256 



Keelin T. Fry and Anthony San Pietro 



EFFECT of PCMB on the VISIBLE SPECTRUM of PPNR 



Fig. 2. Effect of PCMB on the 

 visible spectrum of PPNR. The 

 reaction mixture contained 1. 13 mg 

 of PPNR, determined by the method 

 of Warburg and Christian ( ), in 

 0. 9 ml of 0. 05 M Tris, pH 7. 45. 

 The values were corrected for ab- 

 sorption remaining after complete 

 titration. PCMB concentration was 

 determined spectrophotometrically 



at 232 mp as described by Boyer 



(2l) 



01 0.2 0.3 0.4 0.5 



mMOLES of PCMB ADDED 



the half-cystine content of PPNR from cysteic acid analyses and may reflect 

 a reaction between PCMB and the "labile sulfur" shown previously to be 

 present in the enzyme. 



Similar titrations of PPNR with mercurials have been performed by other 

 investigators (^^' ^^\ Katoh and Takamiya (^^) interpret their results to 

 indicate the presence in PPNR from Brassica leaves of 12 sulfhydryl groups. 

 This interpretation assumes no reaction between mercurial and "labile 

 sulfur". 



TREATMENT WITH o- PHENANTHROLINE (OP) 



(10) 



In addition to iron, PPNR contains "labile sulfur" which is liberated as 

 hydrogen sulfide upon acidification. Assays for the sulfide were performed 

 by applying the method of Fogo and Popowsky '^3^ directly to solutions of the 

 protein. The molar ratio of iron to labile sulfur with several preparations 

 averaged from 0. 9- 1. 1. Control experiments indicate that cysteine groups 

 in the protein are not the source of the labile sulfur. 



To determine the changes resulting from treatment with the iron che- 

 lator OP, the enzyme was incubated with the chelator and then, at varying 

 time intervals, separated from the latter, as well as from the ferrous tri- 

 phenanthrolate complex which formed, by chromatography on Sephadex G-25. 

 Analyses of protein fractions obtained in such an experiment are summarized 

 in Table 3. The data indicate that under conditions of pH and temperature at 

 which PPNR is relatively stable, treatment with OP results in loss of iron 

 from the enzyme as well as a corresponding loss of "labile sulfur", absorp- 

 tion in the visible region of the spectrum and activity of the enzyme in TPN 

 reduction. 



