced for cysteine. Thus a t 12' the ratios of K I : K I I Iare 3700 and 15 for the mercaptides of cysteine and glutathione, respectively. The relation of the dissociation constants K I and KIII of the mercaptides to the hydrogen ion ciissociation constants K'A and K'D of the corresponding species of the free amino acid and peptide is given in Table V. TABLE V
RATIOS(KIIIIK'D) AND ( K I I K ' A ) FOR CYSTEISE AND GLUTATHIONE 120
x
KII I/K'D
120
250
x x
sx 4.6 x
KI/K'A
250
12
PKI pKI I ~KIII PK E PI(F
AFI , kcal. AFII,kcal. A FI11, kcal. AFE, kcal. AFF, kcal. AHI, kcal. AHII , kcal. A H I II , kcal. AHE,kcal. AHr, kcal. A S , cal./deg. ASII, cal./deg. ASIII, cal./deg. ASE, cal./deg. ASF, cal./deg.
oHg(RS)r 25 O
41.82 40.25 45.27 43.60 45.40 43.57 7.39 7.10 10.72 10.48 -54.5 -54.8 -59.1 -59.5 -59.1 -59.3 -9.63 - 9.68 -14.0 -14.3 -46.9 -49.9 -54.5 -8.7 -7.2 +27 $32 +I6 $4 $24
HdGS): 120 250
42.29 40.96 43.54 41.92 43.47 41.58 7.97 7.85 9.28 9.15 -55.2 -55.9 -56.8 -57.1 -56.6 -56.6 -10.4 -10.7 -12.1 -12.5 -39.7 -48.2 -56.1 -3.6 -3.9 54 +30 $2 +24 $29
+
The heat of formation increases from the I- to 111-species for cysteine as well as for glutathione. The ratio K I I I / K ' Dis slightly larger and K I / K ' A The values of AH indicate that the bond energy is slightly smaller for glutathione than for cysteine. between metal and complexing agent is strongest The value of K I for cysteine is of the same order of in the species with the uncharged amino groups, as I The magnitude (about 5 times larger) as that of GSH. also evidenced by the large ~ K I I values. The same order of magnitude was expected, be- values of AH11 are smaller than AHIII indicating cause the stability of the mercaptide is determined that the mercury in complex 111 is bound more almost entirely by the strength of the mercury- firmly than in complex 11, a fact which is not sulfur bonds. The amino groups are in the charged reflected by the PKII and PKIII values which are form and no coordination of mercury with the practically equal. It is interesting to note that the entropy of nitrogen can occur under these conditions. As a formation is larger for the I- and 11-species than for matter of fact because of its positive charge the NH3+ group should decrease the stability. Evi- the 111-species for both cysteine and glutathione. The values of AH and A S for the dissociation of dently, the contribution to stability of the mercaptide by coordination between mercury and the the amino group in the mercaptides are of the same uncharged amino groups is very small, because KII order of magnitude as the corresponding values for and KIII are identical. The large ratio of KI to free cysteine and glutathione, respectively. In conclusion the equilibrium constants of the KIIIfor cysteine as compared to that for glutathione indicates that coordination with the amino groups following reactions in cysteine is more favorable than in glutathione. This is structurally to be expected, but the over-all contribution to the stability is small. From the values of KI, KII and KIII the values of K E and KF can be calculated from equations 18 and 19. From the equality of KII and KIII it follows that K Fis of the same order of magnitude as Kb. can be expressed as The heat of formation of the various species of the mercaptides was calculated from the corresponding dissociation constants a t 12 and 2.5' and the entropies of formation were found from the relation AS = (331 - A F ) / T . The pK values of the five constants, the free energies (RT In K), where the corresponding species of cysteine and RSH: GSH:
1.3 2 2
x
10-85 10-36
0.9 1 7
10-28
IO-^*
G
10-34 10-84
z
5
x
10-82
10 - 2 2
Nov. 20, 1953
PHASE
EQUILIBRIA IN
glutathione are denoted by the indices R and G, respectively. Acknowledgment.-This investigation was sup-
[CONTRIBUTION FROM
THE
POLYMERSOLVENT
SYSTEMS
5681
ported by a research grant from the National Cancer Institute, U. S. Public Health Service. MINNEAPOLIS, MINNESOTA
DEPARTMENT O F CHEMISTRY OF CORNELL UNIVERSITY ]
Phase Equilibria in Polymer-Solvent Systems. 111. Three-component Systems1 BY A. R. SHULTZ AND P. J. FLORY RECEJVED JULY 20, 1953 Phase equilibria data for three ternary solvent-solvent-polystvrene systems have been obtained and interpreted in terms of the statistical-thermodynamic theory of polymer solutions. Thermodynamic interaction parameters calculated from the observed plait points are consistent with those calculated for solvent-solvent and solvent-polymer pairs from binary system liquid-vapor and liquid-liquid phase equilibria data.
Introduction Investigations on liquid-liquid phase equilibria in binary systems consisting of a polymer and a single solvent have yielded results which on the whole agree rather well with the predictions of the statistical-thermodynamic theory of polymer solut i o m 2 Although the theory succeeds only qualitatively in reproducing the observed binodials, the critical miscibility temperatures Tc vary with molecular weight in the manner predicted by the theory.a The polymersolvent interaction parameters calculated from this dependence of Tc on the molecular weights of polymer fractions are remarkably similar to those of chemically analogous small molecule liquid pairs4 This correlation of the thermodynamic functions of large and small molecules is important in providing a common thermodynamic basis for comparing polymers with possibly better characterized small molecule liquids. It may thus be possible to study with greater facility the properties of polymers which are actually peculiar to their great size and chain structure. The practical use of solvent-non-solvent mixtures to effect separation of polymers into less heterogeneous fractions has been reviewed by Cragg and Hammer~chlag.~Powers6 has made several phase studies on polystyrene-mixed solvent systems. Polymer solution theory has been extended to ternary and more complex systems to explain and predict various phenomena dependent upon the free energies of the component^.^-^ It is the purpose of this paper to interpret phase equilibria data for three ternary solvent (1)-solvent (2)-polystyrene (3) systems in the light of present statistical-thermodynamic theory. Methyl ethyl ketone (1)-methanol (2)-polystyrene (3) (system
I) was studied because of its frequent use in fractionation. The interaction parameters for each pair of components in this system are unknown. Consequently, although the data may be used to determine the plait point for the infinite molecular weight polymer species, very little further information is gained in this case. System 11,carbon tetrachloride (l)-cyclohexane (2)-polystyrene (3), on the other hand, is a convenient choice for checking phase equilibria theory. Two of the three interaction parameters are known from vapor pressure and liquid-liquid equilibria mea~urements.~The “co-solvent”-polyrner system, ethylcyclohexane (l)-cyclohexanol (2)-polystyrene (3) (system 111), offered the peculiar advantage that all three pair interaction parameters were theoretically obtainable from liquid-liquid phase equilibria measurements. Experimental
(1) This work constitutes a section of a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Cornell Univereity, by A. R. S. (2) M. L. Huggms, J. Chcm. Phys., 9, 440 (1941); A n n . N . Y . Acad. Sci., 4S, 1 (1942); P. J. Flory, J . Chcm. Phys., 9, 660 (1941); ibid., 10, 51 (1942). (3) A. R. Shultz and P. J. Flory, THISJOURNAL, 74, 4760 (1952). (4) A. R. Shultz and P. J. Flory, ibid., 76, 3888 (1953). (5) L. H . Cragg and H. Hammerschlag, Chcm. Reus., 39, 79 (1946). (6) P. 0. Powers, I n d . Eng. Chcm., 41, 126, 2213 (1949); ibid., 49,
Materials .-The parent polymers were prepared by low conversion polymerizations of styrene monomer in bulk with thermal (for the highest molecular weight polymers) or benzoyl peroxide initiation. Polystyrene fractions were obtained by fractional precipitation of the polymer from dilute solutions in butanone by successive additions of methanol .lo Table I lists the fractions, their intrinsic viscosities in benzene solution at 30°, molecular weights calculated by the relation” log M = 4.013 0.74 log [VI and their percentages of the parent polymers from which they were derived. The intrinsic viscosities were measured in Ubbelohde suspended-level viscometers. Kinetic energy corrections were applied throughout, but rate of shear corrections were unnecessary (rate of shear approximately 2000-2600 sec. -1). Reagent punty solvents were dried and distilled a t least once through a 40-cm . glass helices-packed column. Experimental Procedure.-Phase boundary curves were determined with the aid of a titration apparatus. This apparatus consisted of two Kimble Exax 5-ml. burets, graduated to 0.01 ml., with tips sealed into the head of a solution cell. The tops of the burets were fastened by rubber tube connectors, immersed in mercurv wells, to vertical glass tubes, each having a three-way silicone-lubricated stopcock in the top bend. These vertical tubes were connected by approximately 0.8 meter lengths of rubber tubing to adjacent vertical tubes which could be raised and lowered. The lengths of rubber tubing and vertical glass tubes contained columns of mercury sufficient to prevent contact of vapor with the rubber on the buret side during all manipu-
2558 (1950). (7) R. L. Scott, J . Chcm. Phys., 17, 268, 279 (1949). ( 8 ) H. Tompa, Trans. Paradar Soc., 46, 1142 (1949); ibid., 46, 970 (1950). (9W. H. Stockmayer, J . Chrm. Phys., 17, 588 (1949).
(10) T. G. Fox, Jr.. and P. J. Flory, THISJOURNAL, TO, 2384 (1948). (11) R . H . Ewart. paper presented at the Atlantic City Meeting of the American Chemical Society, April 14, 1947.
+
