VOL. 12 (1953) REVERSIBILITY OF GLUCOSE-6-PHOSPHATE OXIDATION 99 



6-Phosphogluconolacfone. The oxidation of glucose with bromine at neutral pH yields the ^- 

 lactone as the initial reaction product^". Glucose-6-phosphate, which has the pyranose structure, 

 would under similar conditions be expected to yield the ^-lactone. The oxidation was carried out by 

 adding an excess of bromine to a solution of the monopotassium salt in 0.2 M acetate buffer, pH 5.7. 

 During the reaction the pH was maintained between 5.5 and 5.7 by the addition of NaOH; below 

 pH 5.5 the rate of oxidation by bromine was very slow, while above pH 6 the lactone was rapidly 

 hydrolyzed. The oxidation was So-go% complete after about 10 minutes at room temperature and 

 was stopped at this point to avoid extensive hydrolysis of the lactone. Excess bromine was removed 

 by vigorous gassing with helium for 2 minutes and the solution used at once for the enzymic ex- 

 periments. 6-Phosphogluconolactone, presumably the ]^-lactone, was prepared by heating in acid 

 according to Robinson and King". 



Other preparations. Zwischenfevment was prepared from dried brewers yeast by the method of- 

 fvoRNBERG^^ which yields a product freeof phosphogluconic dehydrogenase. Phosphogiuconic dehydro- 

 genase. Phosphogluconic dehydrogenase was prepared from brewer's yeast as previously described^^. 

 This preparation is contaminated with Zwischenfernient. 'TPlS was a chromatographed product which 

 was 79% pure^*. 



/socitric dehydrogenase was prepared from pig heart by the method of Ochoa and Weisz- 

 TaboriI^. 



Determinations. The concentration of lactone was estimated by the colorimetric hydroxamic 

 acid method of Lipmann and Tuttle^^, with D-glucono-(5-lactone as a standard. Sugar lactones were 

 found to vary greatly in this reaction (Table I) and all gave less colour than equivalent amounts of 

 acetyl phosphate. Because of this variation the values given for 6-phosphogluconolactone are only 



TABLE I 



REACTION OF LACTONES IN THE HYDROXAMIC ACID TEST 



Measured with the Coleman Junior Spectrophotometer in 16 mm. I.D. tubes in final volume 

 of 6.0 ml. 



** A commercial sample (Pfizer) obtained from Dr F. Lipmann. 

 *** Furnished by Dr H. C. Isbell. 

 § Furnished by Dr. N. K. Richtmyer. 

 J Furnished by Dr. E. R. Stadtman. 



approximate, although they are in the range expected from the amount of glucose-6-phosphate oxi- 

 dized and the observed rate of hydrolysis of the lactone. With Hestrin's modification" of the hydrox- 

 amic acid reaction lower values were obtained, presumably due to rapid hydrolysis of the lactone at 

 alkaline pH. 



Glucose-6-phosphate was determined spectrophotometrically by measurement of TPN reduction 

 in the presence of ZwiscJieu ferment. The results herein reported depend on the validity of this assay. 

 With the purified Zwischenjerment preparation employed other substances which might be present 

 in the reaction mixtures, such as 6-phosphogluconate, ribulose-5 -phosphate and zsocitrate are com- 

 pletely inactive. Phosphogluconolactone originally present in the reaction mixture does not interfere 

 with the reduction of TPN since it is completely hydrolyzed by the time the determinations are made. 



RESULTS 



The reduction of 6-phosphogluconolactone to glucose-6-phosphate was observed in 

 the presence of TPNH and Zwischenferment, with TPNH generated by reduction with 

 6-phosphogluconate in the presence of phosphogluconic dehydrogenase, according to 

 the following equations : 



References p. 102. 



