TRANSHYDROGENASE REACTIONS AND ESTROGENS 171 



as a stabilizing agent at an early- stage in the purification procedure. 

 The enzvme prepared in tliis way contained hound estradiol-17^, 

 whicli could not be removed conipletc^ly by repeated precipitation 

 of the protein with ammonium sulfate or with acetone. 



Each fraction was tested for dehydrogenase activity with DPN, 

 TPN, deaminoDPN, and various pyridine nucleotide analogues as 

 Indrogen acceptors (Talalay and Williams-Ashman, 1960). Trans- 

 Indrogenase assays which necessitate the addition of auxiliary en- 

 zymes for the generation of TPNH are not trustworthy. Conse- 

 quent! v, transit drogenase activity was determined by measurement 

 of the estradiol-dependent reduction of acetylpyridineDPN by 

 DPNH. Catalvtic amounts of estradiol-17jS or estrone mediate this 

 reaction, apparenth bv the following mechanism: 



Estrone + DPNH + H+^ EstradioI-17/i + DPN + 

 Estradiol-17/3 + acetylpyridineDPN + -^ Estrone + acetylpyridineDPNH f H + 



DPNH + acetylpyridineDPN+^ DPN+ + acetylpyridineDPNH 



Since acetylpyridineDPNH absorbs light strongly at 400 ni/x, 

 whereas DPNH does not (Kaplan and Ciotti, 1956), this reaction 

 can be followed spectrophotometrically at 400 m^u, in the presence 

 of high concentrations of DPNH. However, this assay system is 

 complicated by the fact that a number of soluble flavoprotein en- 

 zymes also catalyze the DPNH-acetylpyridineDPN exchange 

 (Weber and Kaplan, 1957). Such enzymes are highly active in 

 crude placental extracts and contaminate the most purified prepara- 

 tions of the placental enzyme which catalyze estradiol-dependent 

 transhydrogenase reactions. But these "non-specific" DPNH-acetyl- 

 pyridineDPN transfers are unaffected bv TPN, whereas the estradiol- 

 dependent transhvdrogenation between the latter nucleotides is 

 virtually obliterated by low levels of TPN. Thus, the estradiol- 

 dependent DPNH-acetylpyridineDPN exchange could be estimated if 

 suitable controls to which TPN was added were employed. In later 

 experiments (Talalay and Williams-Ashman, 1960), the DPNH- 

 acetylpyridineTPN and the TPNH-acetylpyridineTPN transfer re- 

 actions were also measured at various stages of the purification pro- 

 cedure. 



Talalay and his co-workers purified the placental enzyme more 

 than a hundredfold with retention of a major portion of the orig- 

 inal estradiol-DPN dehydrogenase activity. They were unable to 



