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Biological oxidation. I. The infrared studies on the lipoxidase-catalyzed oxidation of linoleic acid
LETTERS
TO
THE
247
EDITORS
0:&5$& methionine in the diet, in the presence of 0.26% cystine. Similar analysis of d:tt,a by Shelton et al. (9) indicates a methionine requirement of 0.38yo in the presence of 0.31% cystine; hence these reports are in close agreement in respect t 0 total sulfur amino acid requirement of young pigs. The above examples are sufficient to illustrate the broad utility of the mrtho~l of analysis. .4mong other advantages are: (a) bad data are made more conspicuous; (h) requirements can be more readily estimated although no specific data happen to coincide with the full requirements, provided there are sufficient, data t,o estab lish the limits of requirements; (c) full use may be made of the submaximal data to &ablish the response lines. .4s previously stated (l), the principle applies irl many examples of requirement, st.udies on other nutrients such as vitamins ~1111 nlillrr:bls. KEFERENCES
11. J.. J’ottllry Sci. 31,967 (1952) ; ibid. 33, 132 (1953). ALMUUIRT, H. J., .4~1) ~~IERRITT, J. H., Proc. Sot. Jkptl. Biol. Med. 73, 136 (1950). S.4xm.4, 11. C., AND ~f('GINNIS, J., PouZly Sci. 31, 984 (1952). JLTKES. T. H., ASI) STOICSTAD, 15. I,. R., J. Nutrition 43, 459 (1951). RRIws, G. hl., JR., &~ILLS, R. C., ELVEHJEM,C..~., AND JTART, E. B., J. Rio!.
1. i\I.\[QGIST, 2.
3. 4. 5.
f’hwru. 144, 47 (1942). 7. ALEXAXDER, J. C., .4NU HILL,
.J. Nutrition 41, 89 (1950). D. C., .I. N,utrition 48, 149 (1952).
8. (:1lRTIS,
ABRAHAM,
6. (;RAli,
c.
Ii., 1,. v.,
AXD KAMEI, LOOSLI,
I,. .4., .I. Nutrition !). SIIEI.TON,
n.
C.,
AI.,
.I. I<.,
J., wIL1,1.4~,
1.1. Ix.,
.4SD klAPR'ARD,
48, 499 (1952).
BEESOX,
W.
-\I.,
;AvD .\IERTZ,
E. T.,
J.
~lninwl Yci. 10, 57
ilM1).
Biological
Oxidation.
I. The Infrared Studies on the Lipoxidase-Catalyzed Oxidation of Linoleic Acid
Bergstriim and Holman (l-3) concluded from their observations that the ene~ mstic oxidation of linoleic acid in the presence of lipoxidase might follow the same p:lt,t.ern of reactions as ordinary autoxidat,ion. They proposed (4’1 a reaction mrchnnism lvhich indicat,es that liposidase can cause initiation of a free-radical c*huin reaction similar t,o t,hat, operative in the sutoxidntion of methyl linolrtrtc,, giving rise to 9- and 13. conjugated hydroperoxides. Currrntly, Tappel $1 trl. (5) also demonstrated the formation of these conjugated peroxides. They postI]latetl :I process of comples formation of each subskate molecule on the surface of t,hr enzyme and subsequent formation of the peroxide. Recent investigations I)y irlfrared spectroscopy (6,7) gave indications that such spectral analysis would rcxvr:tl somr characteristic features of the peroxides formed by the enzymatic: osi~lation of linoleic acid. The osidations in the presence of lipoxidase were c:trrietl out n-ith dispcrsracl
248
LETTERS
TO
THE
EDITORS
oxygen and continuous agitation at 0°C. in a 24-I. flat-bottomed flask, using sodium linoleate (from 40 g. linoleic acid dissolved as 2 mg./ml. in an equivalent amount of sodium hydroxide solution, keeping the pH at about 9) as substrate (4,s). After 50% oxidation, the soap was acidified with a just sufficient amount of dilute hydrochloric acid, and then sufficient absolute alcohol was added to the mixture to make the alcohol content 20-25%. This mixture was exhaustively extracted with redistilled ethyl ether. After removal of the ether, peroxide was quantitatively concentrated by countercurrent extraction, using two immiscible solvents, 87% alcohol and heptane (Skelly F). Since the acid peroxide (6250
I WAVE
LENGTH,
MICRONS
1. Infrared absorption spectra: A. Isolated peroxide concentrate (reduced) from methyl lineoleate, autoxidized below 0°C. with dispersed oxygen. B. Isolated peroxide concentrate (reduced) from linoleic acid oxidized at 0°C. in the presence of lipoxidase with dispersed osygen. FIG.
mequiv./kg.) is unstable, the peroxide concentrate was at once reduced by stannous chloride to’monohydroxylinoleic acid (OH, 1 mole/mole). For contrast, the ester, methyl linoleate, was autoxidixed below 0°C. with dispersed oxygen to a peroxide value of 509 mequiv./kg. and the peroxides formed were quantitatively concentrated and reduced as hefore. The acid and ester groups in the substrate may influence the initiation process hut not the main autoxidation reaction forming the geometric isomers (9). The infrared spectra of both the reduced materials are shown in Fig. la and b (only the regions indicating cis-truns isomers of the carbon-carbon double bonds
LETTERS
TO
THE
249
EDITORS
are included). Roth the products manifest almost the identical types of absorption at the same wavelengths, proving the presence of the two major isomers: (a) cis-!rccns conjugated (10.55 p, or 948 cm.+) and (b) trans-tram conjugated (10.15 p, or 988 cm.-‘). This confirms the findings on the lipoxidase catalysis in relation to the formation of the conjugated products, and may mean that although lipoxidaso catalysis may have proceeded through the controlled mechanisms proposed by Tappel et nl. (5), it has also stabilized the free radicals at 9- and 13-positions of t,he linolrate system (1, 2, 4), producing the conjugated isomers. In other words, t.1~~free radicals initiated at, the ll-position are able to change over to the morr st:thle forms. Further work is in progress and the det.ails will be published later.
The author wishes to t.hank Drs. R. kind help, as well as Dr. D. H. Wheeler sis. He also acknowledges t,he financial Department of Navy; ant1 the Hormel
T. Holman and W. 0. Lundberg for their and Mr. W. E. Tolberg for infrared analysupport of the office of Naval Research, Institute of thr Pnivcrsity of Minnesota.
REFERENCES BERGSTR~~M, S.? Niafure 166, 717 (1935). BERWTRGM, S., ANU HOIX.~N, It. T., Nature 161, 55 (IWS). ITOLXIN, R. T., d,ch. Riocheti~. 15, -103 (1947). BICRGWR~~M, S., ANU HOLKW, R. T., Advances in E’nqmo?. 8, 449 (1948). T.~PPEI,, A. L., BOYER, P. D., AND LUNDBERG, W. O., J. Biol. (Ihern. 199, 267 (1952). 0. C.~XSOS, J. A., ZILCH, I<. T., BURKET, 8. C., AND DUTTON, H. J., J. .4nr. Oil Cheminfs Sot. 29, 447 (1942). 7. I'RIVETT, 0. S., LUNDBERG, W. O., KHAN, N. A., TOLBERQ, W. E., .~NI) WHEELER, D. H., J. iint. Oil Chemists’ Sot. 30, 61 (1953). 8. TIIIEORELL, H., BERBSTR~M, S., AND AKESON, A., Phnrnt. .4cta Helu. 21, 318 1. 2. 3. 4. 5.
(1916). 9. 1<11.4s. N. A., to be presented at the spring meeting Organic Division, !)I, American Chemical Society, Los Angeles, Calif., 1953. Ho,nwl Institute, .4 ustin, Minnesota Ifwcid Mnrrh 15,1955
x.-k.
The in Vitro Reaction Between Tetraethylthiuram (Antabuse) and Glutathione
Paper E(H.4N'
Disulfide
Recent reports have described the inhibitory effects of tetraet.hylthiuram disulfide1 (TETD) on enzyme systems catalyzing the oxidation of aldehydes. Grah:un (1) found t,hat liver aldehyde dehydrogenase was blocked by this compound, arrtl Nyg:t:trd and Sumner (2) studied the inhibition of 1).glyceraldehydr 3-phosI Research Fellow. ’ I~is(diethylthiocarbamy1)
disulfide; Antabuse.
TO
THE
247
EDITORS
0:&5$& methionine in the diet, in the presence of 0.26% cystine. Similar analysis of d:tt,a by Shelton et al. (9) indicates a methionine requirement of 0.38yo in the presence of 0.31% cystine; hence these reports are in close agreement in respect t 0 total sulfur amino acid requirement of young pigs. The above examples are sufficient to illustrate the broad utility of the mrtho~l of analysis. .4mong other advantages are: (a) bad data are made more conspicuous; (h) requirements can be more readily estimated although no specific data happen to coincide with the full requirements, provided there are sufficient, data t,o estab lish the limits of requirements; (c) full use may be made of the submaximal data to &ablish the response lines. .4s previously stated (l), the principle applies irl many examples of requirement, st.udies on other nutrients such as vitamins ~1111 nlillrr:bls. KEFERENCES
11. J.. J’ottllry Sci. 31,967 (1952) ; ibid. 33, 132 (1953). ALMUUIRT, H. J., .4~1) ~~IERRITT, J. H., Proc. Sot. Jkptl. Biol. Med. 73, 136 (1950). S.4xm.4, 11. C., AND ~f('GINNIS, J., PouZly Sci. 31, 984 (1952). JLTKES. T. H., ASI) STOICSTAD, 15. I,. R., J. Nutrition 43, 459 (1951). RRIws, G. hl., JR., &~ILLS, R. C., ELVEHJEM,C..~., AND JTART, E. B., J. Rio!.
1. i\I.\[QGIST, 2.
3. 4. 5.
f’hwru. 144, 47 (1942). 7. ALEXAXDER, J. C., .4NU HILL,
.J. Nutrition 41, 89 (1950). D. C., .I. N,utrition 48, 149 (1952).
8. (:1lRTIS,
ABRAHAM,
6. (;RAli,
c.
Ii., 1,. v.,
AXD KAMEI, LOOSLI,
I,. .4., .I. Nutrition !). SIIEI.TON,
n.
C.,
AI.,
.I. I<.,
J., wIL1,1.4~,
1.1. Ix.,
.4SD klAPR'ARD,
48, 499 (1952).
BEESOX,
W.
-\I.,
;AvD .\IERTZ,
E. T.,
J.
~lninwl Yci. 10, 57
ilM1).
Biological
Oxidation.
I. The Infrared Studies on the Lipoxidase-Catalyzed Oxidation of Linoleic Acid
Bergstriim and Holman (l-3) concluded from their observations that the ene~ mstic oxidation of linoleic acid in the presence of lipoxidase might follow the same p:lt,t.ern of reactions as ordinary autoxidat,ion. They proposed (4’1 a reaction mrchnnism lvhich indicat,es that liposidase can cause initiation of a free-radical c*huin reaction similar t,o t,hat, operative in the sutoxidntion of methyl linolrtrtc,, giving rise to 9- and 13. conjugated hydroperoxides. Currrntly, Tappel $1 trl. (5) also demonstrated the formation of these conjugated peroxides. They postI]latetl :I process of comples formation of each subskate molecule on the surface of t,hr enzyme and subsequent formation of the peroxide. Recent investigations I)y irlfrared spectroscopy (6,7) gave indications that such spectral analysis would rcxvr:tl somr characteristic features of the peroxides formed by the enzymatic: osi~lation of linoleic acid. The osidations in the presence of lipoxidase were c:trrietl out n-ith dispcrsracl
248
LETTERS
TO
THE
EDITORS
oxygen and continuous agitation at 0°C. in a 24-I. flat-bottomed flask, using sodium linoleate (from 40 g. linoleic acid dissolved as 2 mg./ml. in an equivalent amount of sodium hydroxide solution, keeping the pH at about 9) as substrate (4,s). After 50% oxidation, the soap was acidified with a just sufficient amount of dilute hydrochloric acid, and then sufficient absolute alcohol was added to the mixture to make the alcohol content 20-25%. This mixture was exhaustively extracted with redistilled ethyl ether. After removal of the ether, peroxide was quantitatively concentrated by countercurrent extraction, using two immiscible solvents, 87% alcohol and heptane (Skelly F). Since the acid peroxide (6250
I WAVE
LENGTH,
MICRONS
1. Infrared absorption spectra: A. Isolated peroxide concentrate (reduced) from methyl lineoleate, autoxidized below 0°C. with dispersed oxygen. B. Isolated peroxide concentrate (reduced) from linoleic acid oxidized at 0°C. in the presence of lipoxidase with dispersed osygen. FIG.
mequiv./kg.) is unstable, the peroxide concentrate was at once reduced by stannous chloride to’monohydroxylinoleic acid (OH, 1 mole/mole). For contrast, the ester, methyl linoleate, was autoxidixed below 0°C. with dispersed oxygen to a peroxide value of 509 mequiv./kg. and the peroxides formed were quantitatively concentrated and reduced as hefore. The acid and ester groups in the substrate may influence the initiation process hut not the main autoxidation reaction forming the geometric isomers (9). The infrared spectra of both the reduced materials are shown in Fig. la and b (only the regions indicating cis-truns isomers of the carbon-carbon double bonds
LETTERS
TO
THE
249
EDITORS
are included). Roth the products manifest almost the identical types of absorption at the same wavelengths, proving the presence of the two major isomers: (a) cis-!rccns conjugated (10.55 p, or 948 cm.+) and (b) trans-tram conjugated (10.15 p, or 988 cm.-‘). This confirms the findings on the lipoxidase catalysis in relation to the formation of the conjugated products, and may mean that although lipoxidaso catalysis may have proceeded through the controlled mechanisms proposed by Tappel et nl. (5), it has also stabilized the free radicals at 9- and 13-positions of t,he linolrate system (1, 2, 4), producing the conjugated isomers. In other words, t.1~~free radicals initiated at, the ll-position are able to change over to the morr st:thle forms. Further work is in progress and the det.ails will be published later.
The author wishes to t.hank Drs. R. kind help, as well as Dr. D. H. Wheeler sis. He also acknowledges t,he financial Department of Navy; ant1 the Hormel
T. Holman and W. 0. Lundberg for their and Mr. W. E. Tolberg for infrared analysupport of the office of Naval Research, Institute of thr Pnivcrsity of Minnesota.
REFERENCES BERGSTR~~M, S.? Niafure 166, 717 (1935). BERWTRGM, S., ANU HOIX.~N, It. T., Nature 161, 55 (IWS). ITOLXIN, R. T., d,ch. Riocheti~. 15, -103 (1947). BICRGWR~~M, S., ANU HOLKW, R. T., Advances in E’nqmo?. 8, 449 (1948). T.~PPEI,, A. L., BOYER, P. D., AND LUNDBERG, W. O., J. Biol. (Ihern. 199, 267 (1952). 0. C.~XSOS, J. A., ZILCH, I<. T., BURKET, 8. C., AND DUTTON, H. J., J. .4nr. Oil Cheminfs Sot. 29, 447 (1942). 7. I'RIVETT, 0. S., LUNDBERG, W. O., KHAN, N. A., TOLBERQ, W. E., .~NI) WHEELER, D. H., J. iint. Oil Chemists’ Sot. 30, 61 (1953). 8. TIIIEORELL, H., BERBSTR~M, S., AND AKESON, A., Phnrnt. .4cta Helu. 21, 318 1. 2. 3. 4. 5.
(1916). 9. 1<11.4s. N. A., to be presented at the spring meeting Organic Division, !)I, American Chemical Society, Los Angeles, Calif., 1953. Ho,nwl Institute, .4 ustin, Minnesota Ifwcid Mnrrh 15,1955
x.-k.
The in Vitro Reaction Between Tetraethylthiuram (Antabuse) and Glutathione
Paper E(H.4N'
Disulfide
Recent reports have described the inhibitory effects of tetraet.hylthiuram disulfide1 (TETD) on enzyme systems catalyzing the oxidation of aldehydes. Grah:un (1) found t,hat liver aldehyde dehydrogenase was blocked by this compound, arrtl Nyg:t:trd and Sumner (2) studied the inhibition of 1).glyceraldehydr 3-phosI Research Fellow. ’ I~is(diethylthiocarbamy1)
disulfide; Antabuse.