PYRROLE AS A CATALYST FOR CERTAIN BIOLOGICAL OXIDATIONS

Transcription

1 PYRROLE AS A CATALYST FOR CERTAIN BIOLOGICAL OXIDATIONS BY FREDERICK BERNHEIM AND MARY L. C. BERNHEIM* (From the Departments of Physiology and Biochemistry, Duke University School of Medicine, Durham) (Received for publication, May 5, 1931) Except for some recent work on the pharmacology of pyrrole by Biglioli (1) and Rabbeno (2), little work has been done on the action of this compound with biological systems. Shimizu (3) showed that when it was injected into rabbits, it was methylated and excreted in the urine. Introzai (4) injected pyrrole subcutaneously and produced a pigmentation of the skin and hair after exposure to light. Saccardi (5) and Comini (6) claim that a pigment approximating to a true melanin can be formed in the skin of many animals after the administration of pyrrole. Finally Rondoni (7) showed that incubation of pyrrole with the melanogenic organs of the cuttlefish produced a melanin, and similarly incubation with mammalian tissue, especially liver, produced a dark pigment. It is this last observation that we have investigated more closely with regard to the possible oxidations involved. When pyrrole is added to liver the oxygen uptake of the pyrrole and liver is only 20 to 30 c.mm. greater than that of the liver alone after 5 or 6 hours. If, however, the liver is washed two or three times with buffer, the addition of pyrrole causes an appreciable extra oxygen uptake. This uptake is practically independent of the concentration of pyrrole but is dependent on the concentration of liver. This extra oxygen uptake is further increased by the addition of lactic acid to the mixture. Lactic acid and washed liver in contradistinction to lactic acid and unwashed liver take up little or no oxygen, the amount depending on the thoroughness of the washing. If pyrrole is added to a mixture of lactic acid and washed liver, oxygen is taken up in an amount exactly equal to the * Fellow of Newnham College, Cambridge, England. 461

2 462 Pyrrole as Catalyst for Oxidations quantitative oxidation of lactic acid to pyruvic acid. Within limits the amount of pyrrole present has no influence on the oxygen uptake. Too much pyrrole slows the reaction because of its toxic effects. It is obvious that pyrrole itself is not being oxidized, but is acting as a hydrogen transport system for certain unknown substances in the liver and for lactic acid. It has been shown by Michaelis and Salomon (8) that liver naturally contains such a system which can be extracted by washing and when added to red blood cells will cause them to oxidize lactic acid. This explains why unwashed liver will oxidize lactic acid and washed will not; and why the addition of pyrrole to unwashed liver has so little effect, for it has to compete with a hydrogen transport system already there. It is only when this has been washed away that the effect of the pyrrole can be seen. The mechanism by which pyrrole acts will be discussed below. A dark color is always seen after the liver has been incubated with pyrrole, confirming the observations of Rondoni. But this is a by-product and has nothing to do with the oxygen uptake, for certain livers, such as rabbit liver, will not take up any oxygen in the presence of pyrrole but still form this melanin-like substance. EXPERIMENTAL The liver of the white rat was used in all the following experiments. The fresh liver was chopped as finely as possible with scissors. It was then ground in a mortar with 20 cc. of 0.05 M phosphate buffer, ph 7.3, and centrifuged. This process was repeated three times, after which the washings were free of hemoglobin. It was then ground again with a buffer containing 2 per cent sodium fluoride to prevent bacterial growth and the formation of lactic acid from any remaining glycogen or glucose. This mixture was then squeezed through muslin and the resulting homogeneous liver suspension used. This preparation had a deep red color which was due to the pigment fixed to the protein of the cell, for all the free hemoglobin had been washed away. Occasionally, however, the liver suspension had only a very pale color, in which case it was always inactive. The fixed pigment is therefore necessary for the reaction with pyrrole to take place.

3 F. Bernheim and M. L. C. Bernheim 463 The suspension was then suitably diluted, for if it is too thick the substance in the liver which is activated by pyrrole is present in such a large excess that the addition of lactic acid has little effect. It is possible to get a dilution which with pyrrole alone will take up only 10 to 20 c.mm. of oxygen and is still strong enough to oxidize the lactic acid quantitatively to pyruvic acid. The pyrrole was a synthetic product from the Eastman Kodak Company. It was redistilled and kept at 0 and the solution made up freshly for each experiment by adding 0.1 cc. (94 mg.) to 9.9 cc. of buffer. This amount dissolves completely. From 0.1 to 0.4 cc. of this solution was used in the experiments. The lactic TABLE Experiment 1,a Measurements were made at a temperature of 38. Time Vessel 1 I---- c.mn. 1 hr hrs L min Vessel 2 c.mm. c.mm I Oxygen Vessel minus 2 Vessel 1 uptake Vessel 3 Vessel 4 -- c.mm. e.mm Vessel 4 minus Vessel 3 acid was boiled to insure complete hydrolysis of the anhydride and then neutralized by NaOH, and the concentration estimated by the Friedemann, Cotonio, and Shaffer method (9) as modified by Davenport and Davenport (10). The Barcroft-Warburg apparatus was used to measure the oxygen uptake at temperatures of 28 or 38. Experiment 1,a shows that the oxygen uptake of the washed liver alone is increased by the addition of pyrrole and that a definite amount of some substance in the liver is being oxidized. What this substance is, is not known, but it is likely to be some group in the protein molecule or possibly some lipoid or unsaturated fat for

4 464 Pyrrole as Catalyst for Oxidations all the water-soluble substances in the liver have been washed out. This point is being investigated further. Experiment 1,a Vessel 1 contained 0.5 cc. of washed liver suspension and 1.5 cc. of buffer; Vessel 2 contained 0.5 cc. of liver, 1.3 cc. of buffer, and 0.2 cc. of pyrrole solution (1.9 mg.). Vessels 3 and 4 were like Vessels 1 and 2 respectively except that the liver suspension was diluted three times. The figures given in Table I represent oxygen uptake in c.mm. at a temperature of 38. TABLE Experiment II 1, b Measurements were made at a temperature of 38. Time Oxygen uptake Veaael 1 Vessel 2 Vessel 3 c.nm. c.mm. e.m?n. c.mm. 35 min hr., 5 min I hrs., I Vessel 3 minus Vem?l 1 c.mm. It will be seen that the oxygen uptake is, within experimental error, proportional to the amount of liver present. Moreover, after 5 hours the oxygen uptake due to the presence of pyrrole ceases, thus indicating that a definite amount of some substances in the liver is being oxidized. That pyrrole itself is not being oxidized is shown by the following experiment Experiment 1,b Vessel 1 contained 0.5 cc. of washed liver suspension and 1.5 cc. of buffer; Vessel 2 contained 0.5 cc. of liver, 1.3 cc. of buffer, and 0.2 cc. of pyrrole solution (1.9 mg.); Vessel 3 contained 0.5 CC. of

5 F. Bernheim and M. L. C. Bernheim 465 liver, 1.1 cc. of buffer, and 0.4 cc. of pyrrole solution (3.8 mg.). The liver preparation was more concentrated than that used in Experiment 1,a and the temperature was 38 (Table II). After 6 hours the uptake due to 0.2 cc. of pyrrole is 120 c.mm., and that due to 0.4 cc. of pyrrole 112 c.mm. The amounts of oxygen uptake with the two concentrations are within a few c.mm. of one another. This difference is within the experimental error. Experiments 1,a and 1,b thus prove definitely that the pyrrole itself is not oxidized, but that it catalyzes the oxidation of some substances present in the liver. HOURS FIG. 1. Curve 1 shows the 02 uptake of the liver suspension mg. of lactic acid and 1.9 mg. of pyrrole minus the O2 uptake of the liver and lactic acid alone. Curve 2 shows the same with mg. of lactic acid. Curve 3 shows the O2 uptake of liver mg.of lactic acid minus the 02 uptake of the liver alone. The horizontal lines represent the theoretical uptake for the oxidation of mg. and 0.75 mg. of lactic acid to pyruvic acid. In trying the effect of lactic acid it was necessary to adjust conditions so that the oxygen uptake due to the liver and pyrrole alone was as small as possible and yet that there was present enough to insure the oxidation of the lactic acid. Within limits these conditions were easily obtained, for the pyrrole seemed to act preferentially as a catalyst for the oxidation of the lactic acid, as was shown by the exactly theoretical values obtained for the oxidation of the lactic to pyruvic acid without the small oxygen uptake of the liver and pyrrole alone being taken into account. The justification of this assumption is that the concentration of lactic acid alone determined the oxygen uptake, regardless of the

6 466 Pyrrole as Catalyst for Oxidations concentration of pyrrole and the concentration of liver, unless the liver was there in too large amounts. Fig. 1 shows the oxygen uptake with liver and pyrrole with two concentrations of lactic acid. It will be seen that the theoretical value for the oxidation of lactic acid to pyruvic acid is obtained without taking into account the uptake of the liver and pyrrole which in this case was between 20 and 30 c.mm. This reaction presumably does not take place in the presence of lactic acid for the theoretical uptake has been obtained when the uptake due to the pyrrole and liver has been zero or as much as 50 c.mm. If it is more than 50 c.mm. then the system in the liver competes with the lactic acid system and an indeterminate result is obtained. The theoretical uptake is obtained by subtracting the uptake of liver and lactic acid from that of the liver lactic acid and pyrrole. In a well washed liver preparation the oxygen uptake of liver and lactic acid is only slightly greater than that of liver alone. This slight increase is not due to the oxidation of the lactic acid, for it is independent of the lactic acid concentration, and the curve (see Fig. 1) obtained by subtracting the uptake of liver alone from liver plus lactic acid is a straight line; i.e., it is not a biological oxidation in which the substrate is the limiting factor. It might best be termed a specific dynamic effect. It amounts ordinarily to 20 or 30 c.mm. The oxidation does not start immediately on mixing the pyrrole with the liver and lactic acid. There is a latent period, varying with the temperature, of about 2 hours at 28 and 1 hour at 38. This gives the curves an S shape. The significance of this will be discussed below. As in the case of liver alone, the concentration of pyrrole makes no difference to the oxygen uptake. In fact, the lactic acid system is a little more sensitive to the toxic effects of a high concentration of pyrrole than the liver alone. Whereas with both 0.1 cc. and 0.2 cc. of pyrrole solution there is very little difference in the rate at which the lactic acid is oxidized, with 0.3 cc. there is a marked slowing. Thus after 4 hours with 0.2 cc. there is an oxygen uptake of 89 c.mm. (100 c.mm. are the theoretical uptake), whereas with 0.3 cc. only 68 c.mm. have been taken up. Less than 0.1 cc. of pyrrole also shows a slowing due to the fact that not enough pyrrole is available.

7 F. Bernheim and M. L. C. Bernheim 467 Both KCN and pyrophosphate completely inhibit the oxidations catalyzed by the pyrrole with liver alone and liver and lactic acid. & N KCN was used, and whereas the control had taken up the theoretical amount of oxygen in 6 hours, with KCN only 20 c.mm. had been taken up. These 20 c.mm. were taken up toward the end as the KCN concentration diminished due to distillation into the soda. The same effect was noted with pyrophosphate. 0.1 N pyrophosphate buffer, ph 7.3, was used instead of ordinary phosphate buffer, and there was a complete inhibition for 4 hours, after which, due probably to the hydrolysis of the pyrophosphate, an oxygen uptake began. The liver of the white rat is the most satisfactory tissue for studying the effects of pyrrole. Guinea pig and cat livers react slowly and rabbit livers not at all. It is difficult to obtain active preparations with other tissues. Red blood cells were also tried. It was shown by Harrop and Barron (11) that methylene blue will enable these cells to oxidize lactic acid to pyruvic acid. Later Warburg, Kubowitz, and Christian (12) and Wendel (13) showed that this was due to the formation of methemoglobin by the methylene blue. Evidently pyrrole is unable to do this, for red blood cells will not oxidize lactic acid in its presence. DISCUSSION It is obvious that the action of pyrrole is very like that of methylene blue in restoring the oxygen uptake to washed tissue. It is well known that washed muscle and lactic acid take up no oxygen, but the addition of methylene blue causes a quantitative oxygen uptake. The lactic acid enzyme system reduces the methylene blue to methylene white and the latter is then reoxidized by oxygen to methylene blue again. This last reaction occurs spontaneously, requiring no activation on the part of the tissue. The oxygen uptake catalyzed by methylene blue is not inhibited by KCN or pyrophosphate. By analogy it would be possible to postulate the reduction of pyrrole to pyrroline and the reoxidation of the pyrroline as in the following diagram. The only difference from methylene blue is that the reduction or reoxidation does not appear to take place spontaneously, but requires activation by some iron complex, as shown by the fact that KCN

8 468 Pyrrole as Catalyst for Oxidations and pyrophosphate inhibit it. This iron complex may be necessary for the reduction of pyrrole, the oxidation of pyrroline, or both. i Lactic acid + enzyme + Fe complex+ Fe complex Pyrrole Pyrroline This theory, however, does not explain the latent period before the oxidation begins. This latent period seems to indicate that it is not the pyrrole itself but perhaps some condensation product which acts in the above manner. Whatever the details of the mechanism, this general scheme is correct, as shown by the fact that the oxygen uptake is not a function of the concentration of pyrrole but only of the concentration of the substrate. The pyrrole system is not specific for lactic acid, for citric acid is also oxidized by washed liver in its presence. But due to the fact that it is not known to what citric acid goes, it is difficult to study this reaction quantitatively. SUMMARY 1. Pyrrole added to washed liver cells enables these cells to oxidize lactic and citric acids. It itself is not oxidized. 2. In its presence lactic acid is quantitatively oxidized to pyruvic acid. KCN and pyrophosphate inhibit this oxidation completely. 3. The action of pyrrole is compared with that of methylene blue in restoring the oxygen uptake of washed tissue in the presence of substrates. BIBLIOGRAPHY 1. Biglioli, E., Biochim. e terap. sper., 16, 47 (1929). 2. Rabbeno, A., Boll. sot. ital. biol. sper., 4, 192 (1929). 3. Shimizu, T., Biochem. Z., 117,266 (1921). 4. Introzzi, P., Giorn. ital. dermat. sifilologia, 67,1414 (1926). 5. Saccardi, P., Arch. ital. biol., 72, 208 (1924). 6. Comini, T., Arch. jisiol., 23,247 (1925). 7. Rondoni, P., Sperimentale, 76,33 (1921). 8. Michaelis, L., and Salomon, K., J. Gen. Physiol., 13,683 (1930).

9 F. Bernheim and M. L. C. Bernheim Friedemann, T. E., Cotonio, M., and Shaffer, P. A., J. Biol. Chew, 73, 335 (1927). 10. Davenport, H. A., and Davenport, H. K., J. Biol. Chem., 76,651 (1928). 11. Harrop, C., and Barron, E. S. G., J. Exp. Med., 46, 207 (1928). 12. Warburg, O., Kubowitz, F., and Christian, W., Biochem. Z., 221, 494 (1930). 13. Wendel, W. B., Proc. Sot. Exp. Biol. and Med., 26,865 (1929).

10 PYRROLE AS A CATALYST FOR CERTAIN BIOLOGICAL OXIDATIONS Frederick Bernheim and Mary L. C. Bernheim J. Biol. Chem. 1931, 92: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1