258 



Keelin T. Fry and Anthony San Pietro 



The correlation between the percentage of iron lost and the loss of 

 absorption in the visible region of the spectrum is actually better than is 

 apparent from the values reported in Table 3 since there is apparently some 

 residual absorption in this region even in iron-free enzyme. 



OXIDATION STATE OF IRON 



Although incubation of PPNR with OP results in removal of iron from the 

 protein and formation of the ferrous triphenanthrolate complex, this obser- 

 vation does not constitute proof that the metal in the native enzyme is in the 

 ferrous state since reduction of bound ferric iron could occur before, during, 

 or after release from the protein. Removal of the metal from the native 

 enzyme by the ferric iron chelator Tiron (1, 2-dihydroxybenzene-3, 5- 

 disulfonate) is negligible under comparable conditions. If, however, the 

 PPNR is first treated with the sulfhydryl reagent p-chloromercuriphenyl 

 sulfonic acid (PCMS), there is a rapid reaction with the ferric chelator 

 as shown by the results of an experiment reported in Table 4. In this 



TABLE 4 



Reaction of PPNR with OP or Tiron following addition of PCMS 



Reaction mixture contained PPNR equivalent to 0. 35 jamoles of iron, 

 52 ^moles of Tris- HCl buffer, pH 8, 2. 9 mg of glucose oxidase 

 (Sigma, type II), 29 mg of glucose and 0. 3 mg of crystalline horse 

 liver catalase in a total volume of 4. 5 ml. Additions were 6 pmoles 

 of PCMS in 0. 3 ml of 0. 2 M Tris- HCl buffer, pH 8 and either 4. 5 

 pmoles of OP in 0. 3 ml of 0. 1 M Tris- HCl buffer, pH 8, or 4. 5 

 pmoles of Tiron (adjusted to pH 8) in 0. 3 ml of 0. 1 M Tris- HCl 

 buffer, pH 8. 



experiment the possibility of air oxidation of any ferrous iron present in the 

 protein was minimized by carrying out the reaction with the chelators under 

 anaerobic conditions in glass cuvettes fitted with a ground glass capillary 

 stopper. PPNR and an oxygen trapping system of glucose-glucose oxidase 

 were preincubated in the completely filled cuvettes for 20 min. at 25° before 

 addition of, in order, PCMS and either Tiron or OP. Before use, the PCMS 

 and chelator solutions were freed from oxygen by bubbling with argon for 15 

 min. The mercurial and the chelators were added to the cuvettes from a 

 syringe through the capillary bore of the glass stopper. Thus an amount of 



