THE BIOLOGICAL ACTION OF STRONGLY POSITIVE OXIDATION-REDUCTION SYSTEMS BY ELLA H. FISHBERG AND B. T. DOLIN (From the Biochemical Laboratory of the Beth Israel Hospital, New York) (Received for publication, March 1, 1933) The intermediate metabolism of proteins gives rise to hydroxy- lated phenols from the tyrosine and phenylalanine radicals. These are potential oxidation-reduction systems circulating in the blood, and, as will be proved later, extremely positive from a biological point of view. A survey of the physiological oxidation-reduction systems iden- tified up to the present shows that they have previously been roughly divided into two classes : a more negative, such as cysteine, glutathione, sugar, echinochrome, hermidine, etc., and a more posi- t’ive, such as hemoglobin-methemoglobin, cytochrome, and War- burg’s respiratory ferment. With the exception of the work of Conant and Fieser (1) on hemoglobin and of Ball and Chen on very positive systems (2), the potential of the more positive sys- tems has not been well determined. The cyclic products of pro- tein metabolism mentioned above must now be considered in their relation to other systems in the organism. Their physiologi- cal action can only be really appreciated by a study of their patho- logical manifestations, since the excellent poising effect of the systems of the middle range, such as hemoglobin, makes them im- perceptible. Pathologically the polyhydroxylated phenols make their appearance in two conditions. The first is congenital; an in- herited metabolic anomaly, a total inability to destroy phenyl- alanine and tyrosine, results in the excretion of homogentisic acid as an end-product in the urine, and after many years, the deposition of a black pigment, almost entirely limited to the cartilage and sclerze. Carbolochronosis (3) is a similar condition in which the applicat,ion of phenol over a long period of years results in the deposition of an exactly similar pigmentation. Secondly, there are 159 by guest on June 14, 2020 http://www.jbc.org/ Downloaded from
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THE BIOLOGICAL ACTION OF STRONGLY POSITIVE OXIDATION-REDUCTION SYSTEMS
BY ELLA H. FISHBERG AND B. T. DOLIN
(From the Biochemical Laboratory of the Beth Israel Hospital, New York)
(Received for publication, March 1, 1933)
The intermediate metabolism of proteins gives rise to hydroxy- lated phenols from the tyrosine and phenylalanine radicals. These are potential oxidation-reduction systems circulating in the blood, and, as will be proved later, extremely positive from a biological point of view.
A survey of the physiological oxidation-reduction systems iden- tified up to the present shows that they have previously been roughly divided into two classes : a more negative, such as cysteine, glutathione, sugar, echinochrome, hermidine, etc., and a more posi- t’ive, such as hemoglobin-methemoglobin, cytochrome, and War- burg’s respiratory ferment. With the exception of the work of Conant and Fieser (1) on hemoglobin and of Ball and Chen on very positive systems (2), the potential of the more positive sys- tems has not been well determined. The cyclic products of pro- tein metabolism mentioned above must now be considered in their relation to other systems in the organism. Their physiologi- cal action can only be really appreciated by a study of their patho- logical manifestations, since the excellent poising effect of the systems of the middle range, such as hemoglobin, makes them im- perceptible. Pathologically the polyhydroxylated phenols make their appearance in two conditions. The first is congenital; an in- herited metabolic anomaly, a total inability to destroy phenyl- alanine and tyrosine, results in the excretion of homogentisic acid as an end-product in the urine, and after many years, the deposition of a black pigment, almost entirely limited to the cartilage and sclerze. Carbolochronosis (3) is a similar condition in which the applicat,ion of phenol over a long period of years results in the deposition of an exactly similar pigmentation. Secondly, there are
the cases of poisoning by acetanilide, resorcinol, aniline, nitro- benzene, and similar aromatic substances. Of late years with the development of the aniline dye industry this has become a prob- lem of major importance. The formation of methemoglobin by metabolic products of the pneumococcus, with consequent dimi- nution in the oxygen capacity of the blood in pneumonia patients (Stadie (4)), may perhaps also be included in this list. As demon- strated by Pick (5) for ochronosis, the common etiological factor in all these conditions is the circulation of polyhydroxylated phe- nols and their oxidation products in the blood.
Homogentisic acid is chemically 1,4-p-dihydroxyphenylacetic acid with the side chain ortho to one of the hydroxyl groups. It was isolated from the urine of a patient1 suffering from alkapton- uria by acidifying a 24 hour specimen of urine with 250 cc. of 12 per cent H&SO4 and extracting the acidified urine three times with an equal amount of ether. On evaporation of the extract the dark brown syrupy residue is dissolved in 250 cc. of boiling water. 30 cc. of 20 per cent lead acetate are added and filtered through a fluted filter. On standing, the lead salt of homogentisic acid precipitates. This is filtered, washed with a minimum of ice water, redissolved, H&S is passed in, and the lead precipitated out as sulfide. On evaporating the filtrate in vacua, and on re- crystallizing from boiling water, a pure product (m.p. 145”) is obtained.
The system homogentisic acid-benzoquinone acetic acid fulfils all criteria of a reversible oxidation system.
As is to be seen from Chart 1 and Table I, a typical curve is obtained on oxidation of the reduced phase with stronger oxidizing agents. This was titrated at various pH values between 0.6 and 2.7 with potassium dichromate of the same pH without special precautions for the exclusion of air; also between pH 5.5 and 7.2 with potassium ferricyanide in an atmosphere of nitrogen. (For full details of the technique see Clark et al. (6).) Reduction of the oxidized form with titanous chloride prepared according to Clark gave analogous results.
The insertion of bright platinum electrodes into equimolecu- lar, heavily buffered mixtures of the reduced and oxidized phases
1 We wish to express our thanks to Dr. S. R. Benedict for rendering this work possible by suggesting an alkaptonuric patient to us.
gives sharp potentials as shown in Chart 2 and Table II. Also the potentials obtained are numerically equal to those of the mid- points of the oxidative and reductive titrations.
The form of Chart 2, which is that of a modified bayonet, could have been predicted from a study of the chemical constitution of the molecule. This molecule contains 3 dissociable hydrogen atoms, of which 1 can split off from the carboxyl group at a much
CHART 1. Oxidative titration of homogentisic acid. No precautions taken to exclude air.
lower pH than the other 2 from t,heir respective hydroxyl linkages. The pH of a 0.02 N solution of homogentisic acid was 3.001.
Hence, since (& = m where A is the total acid concentra- tion, K,., = 5.02 X 10e5 and pK, = 4.28 where K,., is the first dissociaution constant of homogentisic acid. Similarly, a quick determination of the pH of 0.02 benzoquinoneacetic acid gave pH 2.490 which corresponds to K, = 5.05 X 1O-4 and pK, = 3.28.
For the oxidized phase we must use (A) = v%?, - (K,/2).
ii 0 pn 1 2 3 4 5 6 7 0 CHART 2. Relation of EI, to pH. pK, and pKPI are defined by the inter-
section of the broken and solid lines.
162 Oxidation-Reduction Systems
(K2,/4 can be neglected in comparison with AK,.) Half titra- tion of the reduced phase gave a value similar to the above, but this method could not be applied to the oxidized phase. The first
pH determination gave a steady potential, but the addition of the least bit of NaOH resulted in the instant formation of highly colored products and unsteady potentials. Blix (7) reports 3.50
for pK, and 4.14 for pK,,. His measurements were made at a temperature of 20°, while ours were made at 25”. An ingenious technique for the determination of such compounds has been de- veloped by Ball and Chen (2) and Ball and Clark (8). Their potential ranges are higher than those reported by Blix and our- selves and they find an increase in potential by introduction of a carboxyl group into the molecule. La Mer and Baker (9) and Biilmann (10) found that the introduction of alkyl groups lowers
TABLE I
Oxidation of Homogentisic Acid with Potassium Dichromate
20 mg. of homogentisic acid in 75 cc. of buffer (NaCl + HCl) with potas- sium dichromate at same pH. End-point 18 cc. Temperature 25”. pH 1.984. No precautions taken to exclude air.
the potential. Hence some of the difference between the poten- tials of gentisinic acid and homogentisinic acid may be explained on the basis of the carboxyl group merely substituting in the side chain instead of in the ring. La Mer and Baker found an increase on introduction of halogen, to which the carboxyl group may be likened. However, the normal potential of dihydroxyphenyl- alanine of 0.797 in comparison with 0.69 of homogentisic acid remains to be explained. The difference in the dissociation con- stants of the oxidized and reduced phases should be apparent from Chart 2.
Applying the method of calculation outlined by Clark et al. (6) to this special case, we have according to the law of mass action
K = h (ox-1 (1) 0 (Hod
+
(2) K,, = (IT)(EHF3 3
Let S, = total reductant, X, = total oxidant.
(5) S, = RH, + RH*- + RH= + R’ (6) 8, = HOx + Ox-
(7) ox- = Kd% (6 4- Ko
Eliminating RHS, RHz-, and RH= from Equations 2,3, 4, and 5 we obtain
When any pair which differs by 2 electrons is taken as the fun- damental oxidation-reduction equation, we have
&! =c-!!q,~ h 2F Ox
If Equations 7 and 8 are substituted in Equation 9, there results
Eh = E. - g In $ f $ In KO
-+ 0 K,1K,2K~3
RT % In
K,1K,zK7a + K,,K,,(&) + Kr,(b -t (ha
6% + Ko
In all our considerations Kr, and K,, may be neglected with re- spect to the other variables. At a fixed ratio of oxidant to reduc- ant, here equimolecular quantities of each, with change in pH only the last term varies and we have
form. This system would have a potential of 0.265 at body pH and an rH of 22.5. From a biological point of view this is extremely positive and must cause changes in any other oxidation-reduc- tion system of lower rH in its vicinity. This is in fact one of the highest systems to be actually shown as present in the body. Quinone itself is still higher and all the other similar compounds formed in the intermediate metabolism of the cyclic rings of pro- tein are within this range. Another substance, adrenalin, has been shown by Ball and Clark (8) to have an rH of 27 (E’o 0.5395, pH 4.40) which also cannot be without biological signifi- cance.
The classic work of Kuester (11) showed that hemoglobin be- haves as a ferrous salt (as also does oxyhemoglobin), while methe- moglobin is a ferric salt. Conant (1) has shown that hemoglobin- methemoglobin is a reversible oxidation-reduction system whose potential at body pH is approximately +0.12 volt. The poten- tial of the hydroxylated phenol systems at this pH is over 0.265 volt. Hence the circulation of these polyhydroxic phenols must cause a shift in the hemoglobin-methemoglobin system in favor of the formation of methemoglobin. The hemoglobin can cope with the normal amount circulating as a result of protein metabo- lism, but the added amount may shift the equilibrium, and on this basis rests the formation of methemoglobin characteristic of poi- soning by aniline derivatives. Van Slyke and Vollmund (12) have shown that the action of aniline on methemoglobin in vitro showed a latent period at the beginning, no methemoglobin being formed for many hours. After methemoglobin formation began, it pro- ceeded slowly and several mols of aniline per mol of hemoglobin were required to complete it. This behavior, they state, accords with the probability that a product of aniline rather than the aniline itself causes the methemoglobin formation. Thus the action of the body is necessary to secure the proper degree of oxidation of the compound to raise it above the hemoglobin- methemoglobin oxidation-reduction system potential. This is an extremely simple example of the familiar action of certain chemotherapeutic agents which cannot act in the test-tube but are potent in the body. A study of some of them, salvarsan, etc., shows that they are also potential oxidation-reduction systems.
We know that there must be a certain minimum of methemoglo- bin present to be measured spectroscopically and we can assume a small quantity as physiological. Various authors give the minimum as 2.5 to 5 per cent as the minimum apparent on spec- troscopic examination. To hemoglobin, in addition to its many other regulatory functions in maintaining body equilibrium, must be added the ability to poise correctly oxidation-reduction systems by virtue of its middle position in the biological oxidation-reduction scale. We know that, if we add dA equivalents of a stronger oxidizing agent to a system in equilibrium where E = RT/nF In a/(S - a) (S is the total concentration of the reversible system and a the concentration of the oxidized form), the potential will
rise dE, dE/da = RT/nF.S/a(S - a). The poising power r* = nFII1T.a. (S - a>/&‘. a can vary only between 0 and S, &r/&S = nF/RT . a2/S2.
For finite values 7r has neither a maximum nor minimum, but the poising effect becomes greater the higher the concentration of the oxidation-reduction system. In other words, when a rever- sible system of middle position, such as Hb-MHb, and another of higher position are brought into contact, one or the other will predominate according to the molecular concentration. If t,he Hb-MHb is much more concentrated in comparison with the Q-&H, then it will completely predominate, which is the usual physiological condition. However, another factor comes into play. The molecular weight of hemoglobin, according to the lat- est and best available methods, assuming 4 iron atoms to the mole- cule, is 67,000:68,000. Since the quinones are approximately 100, small quantities of the quinones can exert enormous effects on the hemoglobin system. Theoretically 1 gm. of quinone (20 mg. per 100 cc. of blood) can convert the entire hemoglobin of the blood into methemoglobin and 1 mg. would be spectroscopically apparent. Hence, the introduction of hydroxylated phenols into the body at a concentration greater than the physiological maxi- mum should cause the formation of methemoglobin. If we dif- ferentiate the poising effect by a, we find &/6a = nF/RT ((5’ - a>/&’ - a/S). At th e maximum dn/da = 0 and a = S/2. The second derivative shows this to be a maximum. Thus Docou (13), in an elaborate study of the effects of injection of acetanilide, resorcinol, and similar products into dogs, reports the formation of approximately 50 per cent methemoglobin.
Another aspect of this problem must be considered; namely, how is this equilibrium affected by the presence of oxygen? Pass- ing O2 or GO into a mixture of hemoglobin and methemoglobin has been found by Conant to increase the oxidation-reduction po- tential of the mixture. He gives 7r (observed) = rn +0.059 log (MHb)/(Hb). If the term (Hb) is greatly diminished by com- bination of hemoglobin with the gas to form oxyhemoglobin or carboxyhemoglobin, and (MHb) stays the same, the potential will rise. Thus the potential at body pH of the system in the
* This is not equal to Clark’s “poising index,” since it is dependent on absolute concentrations.
absence of oxygen is about +0.12 volt compared to the hydrogen electrode, while in the presence of O2 it rises to 0.2 volt. Thus the oxyhemoglobin acts as a bulwark in opposing the action of the highly positive oxidation-reduction systems and shows the reason for the comparative rarity of methemoglobinemia.
A peculiar form of methemoglobin formation first differentiated by Stokvis (14) as a clinical entity, “enterogenous cyanosis methemoglobinemia,” leads to periodic violent intestinal attacks followed by extreme cyanosis with demonstrable high methemo- globin content of the blood. The urine contains a substance which on being exposed to air turns bright red and is able to convert hemoglobin to methemoglobin in vitro. The condition has been referred to absorption into the blood stream of some toxic sub- stances formed in the intestine by putrefactive change. In the intestinal contents there have been reported phenol-p-cresol, p- hydroxyphenylpropionic acid, etc., all of which can be converted into polyhydroxy phenols by the body and hence are potential methemoglobin formers.
An interesting aspect of the equilibrium between hemoglobin- methemoglobin and other oxidation-reduction systems is given in the paper of Michaelis and Salomon (15). The change of oxy- hemoglobin by an oxidizing agent into methemoglobin results in the simultaneous reduction of the oxidant and the liberation of a certain quantity of oxygen from the oxyhemoglobin. 1 mol of oxygen is liberated for every mol of hemoglobin formed. This is the case with ferricyanide and quinone. If, however, the reduc- tion product is autoxidizable, then some of the oxygen is used up in reoxidizing the dyestuff. Hence, when an organic dye is used, three-quarters of the oxygen should be produced which is formed by ferricyanide, if all the hemoglobin is converted into methemo- globin. At least, this happens when an excess of dye is used and every molecule acts only once as an oxidant of the hemoglobin. However, in making the list of the amounts liberated by the vari- ous commonly used oxidation-reduction systems, they find that less than three-quarters is liberated (at pH 6) except in the case of chlorophenolindophenol (E’o = +0.295), phenolindophenol (+0.28). Methylene blue (+0.047) gave only half this quan- tity “in nicht gut reproduzierbarer Weise;” indigo sulfonate (-0.006) only a fifth, safranine (-0.2), and rosinduline practi-
tally none at all. This is to be expected because chlorophenolin- dophenol, phenolindophenol, and quinone are the only systems whose E’o exceeds that of the hemoglobin-methemoglobin system (approximately +0.09).
The case of methylene blue is exceedingly interesting. As can be seen from the above, it is practically in the potential range of the hemoglobin-methemoglobin system, and if we take Conant’s measurements as of the precision of other more easily investigated systems, a little lower. It can be easily calculated how much methemoglobin can be formed. Methylene blue as commonly used has a minimum amount of leuco product. Let us assume that at equilibrium between the two systems, which start at equimolec- ular concentrations of hemoglobin and methylene blue, z equiva- lents of methemoglobin are formed. We have
s Mbf = ‘Mbi - x s MHbr = ‘MHbi fx _I
s Lmb, = x Lmbi +x s Hbf = ‘Hbi - ’
where MHb is methemoglobin, Hb hemoglobin, Lmb leucomethyl- ene blue, and Mb methylene blue, and i and f are the initial and final concentrations respectively.
At constant pH the systems are separately defined by
(sMb)i Em = E’m + 0.03 log (sL,b)i
(‘MHb) Ea = E’b + 0.06 log oi’
The systems react to a common potential E, = Et,. Hence
where @Lrnb> = 0 and (X&n& i = 0. Using the values given by Conant for Elb and the equivalent value at a definite pH of methyl- ene blue, we have x = 0.106 concentration of hemoglobin. Hence approximately 10 per cent would be converted into methem- oglobin, and the reaction would stop. Another factor enters here; when air is admitted, the leucomethylene blue is instantly reoxidized to methylene blue and the reaction goes on continu-
ously. Thus methylene blue can be regarded as a positive system only in the presence of air; it is a facultative positive system in contrast to the obligate systems previously discussed. The sys- tems of this range, the facultative positive, are the systems which play the greatest role in catalyzing respiration. In contrast to the obligate systems, the strongly positive ones spoken of pre- viously, they are dependent on the concentration of the reacting systems to a greater extent. Expressed otherwise, the important factor is not the absolute difference in the characteristic potential (E’m and E’b in the last few equat,ions), but the spread between the systems achieved by having the oxidant of the one system differ so extremely in its proportions from its reductant, and the reductant of the other system differ so much from its own oxidant. These must be extremely unstable systems and in a state of con- stant flux, because we know that these are the regions of great in- stability, as stability is really only achieved where the oxidant and reductant tend to approach each other in relative concentra- tion. This is admirably adapted to respiratory needs. Warburg (16) found that the methemoglobin formed by the action of the very positive systems differs very much from that formed by methylene blue in its ability to catalyze oxidation, and that a much higher concentration of the former was required to attain the same velocity of oxidation as with the latter.
Some light is thrown on the old question as to why the pigment in ochronosis is so strictly localized in the cartilages and scler=. Virchow states in the original description, “I will name this con- dition ochronosis. It is strictly localized in those parts that are without nerves and blood vessels.” These are the regions where there can be no poising effect of hemoglobin. Hence the quinone from the homogentisic acid can be formed and at the pH of the body fluids instantly form the colored product characteristic of ochronosis. “The natural question is whether any physiological analogy can be found. It seems to me it can be found in the hair, which is dependent on the same condition.”
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3. Fishberg, E. H., Virchows Arch. path. Anat., 261, 376 (1924). 4. Stadie, W., J. Exp. Med., 33, 627 (1921). 5. Pick, L., Berl. klin. Woch., 43, 509, 556, 591 (1906). 6. Clark, W. M., et al., Bull. Hyg. Lab., U. S. P. H. S., No. 161 (1928). 7. Blix, G., 2. physiol. Chem., 210, 87 (1932). 8. Ball, E. G., and Clark, W. M., Proc. Nat. Acad. Xc., 17, 347 (1931). 9. La Mer, V. K., and Baker, L. F., J. Am. Chem. Sot., 44,1954 (1922).
10. Biilmann, E., Ann. chim., series 9, 16, 190 (1921). 11. Kuester, W., 2. physiol. Chem., 66, 244 (1910). 12. Van Slyke, D. D., and Vollmund, E., J. Biol. Chem., 66, 415 (1925). 13. DOCOU, P., Considerations sur la methemoglobine: Son dosage dans le
sang, Thesis, University of Paris (1925). 14. Stokvis, B. J., Nederl. Tijdschr. Geneesk., 2, 678 (1902). 15. Michaelis, L., and Salomon, K., Biochem. Z., 234,107 (1931). 16. Warburg, O., Kubowitz, F., and Christian, W., Biochem. Z., 227,
