255 



Keelin T». Fry and Anthony San Pietro 



TABLE 2 



Amino Acid Analysis of Spinach PPNR 



Amino Acid Residues per Mole 



For the determination of tryptophane, samples of protein were hydro- 

 lyzed with 5 N NaOH containing basic lead acetate ^^^> by heating for 18 hours 

 at 100° in evacuated glass ampules. Tryptophane content of the hydrolvsates 

 was determined by the method of Spies and Chambers (Procedure Q) °^' 



Preliminary analyses of the cysteine content of the protein indicate the 

 presence of about six half-cystine residues determined as cystein acid follow- 

 ing oxidation with performic acid ^ ). 



REACTION WITH p-CHLQROMERCURlBENZOATE (PCMB) 



Treatment of PPNR with PCMB results in a rapid loss in the visible 

 spectrum of the enzyme. As shown in Figure 2, there is a parallel loss in 

 absorbance at each of the three absorption maxima in the visible region. 

 After complete reaction, the percentage of the original absorbance remaining 

 at 330, 420 and 465 mp was 40%, 10% and 5%, respectively. 



The decrease in color with addition of mercurial can most easily be 

 interpreted as resulting from the disruption of the iron- protein chromophore. 

 The amount of PCMB required for complete titration (Figure 2) was 0. 47 

 pmole. This amount of PCMB corresponds to about 8-9 equivalents of PCMB 

 reactive groups per mole of enzyme. The same result was obtained by a 

 sulfhydryl titration of PPNR according to the method of Boyer ^^l). These 

 values are higher than expected on the basis of the preliminary estimate for 



