426 TX. HEMATIN ENZYMES, II 



tyrosine hydroxy! group, the Hnkage, an ester linkage. The structure 

 of peroxidase at ;>H 7 would thus be that given in Figure 2. 



protein 



■CO2 Fe'+OH 



COjH 



Fig. 2. Structure of peroxidase (for explanation 

 of figure cj. Section 3..*}, Chapter V). 



An alternative possibility envisages linkage of the hematin iron to 

 a hydroxy 1 group of the protein, with two ester linkages of the 

 carboxyl groups to protein hydroxyl groups. 



Spectrophotometric evidence was adduced for dissociation, with inacti- 

 vation of the enzyme, at pH 4.0 and for a pK of o.O. The latter is in agree- 

 ment with the pK value of 5.0 for the dissociation of (FeOH) — > (Fe)+ + 

 (0H)~ found from dissociation experiments on fluoride peroxidase. Finally 

 in peroxidase another pK value of about 11.0 replacing a pK value of 10.2 

 in the apoenzyme, is found from differential titration of enzyme and apo- 

 enzyme (c/. Table VI) and of ferro- and ferriperoxidase. This is supported 

 by magnetochemical evidence showing transformation of peroxidase with 

 ionic type of linkages into an alkaline peroxidase with covalent type of 

 linkages. Theorell and Paul {2789) arrive at the scheme in Figure 3. 

 This is supported by the following evidence: 



(/) Peroxidase has only two moles of histidine per mole and both imidazole 

 groups are titrated within their normal range of 5.5 to 8. 



{2) Between />H 8 and 9.5 there is a titration difference of one equivalent 

 between ferric and ferrous enzyme. 



[3) There is no difference between the titration curves of ferroperoxidase 

 and carl)on monoxide ferroperoxidase, although the type of linkage changes 

 from ionic to covalent. In hemoglobin, in which the iron is linked to imida- 

 zole groups, the combination with carbon monoxide influences the titration 

 curve. 



(4) Between pYi 5.5 and 9 the differential titration between enzyme and 

 apoenzyme indicates a difference of two equivalents per mole. In his first 

 paper Theorell (2770) claimed that a difference of three equivalents should 

 be found, if the pK of the acid group of the protein before combination was 

 above 4.5 — leaving out of consideration the (FeOH) formation. Later he 

 stressed that the difference should be three, in consideration of the dissocia- 



