232 ASCORBIC ACID 



bic acid. The preparation thus contained a diaphorase I and II similar in 

 character to those reported in peas. The hydrogen acceptor in the reaction 

 Avas not dehydroascorbic acid, prepared l)y the oxidation of ascorbic acid 

 with iodine. The suggestion was made that the hydrogen acceptor is a 

 semiquinone intermediate (monodehydroascorbic acid) of ascorbic acid. 

 If this suggestion is correct, it may well be the reason why the present 

 writer was unable to demonstrate the reduction of dehydroascorbic acid 

 by either coenzyme I-specific formate or ethanol dehydrogenase enzymes 

 from peas with added coenzyme I. 



a. Role of Glutathione (GSH) 



There has been much work indicating the close association between GSH 

 and ascorbic acid in plant and animal tissue. ^^* In germinating seeds and 

 sprouting potato tubers, both these substances appear at the same time. 

 When potato tubers are cut or subjected to ethylene chlorohydrin,^-'' their 

 content of both GSH and ascorbic acid increases, and the formation of 

 both substances is abolished under anaerobic conditions. In animal tissue, 

 Szent-Gyorgyi (1928) first showed^-^ that in minced rat kidney dehydro- 

 ascorbic acid could be reduced and that this reduction could be effected 

 by GSH in buffer solutions. This work was extended to liver tissue in which 

 it was found that GSH protected ascorbic acid from oxidation.^"* The pro- 

 tective effect of GSH on the oxidation of ascorbic acid has been observed 

 by several workers, but the explanation of this has been ascribed to different 

 causes. The fact that GSH combines readily with copper and that ascorbic 

 acid may be protected from oxidation catalyzed by copper in the presence 

 of sulfhydryl compounds is believed by some workers to be one explanation 

 for the stabilizing effect of GSH on ascorbic acid. 



That this is not the sole explanation is shown by the fact that GSH will 

 reduce dehydroascorbic acid (DHA) in solution without addition of added 

 catalysts. ^-^ The rate of this uncatalyzed reaction is, however, too slow to 

 be of much consequence either in maintaining ascorbic acid in the reduced 

 form in tissues, or as part of a respiratory cycle. The "half-time" period for 

 the reduction of DHA by GSH at physiological temperatures and pH and in 

 concentrations usually found in vivo is of the order of 15 minutes ;^''^ on the 

 other hand, under the same conditions the conversion of DHA to 2,3- 

 diketogulonic acid itself has a half-life of only 2 minutes.^-'' 



Pfankuch^-^ was the first to describe the catalytic reduction of DHA by 



"3 L. B. Pett, Biochem. J. 30, 1228 (1936); F. G. Hopkins and E. J. Morgan, Nature 



152, 288 (1943). 

 ^^^ J. Guthrie, Contribs. Boyce Thompson Insi. 9, 17 (1937). 

 "6 A. Szent-Gyorgyi, Biochem. J. 22, 1387 (1928). 

 126 E. M. Crook and F. G. Hopkins, Biochem. J. 32, 135G (1938). 

 12^ M. Ball, J. Biol. Chem. 118, 219 (1937). 



