PAULW. PRE~SLER AND MARGARET M. BATEMAN
2632
dZ-r- (,b-Hydroxyphenyl) - 4 - (4t-hyclroxycycloIiexyl)-hcxatie has been isolated from the alkali soluble inaterial and its structurc has bcen establishcd. 'The remaining mixture of cis- and trans-
VOl. 69
isomers appears to be resistant to the usual methods of separation. I t contains small amounts of isomers belonging to the meso series. CDLUMRIA, MISSOURI RECEIVED NOVEMLIER7, 1946
[CONTRIBUTION PKUM THE 1)EPAKTMliNT O Y ~ I O L O G I C A I . CHEMISTRY,
WASHINGTON UNIVERSITY
SCHOOL OF
MEDICINE]
Oxidation-Reduction Potentials of Thiol-Disulfide Systems. II. Dithiobiuret3,5-Diimino- 1,2,4-dithiazoline B Y PAUL
w.PREISLER AND MARGARET M. IJATEMAN
The activation of certain enzymes by the forma- The recorded potentials have been converted into 13 (normal hydrogen electrode taken as zero). The tion or liberation of the thiol (-SH) groups of the values poteiitials of the two electrodes usually were within 0.0001 enzyme molecule or their inactivation by altera- volt and, after the first two minutes allowed for mixing tion or blocking of these groups has been indicated and reaction, did not change more than 0.0002 in thirty minutes. in the recent literature. The materials and solutions were prepared from comSince tlic reduction of ( - S S ) to two (-SH) mercial analytical grade reagents. is a possible mode of activation and the reverse Dithiobiuret' was recrystallized from hot 0.01 N hydrooxidation a possible mode of inactivation, the oxi- chloric acid. At this acidity the slight amount of oxidized dation-reduction potentials of these components product formed by air oxidation remains in solution and not decompose significantly into sulfur and other of the enzyme molecule or the potentials of the does undesired products. Analysis of the material showed : reagents which react with them are of interest. N, 30.5, 30.7; calculated, 31.1. The measurement of the simpler systems in3,5-Diimino-l,2,4-dithiazoline,the oxidation product has been undertaken of dithiobiuret was isolated as the hydrochloride. Twentyvolving (-SH) and (-SS-) grams of recrystallized dithiohiurct was suspended as one approach to the study of the mechanism of five in 350 cc. of N hydrochloric acid. Whilc cooling in an such reactions. A further aim is to establish a ice-bath, 21.5 cc. of 30% hydrogen peroxide was added graded series of reagents which might be useful tlropwise and stirring was continue11 for about a halfhour or until, by microscopic examination, all of the for such studies. needle-like crystals of dithiobiurct had disappeared and The following simple type system is the first to only the granular crystals of the oxirlation product hydrobe reported which involves the opening and clos- chloride were present. These were filtered off by suction, ing of a ring structure as part of the oxidation- washed with cold N hydrochloric acid, acidified ethanol, ethanol, and finally ether. The ,570water remaining after reduction equilibrium. H
H
s
S
I
I
HN=C---N-C=NH
I
-S
- 2e - 2H
S
I
+
I
= HN=C-N-C==KH
I
H 3,5-Diimino1,2,4-dithiazoline (oxidant)
H Dithiobiuret (reductant)
The potentials, within the PH range studied, are stable and follow the equations applicable to systems of the type: reductant - 2 H + = oxidant. The relatively low position of the system on the oxidation-reduction scale (E6 = +0.251 a t pH 0 and Ed = +0.102 a t PH 5 ) indicates that dithiobiuret is a moderately strong reducing agent and suggests its use for this purpose.
Experimental The oxidation-reduction potential apparatus and procedure generally used For such measurements were employed. The potentials of platinum wire electrodes immersed in the test solution were measured against a saturated potassium chloride calomel half-cell connected by a saturated potassium chloride-4% agar bridge. A Leeds and Northrup Type K potentiometer and a No. 2420-C galvanometer were used. Liquid junction potentials were considered negligible. The electrode, vessel and calomel cell were kept in a water-bath a t 30" and the test solution was deoxygenated and stirred with cylinder nitrogen gas previously purified by passing over hot copper.
drying over calcium chloride was removed by heating a t 110' for about two hours. Analyscs showed: N, 22.4, 24.4; C1, 21.1, 21.0; calculated N, 21.7; C1, 20.9, indicating the monohydrochloridc, Cz,HdN&C!. Ceric or thallic sulfate solutions in sulfuric acid were prepared from the corrcspontling oxides and standardized. Formamidine disulfide solutions in N hydrochloric acid were prepared from weighed amounts of the di-trichloroacetate. The pH of the acid solutions or buffers were calculated from their corresponding quinhytlroiie electrode potentials. Their composition, within 1% accuracy, was as follows: PH 0.05--1 N HCl; PH 0.73-0.2 N HCI, 0.8 M KCl; QH 1.32-0.05 N ----, HCI. 0.95 MKC1: bII 1.99-0.1 N HCI. - - -~ -~ 6:i k-iycine, 6.9 M KCI; PH 2.60x0.06 N HCI,; 0.14 A i glycine.O.92 MKCI; PH 3.36-0.02 NHCI, 0.18 Mglycine, 0.98 M KCl; PH 3.97--').16 N HAC, 0.04 M KAc, 0.96 M KCI; .bH 4.58-').1 N HAC, 0.1 M KAc, 0.9 MKCI; pH .5.19--kU4 N HAC, 0.16 M KAc, 0.84 M KCl. Quantitative solutions of the reductant or oxidant were made with deoxygenated solvents and protected by the passage of a stream of purified nitrogen. Moderate heating was usually necessary to facilitate solution of the reductant.
Calculations The mathematical equations applicable to the potentials of the system may be developed in the conventional mannerZ from the fundamental electrode equations.
(1) Obtained through the courtesy of the American Cyanurnid Company. 30 Rockefeller Plaza, New York 20, N. Y. (2) W. M . Clark und B. Cohen, U. S Public Health Repts., 811, 670 (1023); 40, 1168 (1925).
Nov., 1947
DITHIOBI~T-3,5-DIIMINO-1,2,4-DlTHIAZOLINE POTENTIALS
2633
mixture of oxidant and reductant a t some specified pH.) Graphically EOmay be considered as the intersection of the projected 0.00 slope portion + I )r ir + H n N = C - - IN - L H of the Eo' - pH with the pH = 0 ordinate line. I Since K., = lo+' was assumed for convenience for B illustrative purposes, no particular significance +S should be attached to the magnitude of the value I I I so selected nor to the EOso obtained. H2~=&--N-c=NH :1_ 'H + HN=C-N--C=NH The effect of pH or acidity upon the potentials I IH H (2) of an equi-molar mixture of oxidant and reducthe first being a strong acid of K = lo+' or greater, tant is shown in Fig. 1, where Eo' is plotted assumed for convenience in developing the equa- against pH. The curve follows the equation (8) tions to be Ka, = lo+', and the second measured and is a straight line from pH 0 to 5, except for as P K a y = 7.4, or .Kay= 4 x The reductant slight deviation at each end due to the effect of does not ionize appreciably within or near the #H the ionizations. range studied. The sum, So, of the concentrations of all of the species of the oxidant present may be expressed ++ The oxidant has two possible ionizations b S
!TO = (OxHz)
+ (OgH) + (Ox)
(3)
Since the reductant has essentiFlly only one species, its concentration may be represented s'lmply by S,. The potential of one of the equilibria participating may be ca:lculated by
where E h is the potential of the platinum oxidation-reduction electrode referred to the normal hydrogen electrode as zero; Eo is a constant characteristic of this system and based on the values of the specific ionization constants as designated and assutned above; and R, T In and F have their usual significance.. At 30°, (RT/nF) log, ( ) becomes 0.03 logllD( ).
-0.200 so that the succlessive elimination of (Ox)and + (OxH) by a combination of (6) then ( 5 ) with (3) results in (OS,) =
SO
(b9 + K.,H) + (U'LJ (b ++
(7)
Then by substituting (OxHn) in (7) into (2) and separating terms the final complete equation becomes Eb m Eo
+ 0.03 1Og:io-SSO, + 0.03log,,
(618
(b+ (K.,d) + (Kn,Ks*)
(8)
It should be noted that EO is not the value of + E6 a t (H) = 1, or pH = 0, as is the case with many systems but it is the Eo' of that portion of + the Eo' -pH cunre where H is not involved in the equilibria, and the slope is zero. (E4 has its customary definition as the E h of an equi-molar
L2L-J
3 4 5 PH. Fig. 1.-Relationship of the &' of common oxidationreduction systems at various PH to the thiol-disulfide systems. 1
2
The potentials of mixtures of oxidant and reductant are shown in Tables I and 11. The observed values agree with those calculated by the appropriate equation usually to within 0.0001 volt. The calculated value of Eo for each pH is shown in Table 11; the arithmetical average is 0.2520 with a maximum variation of *0.0013. Using this average, E; at pH = 0 may be calculated by equation (8) as 4-0.2510 volt. "Semiquinone" formation for this system is negligible. A "semiquinone" is a structure3intermediate between the fully oxidized and fully reduced form of a two-valent change reversible oxidation-reduction system which differs from each (3) L. Michaelis, Chsm. Rco., 16, 243 (1935); L. Michaelis and M.P. Shubert, ibid.. 44, 437 (1938).
2634
PAUL
% Total e!quivalents
w.PREISLER AND MARGARET M . BATEMAN
-Eh
Reductant
Oxidant
Eh Exptl.
90 80 75 70 60 50 40 30 25 20 10
10 20 25 30 40 50 60 70 75 80 90
0.2201 ,2310 .2336 .2377 ,2428 .2481 .2532 .2594 .2624 .2664 .2775
Calcd.
-
+ + + + +
+
- E;-Theor.
-0.0280 .0171 ,0145 .0104 - .0053 0000 .0051 .0113 .0143 .0183 ,0294
.
Vol. 69
tically equal to (OxH). It follows from equation
+
-0.0286 .0181 .0143 .0110 .0053 .om0 .0053 .0110 .0143 .0181 .0286
-
(2) that (Ox) = (H). Therefore, equation (6) becomes
-
-
+ + +
+ +
- (HI2 (salt) (salt) log K,, = -log (H)* 4-log (salt) pK., = 2 PH log (salt) (K) X (H)
Ks*=
+
(9)
(10) (11)
The PH of a 0.1 M solution of oxidant hydrochloride was found to be 4.2 and that of a 0.01 M solution 4.7, when brom cresol green was employed with the usual indicator techniques. pKa, was calculated a t 7.4 for both cases.
TABLE I1 MIXTURES OF DITHIOBIURET AND 3,5-DIIMINO-1,2,4-DITHraZoLINE I N BUFFERS AT VARIOUS fiH Total concentration of mixture, oxidant f reductant, = 0.01 N (0.005 M ) : ionic strength = 1.0
POTENTIALS OF
Ea/,
El/,
(76% oxid. 25% red.)
6H
(E3
(0.00) .Oti .7:1 1.32 1.99 2.60 3.36 3.97 4.58 5.19
(+0.2510) .2481 ,2296 .2124 .1916 .1745 .1525 .1331 .1144 ,0958
+
+0.2624 .2439 .2268 .206 1 .1884 ,1667 .1480 .1290 ,1101
- E I/;
(25% oxid. 75% red.)
4-0.0143 .0143 .0144 .0145 ,0139 ..0142 .0149 .0146 .0143
+0.2336 .2155 .1982 .1774 ,1599 .1382 .1189 .lo03 .0810
of these Sorms by one equivalent. A structural formula ,with completely satisfied valences for each element cannot be written for semiquinones. Their presence may be best detected by the deviation of the slope of the potential curve from that of the theoretical curve when n = 2. The slope a t each PH (see Table 11)is estimated from the difference between the value a t 75y0 or a t 25% oxidized and that a t 50% oxidized. The theoretical or - 0.0143 v. and difference is respectively there is no significant deviation from this value. The ionization constants of the components of the system could not be determined from the changes in slope of the EA-pH curve, since only one distinctive slope portion was measureable. The rate of decomposition of the oxidant a t pH higher than 5.2 prevents reliable measurement of potentials for the region where both components are un-ionized and the uncertainty of the liquid junction potentials with concentrated acids rendered dificult the intcrpretation of results with such solutions. The second acidic dissociation constant of 3,5 diimino-l,2,4-dithiazoline,PKal, could be estimated from the measurements of the pH of aqueous solutions of the oxidant hydrochloride salt by employing the equation (11) below.
+
-
El/,
(calcd.)
Eo
(calcd.)
(calcd.)
-0.0146
(4-0.2820) .2807 .2818 .2820 .2814 .2825 .2833 .2822 .2818 .2815
-
-
-
.0141 .0142 .0142 .0146 .0143 .0142 .0141 '.0148
+
Dithiobiuret does now ionize appreciably within the PH range of 0 to 5. The effect of total concentration was negligihle or within reasonable experimental error. For a total concentration of 0.01 N with ratio of oxidant to reductant concentration of one, in 1 N hydrochloric acid Eh = f0.2481 volt; for 0.001 N concentration Eh = +0.2493. The potentials a t 0.0001 N concentration were quite unstable (unpoised) while stirring the solution with nitrogen gas but attained the value of +0.251 within a few minutes after stirring was discontinued. Titration of the reductant with ceric or with thallic sulfate in 1 N sulfuric acid solutions, or with formamidine disulfide in AT hydrochloric acid, gave potentials which were within 0.001 volt of the theoretical values a t all of a great number of measured points between 10 and 90% reduced or oxidized. Titrations of the oxidant with titanous sulfate, or of the reductant with vanadic (5+)sulfate or with molybdicyanide, in acid solutions were unsuccessful because of the slow rate of reaction with the reagents employed. Discussion Because of the importance of the (-SH) and (-SS-) groups in biological materials, many at-
Nov., 1947
D I T H I O B ~ 3 , 5 - D I ~ O - 1 , 2 , 4 - D I T H I A Z O L I N POTENTIALS E
2635
tempts have been made in the past twenty years consideration must be taken of the total combined to measure the oxidation-reduction potentials of concentration of the components, as well as the 2H = RSSR. ratio of the reductant to the oxidant, because the the apparent equilibrium, BRSH The potentials were generally unstable and diffi- equation for such systems involves the square of cult to duplicate and did not follow with sufficient the RSH c~ncentration.~J At low PH, dithiobiuret is among the strongest accuracy the equations believed to be applicable to the systems. of the common organic reducing agents. When Recently some results for cysteine-ystine and the side products of the decomposition of the unother thiol-dithio systems were reported which ionized oxidant ( P K a , = 7.4) are not objectionwere obtained under special conditions of titrat- able, dithiobiuret may also be useful for reducing ing agents and ~ a t a l y s t s . These ~ results, while purposes above PH 5.2. Formamidine disulfide, the oxidant of thioindicating apparent agreement with the theoretical calculations, are not in accord with the many urea, readily oxidizes dithiobiuret as predicted by prior investigations cited in the same publication4 the potentials of the two systems, the expected with regard to the reliability and possible sig- potentials being rapidly and accurately attained, nificance of the ineasurements of these systems; This result demonstrates that systems of the further study and confirmation seem desirable to (-SH) and (-SS-) type can react with each definitely establish the potentials of these systems. other and suggests the extension of the present use The potentials of the thiourea-formamidine di- of (-SH) and (-SS-) compounds for the intersulfide system, measured under a variety of condi- action with similar groups when these are part of tions of acidity and concentration,s agree with the the enzyme molecule for activations or inactivacalculated values well within the tolerances usu- tions by oxidative or reductive reactions. ally set for organic systems and demonstrate the The measurements are being extended to inreversibility of the thiol-dithio equilibrium. clude the potentials of various thiol-disulfide sysThe measurement of the oxidation of dithio- tems in order to prepare a series of graded reagents biuret to its oxidant, 3,5-diimino-1,2,4-dithiazo-of this type and to study the effect of substitutions. line, seemed a desirable step in the development of Summary this subject because of the two thiol groups on The oxidation-reduction potentials of the thiolthe same molecule which offered the possibility either of the formation on oxidation of a ring struc- disulfide system, dithiobiuret-3,5-diimino-1,2,4ture with two sulfur atoms or of the formation of a dithiazoline have been measured in the pH region chain compound with two or more sections con- of 0.05 to 5.2 and found to follow the equations aplinkages. The conformity of the plying to systems involving a reversible twonected by 4% potentials to the simple equation for a two-valent equivalent change from reductant to oxidant. step oxidant-reduction affected by changes in the This result establishes the oxidation step as the ratio of oxidant to reductant but unaffected by removal of 2H from the 2 -SH of dithiobiuret to changes in the total combined concentrations and form a five-membered ring structure with two adalso the symmetry of the curve shows that the di- jacent S atoms, the first system of this type whose potentials have been measured. sulfide formed is the single ring structure. The E; of the system a t PH 0 was estimated as The relative position of the Ei-PH curve of diEh = +0.251 volt and the second acidic ionization thiobiuret-3,5-dii mino-1,2,4-dithiazoline system toward several other common reversible systems is constant of Lhe oxidant as PKa, = 7.4. The low shown in Fig. 1. When comparing potentials of a E6 suggests the use of dithiobiuret as a reducing particular mixture of reductant and oxidant of a agent. Formamidine disulfide oxidizes dithiobiuret givsystem of the type BRSH 2H = RSSR (thiourea-formamidine disulfide) with any other, due ing the theoretical potentials and showing that (-SH) (-SS-) type systems can react rapidly (4) L R. Ryklan and C. L. A. Schmidt, University of California and reversibly with each other. publications in Physrology, 8, 257 (1944).
-
+
(5) P. W. Preisler and L. Berger. THIS JOURNAL, 69, 322 (1947).
ST.LOUIS,
MO.
RECEIVED MAY23, 1947
