Sept. 5, 1954
POLAROGRAPHY OF VITAMINBlZrAND BlZa
4345
no1 comparably labeled with deuterium or tritium. studies, to make an unequivocal deduction with Thus, in metabolic studies involving reactions where regard to a particular intermediate. It should be pointed out that the multiple lasuch fractionation may occur, conclusions regarding the possible pathways of a compound depend on the beling of carbon and hydrogen employed in the above particular isotope of hydrogen chosen as the tracer, mentioned experiments was intermolecular as a resince the data obtained under identical conditions sult of admixture of separately labeled species of with a compound labeled identically with either methanol. The double labeling of a carbon and its deuterium or tritium allow different interpreta- bonded hydrogen within the same molecule, i.e., intramolecularly, should minimize the influence of tions. The second significant point, related closely to hydrogen isotope selection on the ratio of isotopic the isotope effect, is concerned with the ambiguity hydrogen to isotopic carbon. Any change in this that can arise from replacing different numbers of last named ratio should be more an indication of equivalent hydrogen atoms in the individual mole- the nature of intermediates and less a reflection of cules of a metabolite with the same labeling isotope isotopic selection. However, if more than one hyof hydrogen. If one allows that the change in the drogen atom is linked to the carbon-labeling atom, D :CI4ratio observed between the methyl of metha- e . g . , C14,in a molecular grouping under study, the nol and the methyl of tissue choline and creatine possibility of fractionation between the lighter and is due primarily to a metabolic change in the hydro- the heavier isotopes of hydrogen within the molecgen-carbon ratios during intermediary steps, then ular grouping, and therefore between labeled carthe results of the previous utilizing bon and labeled hydrogen, still can exist. The ideal the CDs-species of methanol indicate the possibility case would be that in which the carbon-labeling that “formate” is an intermediate in the neogenesis atoms only are directly linked to only hydrogenof methyl from methanol whereas the present ex- labeling atoms in pertinent carbon-hydrogen bonds. periment with the CHID- species shows that labile At the present time, this objective in labeling can be methyl arises de novo from a “formaldehyde” inter- approached most closely with the available concenmediate without passage through a “formate” trations of deuterium, tritium and C13, and to a step. On the other hand, i t can be seen that the lesser degree with the available C14. Experiments deuterium content of formate isolated in this ex- in which metabolites are doubly labeled with isoperiment from the total pooled urine has been in- topes of carbon and hydrogen within the same molecreased relative to C1*. The ratio of D :C14 in the cule have been undertaken in one of these laboramethyl of tissue choline, that is suggestive of a tories (CUMC). “formaldehyde” precursor, could be a reflection of Acknowledgment.-The authors wish to express deuterium enrichment a t all intermediary steps in- their thanks and appreciation to Dr. Vincent du cluding a “formate” level. Thus “formate” as a Vigneaud for his interest and counsel during the required intermediate would not be ruled out. In course of the experiments and in the preparation of view of the large degree of selection among the hy- this manuscript. The work a t Sloan-Kettering Indrogen isotopes during these metabolic reactions, it stitute was supported in part by grants-in-aid from is not possible, from the type of experimentation the U. S. Atomic Energy Commission AT(30-1)-910. employed in the present or the previous isotopic NEW YORK,N. Y.
[CONTRIBUTION FROM
THE
DEPARTMENT OF CHEMISTRY, IOWASTATECor2rxc.rs]
The Polarography of Vitamins Bllr and Bils BY BRUNOJASELSKIS
AND
HARVEY DIEHL
RECEIVED MARCH26, 1954
Unlike vitamin B11, which undergoes a single two-electron, polarographic reduction, B12a undergoes two one-electron reductions corresponding to changes in valence of the cobalt from three to two and from two to one. Vitamin B1z,, in which the cobalt is bivalent, shows one anodic and one cathodic wave agreeing in position and height with the waves of B,,,.
The catalytic hydrogenation of an aqueous solution of vitamin Blz yields a brown solution containing a reduced cobalt compound’ which has been designated as B12r. Potentiometric titration of BlZrwith potassium ferricyanide in a completely oxygen-free atmosphere has indicated that one equivalent is required to oxidize the BIZr: the cobalt atom is thus in the bivalent state.2 During hydrogenation the cyanide group of BIZis reduced to methylamine. The polarography of vitamin BlZr (1) E. Kaczka, E. E. Wolf and K. Folkers, THISJOURNAL, 71, 1514 (1949). (2) H. Diehl and R. Murie, Iowa Sfofa Coli. J . Sci., 24, 555 (1952). (3) J. L. Ellingboe, J. I. Momson and H. Diehl, in press.
has been reported briefly4 and there also has been described the polarographic behavior of a related but distinctly different product resulting from the action of chromous ethylenediaminetetraacetate on B12r.5
In the present paper the polarographic behavior of BIZr is given in further detail. In particular the polarogram has been extended into the region positive to the saturated calomel electrode by using potassium sulfate as the supporting electrolyte and another wave has been found having a half(4) H. Diehl and I. I. Morrison, Record of Chcm. Progress, KresgeHooker Library, 18, 17 (1952). (5) R . N. Boos, J. E. Carr and J. B. Conn, Science, 117, 603 (1953).
BRUNOJASELSKIS
4346
AND HARVEY
wave potential -0.04 v. toward the S.C.E. This makes a total of three waves in the polarogram of B I ~the , half-wave potentials being -0.04, -0.95 and -1.55 Y., respectively, toward the S.C.E. The first wave is anodic and the second and third cathodic in nature. The first two waves are oneelectron steps. The height of the third wave depends on the time of hydrogenation and other conditions. The characteristics of these waves and their relation to those of B12 and B1a were examined. In this connection new values for the diffusion constants of vitamins Blz and B12a6 have been used.
Experimental Work
DIEHL
Vol. 76
the polarogram obtained in the usual manner. Another aliquot of the solution was transferred t o the hydrogenation vessel! platinum oxide equal to about half the weight of the vitamm was added, and hydrogen was passed through the solution for seven h m after which no further change in pH occurred.* The resulting solution of BI*, was transferred through the fritted glass filter t o the polarograph cell, the entire system being well flushed with oxygen-free nitrogen. The polarogram a s then taken. An aliquot of the residual solution was then taken for a cobalt determination; this obviated any error caused by the slight changes in concentration which occurred during hydrogenation. In another experiment this same procedure was followed but polarograph cell was used having a wide mouth rubber sheet pierced t o accommodate glass and calomel electrodes, capillary, and the tip of a Machlett buret. Standard hydrochlonc acid was added and the polarogram of BI?, obtained a t various values of pH. Polarograms of Vitamins Biza and B1z,.--Starting with crystalline B1za polarograms were obtained using the procedure described in the preceding section.
Materials .-Vitamin B1l,obtained from the Squibb Institute for Medical Research, New Brunswick, New Jersey, was recrystallized from water and dried in a vacuum over anhydrous magnesium perchlorate. BIZ. was prepared by Results and Discussion hydrogenation of recrystallized Blt over platinum, oxidation by air, and precipitation by acetone. Moisture determinaThe polarogram obtained for B12, identical with tions were made on the preparations of both BIZand BIZ^, and samples were weighed out accurately on a semi-micro that reported earlier,',* and that of the B12r derived balance; nevertheless, the concentrations of both BIZand B I ~ from the Bla are shown in Fig. 1. As will be seen given in the following work were obtained by direct colori- the Polarogram of Bla shows three waves, the first metric determination of the cobalt following ashing with wave being anodic and the second and third cathoperchloric acid. Potassium sulfate, A.C.S. grade, was recrystallized from dic. The first and second waves are each just half distilled, carbonate-free water. Solutions exactly 0.1 N the height of the B12 wave, and by inference each and free of carbonate were prepared from this potassium involves a one-electron change. This is borne out sulfate for use as the supporting electrolyte. Cylinder nitrogen was freed of oxygen by passage through by an application of the Ilkovir equation, using for a vanadous sulfate train, alkali and water. Glass tubing the diffusion constant of BlZr,the recently obtained was used in connecting the scrubbing train to the apparatus. value for the diffusion constant of Biz. The pertiApparatus.-Polarograms were recorded on a Sargent nent data are given in Table I. Model XXI polarograph. The functional operations of the polarograph were checked frequently by calibration against a TABLEI standard resistance. A saturated calomel electrode having POLAROGRAPHIC CHARACTERISTICS OF VITAMINS Bu, Birr an agar-potassium chloride salt bridge was used as anode; the internal resistance of this electrode was shown t o be AND Biia sufficiently low t o have no effect on the polarograms. Supporting electrolyte: 0.100 N potassium sulfate Hydrogenation of vitamin BIZand vitamin BIP.was car005 rn'/$f''fi ried out in the hydrogenation apparatus described in a pre(cod.) vious publication.2 (cm.) The hydrogen used was cylinder hydrogen purified by El/a D (equiv.) passage through alkaline permanganate and ascarite. MaConcn., v. os id, cm.a/ser. (sec.)l/z X 10-8 n terial mM S.C.E. amp. X 106 Polarograms of Vitamins BIZand BlZ,.-A weighed amount of vitamin BIZwas dissolved in a definite volume of 0.1 N 1.44 2.95" 0.416 -1.11 1,002 1.98 B,r potassium sulfate to give a solution approximately 5 X 10-4 0 . 8 1 2.95d 1.003 1.06 Bm" ,434 -0.04 M. A 1.00-ml. aliquot was taken for a cobalt analysis. (anodic) Another aliquot was transferred t o the polarograph cell and -0.95 0.76 2.95 1.002 1.01 -1.55 BI?' ,303 -0.06 0.39 2.33" 1.003 0.84 .12 2.33 -0.55' 1,002 .25 -1.02 .45 2.33 1.002 .96 B12r) ,306 -0.04 .40 2.33" 1.003 .85 -0.94 .50 2.33 1.002 1.05 a Obtained by hydrogenation of vitamin BIZfor 7 hours. b Obtained by hydrogenation of vitamin Bizs for 6 hours. Ref. 6. Assumed t o be the same as that of the BIZfrom which derived. oAssumed t o be the same as that of the BIZ. from which derived. Diffusion current depends on time of hydrogenation. 0 Probably due t o impurity.
'
;-811,
-0.50 -1.00 -1.50 -2.00 Potential, volts us. S.C.E. Fig. 1.-Polarograms of vitamins BIZand Bizr; potassium sulfate, 0.100 N as supporting electrolyte. 0.0
(6) B. JaeelsLii and H. Diehl, unpublished work.
The height of the third wave of B1zr depends on various factors which have not been elucidated; it is possible, as happened in our very early work with B1%,to have this wave the height of a one-dectron reduction but this is quite accidental. This wave apparently involves the aeduction of the organic portion of the molecule and probably also (7) H. Diehl, R. R. Sealock and J. I. Morrison, Iowa Stole CoZL J . Sci., SI, 433 (1950). (8) R. Diehl, J. I. Morrison and R. R. Sealock. B%#erieanlh,7, 60
(1961).
Sept. 5, 1954
POLAROGRAPHY O F VITAhlIN
B12rA N D R1za
4347
the further reduction of the cobalt as we postulated earlier. The first two waves of BlZrare independent of PH ; the data are presented in Table 11. I n preliminary experiments t o determine the PH dependence of the half-wave potentials of B12r9 various buffer solutions were used and difficulty was encountered in filtering off completely the platinum catalyst. I n the presence of platinum both the wave height and half wave potential were altered and in view of this, the same supporting electrolyte, 0.1 N potassium sulfate with which no such difficulty was encountered was used throughout the work. Admittedly the solutions were not well buffered but it may be definitely concluded that the half wave potential is independent of pH. TABLE I1 POLAROGRAPHIC CHARACTERISTICS OF VITAMIN Bur Supporting electrolyte: 0.1000 N potassium sulfate; pH adjusted by addition of oxygen-free hydrochloric acid. OH 10 08 8.38 6.85 5 32
El/2
E’/z
-0.06 - .04 - .04
-0.96 - .94 - .95 - .93
-
.04
The polarogram of BIza shows two one-electron cathodic waves, Fig. 2 . The half-wave potential of the first wave is the same as that of the first wave of Bizr. Moreover the polarogram of Blzr prepared from Biza is identical with that of Blzr prepared from BIZ; Table I. The addition of methylamine to the solutions did not affect the polarograms. The anodic, first wave of B12r and the cathodic, first wave of B12a are therefore for the same couple RCo(III)+
+ e-
= RCo(1I)
The small wave falling between the first and second waves in the polarogram of BIZa is quite likely due t o an impurity, produced during the air oxidation of BlZr. This small wave decreases in height and shifts in the negative direction with decreasing PH. The height of the wave varies from one preparation to another. The second wave of BlZa,El/, = -1.02 v. os. S.C.E., is a one-electron step. Like the first wave of Biza it is independent of #H. It has about the same half-wave potential s the second wave of Bizr. Both waves correspond to the reduction of cobalt from two to one RCo(I1)
+ e-
= RCo(1)-
the variation in half wave potential resulting from the difference in the composition of the main body of the electrolyte. The most negative wave of BIZa is a multi-electron step the nature of which, as with the corresponding waves of BIZand Bizr, is not understood. Quite possibly it is a catalytic hydrogen wave similar t o those found with certain other cobalt complexes. In another paper3we have shown that the hydrogenation of BIZtakes place according to the reaction RCOCN Biz
+
6/2H2
RCO
+ CHaNHs
Biz7
We have confirmed also our earlier2finding that one equivalent is required t o oxidize Bizr to BlZa,both by repetition of the ferricyanide titration and by
-0.50 -1.00 -1.50 -2.00 Potential, volts os. S.C.E. Fig. 2.-Polarograms of vitamin Blzs and Bm; potassium sulfate, 0.100 N as supporting electrolyte. 0.00
titration in acid solution with i ~ d i n e . ~ Inasmuch as the hydrogen absorbed is all accounted for and again t h a t only one equivalent of oxidizing agent is necessary under different conditions, it is likely that the Bizr solutions contain only a single material. The poor yield of Blla obtained by the hydrogenation of Blz and air oxidation of BlZrresults from side reactions in the oxidation step.3 In a cyanide solution the two-electron wave of Blz shifts in half-wave potential (but not in height) from - 1.12 to - 1.35 v. us. S.C.E.,sa change which is consonant only with the explanation that this wave involves cobalt and results from the reduction of cobalt from three t o one. The same shift, of course, happens with B12and BlZrwhich are converted to the BIZ-cyanide purple on treatment with cyanide, the conversion of BIZ, happening with great speed and without the benefit of oxygen. That the waves of B1zr and Biza having half-wave potentials at -1.0 v. involve the reduction of cobalt to the univalent state is perfectly reasonable in view of the firmness with which cobalt is attached to the organic portion of the molecule. In this connection i t is of interest to note that univalent and zero-valent cobalt compounds have now been actually isolated. lo The reduced Blz compound reported by Boos and others! prepared by the reduction of Blz by bivalent chromium, is a distinctly different material than the B1zr studied in this paper; the numbers of equivalents of reducing agent required in the preparations differ and the absorption spectra are markedly different. Probably cyanide is still present in the Boos compound and the material would be expected to show a reduction wave more negative than the -0.95 v. wave of B12r. It is unfortunate that Boos and co-workers did not report the complete polarogram of this material. The great difference in the reactivities of Biza and Bl2 is nowhere more evident than in the ease with which they are reduced as evidenced by their po(9) J. M. Brierly, J. L. Ellingboe and H. Diehl, Iowa State Coil. J s ~ i .a, i , 428 (1953). (10) W. Hieber and C. Bartenstein, Nalzcrwissenschuflcn, 89, 300 (1952).
4348
J. A. BARLTROP, P. M. HAYES AND M. CALVIN
larography. Whereas the cobalt of B,za is easily reduced to the bivalent state, El/, = -0.04 v., and at a more negative potential reduced to the univalent state, E l / , = -1.02 v., B I ~ undergoes only a single two-electron reduction, Ell2 = -1.12 v. vs. S.C.E. We have observed also that Blzacan be hydrogenated to BIZr without a catalyst. There appear, then, to be four classes of cobaltic ammines with respect to their polarography: Class
I
Ha;lf-wave potential
E
Material
Most ammines,11 e.g., Co(NH3)8(11) J. B. Willis, J
E 1,
-0.00 to
n
-1.3 to
Valence change
1 3 to2
.4.Friend and D P. Mellor, T H IJOURNAL. ~ 67,
-0.04 -0.04
11
B12a Bna,
I11
B127
IV
Bi*(CN)-* KICO(CN)sH*O 1' KaCo( CN)sHzO I*
-1.4
-1.02
B~zr
-1.12 -1.35 - 1.3 - 1.45
2 1 1 2 2 1 2
2t0O 3to2 2to1 3to1 3to1 2to1 3to1
Acknowledgment.-The authors wish to express their appreciation of the aid they have received from The Squibb Institute for Medical Research. (13) D. N. Hume and I. M. Kolthoff, i b i d . . 71, 867 (1949)
AMES. Iowa
lii80 (1945).
[CONTRIBUTION FROM
CIS
Vol. TC,
THE
RADIATION LABORATORY AND DEPARTMENT OF CHEMISTRY, UNIVERSITYOF CALIFORNIA, BERKELEY]
The Chemistry of 1,2-Dithiolane (Trimethylene Disulfide) as a Model for the Primary Quantum Conversion Act in Photosynthesis'" BY J. A. B A R L T R O PP., ~M. ~ HAYESAND M. CALVIN RECEIVED MARCH 24, 1954 Some chemical and photochemical observations on 1,a-dithiolane and its derivatives with particular reference to the pos sible mode of function of the naturally occurring system, 6-thioctic acid, are discussed. Experimental evidence is presented to demonstrate that the strain energy in this 5-membered ring is not less than 6.5 kcal. and probably larger. Reagents which both oxidize and reduce this ring are described together with the conditions required for its reformation from the corresponding dithiol. Evidence is adduced to indicate that the primary product of photolysis of this ring in acidic media is very likely a thiol and sulfenic acid or derivative thereof.
'
o'R 1
Although plants which are allowed 4/R A/R CoA to photosynthesize in C1402 rapidly --e+ Ac-CoA assimilate labeled carbon into a series + CHaCoCooH 1 of compounds, very little of the C14 S-s S SH + COZ SH SH I finds its way into the intermediates CO-CHI of the Krebs tricarboxylic acid cycle DPN durinp illumination. If. after a periodv of photosynthesis, the light is turned off, the bloreover, since the coenzyme must be present compounds of the Krebs cycle rapidly become in its oxidized form in order that the oxidation of labeled.2 Thus there is a reaction path linking the pyruvic acid may proceed, these authors suggested photosynthesis and Krebs cycles which becomes that the reducing power formed in the presence of blocked during illumination. With the d i s ~ o v e r y ~ -light ~ shifted the steady-state conditions of the cothat 6-thioctic acid is a coenzyme for the oxidative enzyme toward its reduced (dithiol) form and thus decarboxylation of pyruvate to active acetyl groups, reduced the rate a t which intermediates of the phowhich through C o 4 feed carbon into the Krebs tosynthetic cycle entered the TCX system. At this cycle.718 Calvin and blassinig suggested that the point, specimens of the isomeric 4-, 5- and 6-thioctic acids became available to us through the courtesy process could be formulated as of Dr. T. H. Jukes of Lederle Laboratories. The (1) (a) T h e work described in this paper was sponsored by t h e U. S. ultraviolet absorption spectra of these compounds Atomic Energy Commission; (b) Rockefeller Fellow, 1952-19.53, while on leave of absence from Brasenose College and t h e Dyson Per(Fig. 1) showed a displacement of the absorption rins Laboratory, Oxford University, England. peak to progressively longer wave lengths as the ( 2 ) A. A. Benson and M. Calvin, J. Exptl. Bot.. 1, 63 (1950). size of the disulfide ring diminished, a phenomenon ( 3 ) ( a ) L. J . Reed, I. C. Gunsalus, el a l . , THISJ O U R N A L , 73, ,5920 which might well be due to ring-strain. If one sup(19.511; ( b ) E: I.. Patterion. rl a i . , ibid., 73, 5919 (1951). I C. G u n i t i l u i . 1.. Striiglia and D . I. O'Kane, J. Bioi. Chrm., poses that the absorption band is due to a transi1 9 4 , P5tl (l%-,?r tion ( 5 ) L. J . Reed and B. G. DeBusk, THIS JOURXAL, 74, 3457
1
i
f
t
(19.52). (6) M. W. Bullock, el d . ,ibid., 74, 34R.5 (1952). ( 7 ) S. Ochoa, J . R. Stern and R I . C. Schneider, J . Bioi. Chpm., 193, 691 (1951). ( 8 ) S. Korkes. .i DrlCarnilo, I. C. Gunsalus and S. Ochoa, ibid., 193,
721 (1951). (9) >I Calvin . and P. Massini, Experientia, 8 , 445 (1952).
c ( C H 3 2 --+ hv ('s'H2);l S-
.
.
then assuming to a first approximation that the excited states of all the disulfides have the same energy
