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The photoinduced oxidation of manganous ions
584
LEITERS
TO
THE
EDITORS
J3iochemical Genetics Laboratory, Department of Zoology, University of Texas, Austin, Texas Received February 23, 1966
’ Present address: Biology Ridge, Tennessee.
C. 0. DOUDPCEY~
R. P.
Division,
The Photoindnced
Oak Ridge Sational
WAGNER
Laboratory,
Oak
Oxidation of Manganous Ions1
Kenten and Mann (1) have shown that in the presence of peroxidase, hydrogen peroxide, pyrophosphate, and a mononhydric phenol manganous ions are oxidized to manganic ions which accumulate as manganipyrophosphate. It has now been found that in the presence of a suit.able light sensitizer and catalytic am0unt.s of a hydrogen donor manganous ions can undergo n photoinduced oxidation. This can be demonstrated by carrying out the reaction in pyrophosphate and estimating t.he concentrat.ion of manganipyrophosphate qualitatively by the benzidine test (2) or quantitatively by spectrographic or polarographic analysis? The reaction can also be followed by titrating the residual manganous ions (3) and by measuring the oxygen uptake. Similar results were obtained by all t.hesc methods. The reaction mixture was prepared as follows: 5 ml. of 0.2 dl sodium pyrophosphate adjusted to pH 7 with sulfuric acid, 0.5 ml. of 0.04 JV manganous sulphate, 2 ml. of an aqueous solution of the light sensitizer, 1 ml. of IO-3 ,li solution of a hydrogen donor, and 0.2 ml. of purified horse liver catalase prepared by the method of Keilin and IIartree (4) were placed in a Xl ml. beaker. The beaker was then set in an ice bath on a mechanical shaker and exposed to the light of a 500 w. incandescent lamp at a distance of 40 cm. This provided a light intensity of about Wtl foot candles. Of the photosensitizers studied riboflavin was found to be the most effective, followed in order of decreasing activity by fluorcscine, eosine, scopolctin, methylumbelliferone, and quinine sulfate. As riboflavin was the most active agent further studies were carried out primarily with this substance. Table I indicat.es that there is an appreciable accumulation of manganic ions only when all compor1ent.s of the reaction mixt.ure are present. No formation of manganipyrophosphate occurs if the reaction is carried out under nit,rogen. If the reaction mixture is placed in a narrow test tube in air and not agitated, the accumulation of manganic ions is markedly decreased and the solution turns colorless indicating reduction of riboflavin to the leuco form. On subsequent shaking the yellow color of riboflavin is restored. In the absence of catalase the accumulation of manganic ions is decreased due to the reaction of the manganic ions with the hydrogen peroxide produced on the oxidation of t.he leuco riboflavin; thus 11nnOa + HP02 + 2M110 + Hz0 + 02
-.
’ Contribution No. 46, Science Service I,aboratory, Canada Department Agriculture, University Sub Post Office, I,ondon, Ontnrio. * D. M. Miller, unpublished.
of
LETTERS
TO
THE
TABLE Factors
Affecting
the Photoinduced
I Oxidation
nlanganous sulfate, pyrophosphate, air, hydrogen donor (p-cresol), catalase, light sensitizer (riboflavin 3 mg./lOO ml. aqueous solution) So catalase So p-cresol So catalase and p-cresol So air No pyrophosphat,e No riboflavin So light, by the benzidine
of
Man,ganows
Zons
Accumulation of manganipyrophosphate”
Reaction mixture
(1IMimated 20 min.
585
EDITORS
reaction
(2) after
++++
+-+ + + an cxperimentjal
period
01
h large number of substances were found to catalyze the reaction. ,411 the monohydric and polyhydric phenols studied which do not readily form quinones on oxidation catalyzed the oxidation of manganous ions when riboflavin was used as the light sensitizer but not when the light sensitizer itself was a monohydric phenol, e.g. scopoletin. Aromatic amines and related substances such as tryptophane and indoleacetic acid also catalyzed the reaction and so did maleic hydrazide. Quinone-forming phenols such as catechol and hydroquinone completely inhibit the reaction. In this connection it is interesting to note that t,he dihydric phenolic coumarin derivative es-uletin, although closely related to t,he monohydric light-sensitizer scopoletin, does not art as a photosensitizer and inhibits the riboflavin sensitized react,ion. According to Kenten and Mann (1,5) monohydric phenols can undergo a cyclic oxidation-reduction, being oxidized by peroxidase and hydrogen peroxide to :L highl?- reactive intermediary which can then undergo reduction by manganous ions. .4 similar oxidation-reduction reaction appears to be operative in the present spst,em for it was observed that p-cresol, indoleacetic acid, or maleic hydrazidc all undergo photooxidation in the presence of riboflavin but that the rate of disappearance of these substances is decreased if manganous ions are added t,o the reaction mixture at the commencement of the experiment. If the manganous ions are not added until these substances have undergone photooxidation, they have no effect. The results suggest then that riboflavin first brings about a photooxidation of the hydrogen donor to a reactive intermediary which is then rapidly reduced by manganous ions. The fact that manganous ions do undergo a slow oxidntion in t,hc absence of an intermediary suggests that) either riboflavin can cause a direct but slow photooxidat,ion of manganous ions or that there are impurities presentj which may act as intermediaries. Tal)lr I also shows that no reaction is detectable if pyrophosphate is omitted from the reaction mixture. The importance of this subst ante appears to lie in its
586
LETTERS
TO THE
EDITORS
ability to form a stable complex with manganic ions thus preventing the dismutation of the trivalent manganese into the di- and tetravalent forms. Citrate, which also forms a complex with manganic ions, was much less effective than pyrophosphate. The results of these studies on the photoinduced oxidation of manganous ions by riboflavin appear to be compatible with the following scheme: Rbf Rbf* oxidation
product
light > Rbf*
+ D -+ Rbf Hz + oxidation
of D + 2Mn++
+ 2H+
product
of D
(2)
pyrophosphate f D + 2Mn+++ pyrophosphate
Rbf Hz + 02 + Rbf + HzOz
(3) (4) (5)
over-all
reaction
: 2Mn++
+ 02 + 2H++ -+ 2Mn+++
+ Hz0
+ W 02
riboflavin, = activated = hydrogen donor, an aromatic phenol or amine which does not readily form a quinoid structure on oxidation. The question arises as to whether the observed photoinduced oxidation of manganous ions can be related to the action of manganese in illuminated biological systems. Gerretsen (6) and Mehler (7) working with chloroplast suspensions have shown that the addition of manganous ions causes an increase in oxygen uptake in light. If manganous ions do undergo a photoinduced oxidation in such chloroplast suspensions, one would not expect manganic ions to accumulate, due to the presence of plant metabolites which would bring about their rapid reduction. The existence of such a cyclic oxidation-reduction of manganese might help to explain the observed effect on oxygen uptake in illuminated chloroplast suspensions. where
Rbf* D
REFERENCES 1. 2. 3. 4. 5. 6. 7.
KENTEN, R. KENTEN, R. LINGANE, J. KEILIN, D., KENTEN, R. GERRETSEN, MEHLER, A.
H., AND MANN, P. J. G., Biochem. J. 46, 255 (1949). H., AND MANN, P. J. G., Biochem. J. 60, 360 (1951). G., AND KARPLUS, R., Znd. Eng. Chem., Anal. Ed. 18, 191 (1946). AND HARTREE, E. F., Biochem. J. 39, 148 (1945). H., AND MANN, P. J. G., Biochem. J. 48, 67 (1950). F. C., Plant and Soil 2, 323 (1945-50). H., Arch. Biocfwm. and Biophys. 34, 339 (1951).
Science Service Laboratory, Canada Department of Agriculture, University Sub Poet Ofice, London, Ontario, Canada. Received January 29, 1966
W. A. ANDREAE
LEITERS
TO
THE
EDITORS
J3iochemical Genetics Laboratory, Department of Zoology, University of Texas, Austin, Texas Received February 23, 1966
’ Present address: Biology Ridge, Tennessee.
C. 0. DOUDPCEY~
R. P.
Division,
The Photoindnced
Oak Ridge Sational
WAGNER
Laboratory,
Oak
Oxidation of Manganous Ions1
Kenten and Mann (1) have shown that in the presence of peroxidase, hydrogen peroxide, pyrophosphate, and a mononhydric phenol manganous ions are oxidized to manganic ions which accumulate as manganipyrophosphate. It has now been found that in the presence of a suit.able light sensitizer and catalytic am0unt.s of a hydrogen donor manganous ions can undergo n photoinduced oxidation. This can be demonstrated by carrying out the reaction in pyrophosphate and estimating t.he concentrat.ion of manganipyrophosphate qualitatively by the benzidine test (2) or quantitatively by spectrographic or polarographic analysis? The reaction can also be followed by titrating the residual manganous ions (3) and by measuring the oxygen uptake. Similar results were obtained by all t.hesc methods. The reaction mixture was prepared as follows: 5 ml. of 0.2 dl sodium pyrophosphate adjusted to pH 7 with sulfuric acid, 0.5 ml. of 0.04 JV manganous sulphate, 2 ml. of an aqueous solution of the light sensitizer, 1 ml. of IO-3 ,li solution of a hydrogen donor, and 0.2 ml. of purified horse liver catalase prepared by the method of Keilin and IIartree (4) were placed in a Xl ml. beaker. The beaker was then set in an ice bath on a mechanical shaker and exposed to the light of a 500 w. incandescent lamp at a distance of 40 cm. This provided a light intensity of about Wtl foot candles. Of the photosensitizers studied riboflavin was found to be the most effective, followed in order of decreasing activity by fluorcscine, eosine, scopolctin, methylumbelliferone, and quinine sulfate. As riboflavin was the most active agent further studies were carried out primarily with this substance. Table I indicat.es that there is an appreciable accumulation of manganic ions only when all compor1ent.s of the reaction mixt.ure are present. No formation of manganipyrophosphate occurs if the reaction is carried out under nit,rogen. If the reaction mixture is placed in a narrow test tube in air and not agitated, the accumulation of manganic ions is markedly decreased and the solution turns colorless indicating reduction of riboflavin to the leuco form. On subsequent shaking the yellow color of riboflavin is restored. In the absence of catalase the accumulation of manganic ions is decreased due to the reaction of the manganic ions with the hydrogen peroxide produced on the oxidation of t.he leuco riboflavin; thus 11nnOa + HP02 + 2M110 + Hz0 + 02
-.
’ Contribution No. 46, Science Service I,aboratory, Canada Department Agriculture, University Sub Post Office, I,ondon, Ontnrio. * D. M. Miller, unpublished.
of
LETTERS
TO
THE
TABLE Factors
Affecting
the Photoinduced
I Oxidation
nlanganous sulfate, pyrophosphate, air, hydrogen donor (p-cresol), catalase, light sensitizer (riboflavin 3 mg./lOO ml. aqueous solution) So catalase So p-cresol So catalase and p-cresol So air No pyrophosphat,e No riboflavin So light, by the benzidine
of
Man,ganows
Zons
Accumulation of manganipyrophosphate”
Reaction mixture
(1IMimated 20 min.
585
EDITORS
reaction
(2) after
++++
+-+ + + an cxperimentjal
period
01
h large number of substances were found to catalyze the reaction. ,411 the monohydric and polyhydric phenols studied which do not readily form quinones on oxidation catalyzed the oxidation of manganous ions when riboflavin was used as the light sensitizer but not when the light sensitizer itself was a monohydric phenol, e.g. scopoletin. Aromatic amines and related substances such as tryptophane and indoleacetic acid also catalyzed the reaction and so did maleic hydrazide. Quinone-forming phenols such as catechol and hydroquinone completely inhibit the reaction. In this connection it is interesting to note that t,he dihydric phenolic coumarin derivative es-uletin, although closely related to t,he monohydric light-sensitizer scopoletin, does not art as a photosensitizer and inhibits the riboflavin sensitized react,ion. According to Kenten and Mann (1,5) monohydric phenols can undergo a cyclic oxidation-reduction, being oxidized by peroxidase and hydrogen peroxide to :L highl?- reactive intermediary which can then undergo reduction by manganous ions. .4 similar oxidation-reduction reaction appears to be operative in the present spst,em for it was observed that p-cresol, indoleacetic acid, or maleic hydrazidc all undergo photooxidation in the presence of riboflavin but that the rate of disappearance of these substances is decreased if manganous ions are added t,o the reaction mixture at the commencement of the experiment. If the manganous ions are not added until these substances have undergone photooxidation, they have no effect. The results suggest then that riboflavin first brings about a photooxidation of the hydrogen donor to a reactive intermediary which is then rapidly reduced by manganous ions. The fact that manganous ions do undergo a slow oxidntion in t,hc absence of an intermediary suggests that) either riboflavin can cause a direct but slow photooxidat,ion of manganous ions or that there are impurities presentj which may act as intermediaries. Tal)lr I also shows that no reaction is detectable if pyrophosphate is omitted from the reaction mixture. The importance of this subst ante appears to lie in its
586
LETTERS
TO THE
EDITORS
ability to form a stable complex with manganic ions thus preventing the dismutation of the trivalent manganese into the di- and tetravalent forms. Citrate, which also forms a complex with manganic ions, was much less effective than pyrophosphate. The results of these studies on the photoinduced oxidation of manganous ions by riboflavin appear to be compatible with the following scheme: Rbf Rbf* oxidation
product
light > Rbf*
+ D -+ Rbf Hz + oxidation
of D + 2Mn++
+ 2H+
product
of D
(2)
pyrophosphate f D + 2Mn+++ pyrophosphate
Rbf Hz + 02 + Rbf + HzOz
(3) (4) (5)
over-all
reaction
: 2Mn++
+ 02 + 2H++ -+ 2Mn+++
+ Hz0
+ W 02
riboflavin, = activated = hydrogen donor, an aromatic phenol or amine which does not readily form a quinoid structure on oxidation. The question arises as to whether the observed photoinduced oxidation of manganous ions can be related to the action of manganese in illuminated biological systems. Gerretsen (6) and Mehler (7) working with chloroplast suspensions have shown that the addition of manganous ions causes an increase in oxygen uptake in light. If manganous ions do undergo a photoinduced oxidation in such chloroplast suspensions, one would not expect manganic ions to accumulate, due to the presence of plant metabolites which would bring about their rapid reduction. The existence of such a cyclic oxidation-reduction of manganese might help to explain the observed effect on oxygen uptake in illuminated chloroplast suspensions. where
Rbf* D
REFERENCES 1. 2. 3. 4. 5. 6. 7.
KENTEN, R. KENTEN, R. LINGANE, J. KEILIN, D., KENTEN, R. GERRETSEN, MEHLER, A.
H., AND MANN, P. J. G., Biochem. J. 46, 255 (1949). H., AND MANN, P. J. G., Biochem. J. 60, 360 (1951). G., AND KARPLUS, R., Znd. Eng. Chem., Anal. Ed. 18, 191 (1946). AND HARTREE, E. F., Biochem. J. 39, 148 (1945). H., AND MANN, P. J. G., Biochem. J. 48, 67 (1950). F. C., Plant and Soil 2, 323 (1945-50). H., Arch. Biocfwm. and Biophys. 34, 339 (1951).
Science Service Laboratory, Canada Department of Agriculture, University Sub Poet Ofice, London, Ontario, Canada. Received January 29, 1966
W. A. ANDREAE