1. The amounts of O
2 consumed and CO
2 evolved in the oxidation of glycolic acid by the salt-precipitated enzyme were found to be dependent upon the amount of catalase contaminated in the enzyme sample. When catalase was practically absent, the amount of O
2 consumed per mole of glycolic acid oxidized was one mole. When excess of catalase was present the O
2-uptake was reduced to 1/2 of this value.
2. The addition of cyanide caused the accumulation of H
2O
2 together with the disappearance of CO
2-formation without changing the amount of O
2-uptake. The addition of azide, on the other hand, caused the increase in CO
2-formation with no H
2O
2-accumulation, but the same O
2-uptake was brought about as in the case of CN-addition.
3. The addition of peroxidase and its hydrogen donor (reduced Bindschedler's green) caused the disappearance of CO
2-formation and of H
2O
2-accumulation without changing the amount of O
2-uptake.
4. H
2O
2 was proved to react spontaneously with the intermediate as well as with synthetic glyoxylic acid to form CO
2. Some additional evidence was aduced in support of the conclusion that glyoxylic acid is the intermediary product in the oxidation of glycolic acid.
5. Glycolic acid is oxidized by plant sap with an equivalent amount of O
2-uptake but only with a little CO, -formation. When the sap was pretreated with H
2O
2, CO
2-formation was remarkably increased.
6. The formation of H
2O
2 and accumulation of pyruvic acid were confirmed in the oxidation of lactic acid by the same enzyme in the presence of catalase in excess.
7. Based on these observations it was concluded that the oxidation of glycolic and lactic acids proceeds as follows:
In the enzyme solution,
Glycolic acid+O
2→
oxidaseglyoxylic acid+H
2O
2→
spontaneousformic acid+CO
2+H
2O,
Lactic acid+O
2→
oxidasepyruvic acid-+H
2O
2→
spontaneousacetic acid+CO
2+H
2O.
In plant sap,
Glycolic acid+O
2+AH
2oxidase→
peroxidaseglyoxylic acid+A+2H
2O,
Lactic acid+O
2+AH
2oxidase→
peroxidasepyruvic+A+2H
2O,
where AH
2 represents some unknown, presumably phenolic, substances present in the plant sap.
The authors wish to thank Prof. Dr. Hiroshi Tamiya for his kind advice and interest in this work. The authors are also indebted to Dr. Yasuyuki Ogura for furnishing purified equine liver catalase used in this study, and to Dr. Takeshi Mori for his helpful suggestions in the purification of the enzyme.
This work was aided by a Grant from the Scientific Research Fund of the Ministry of Education.
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