HORSE-RADISH PEROXIDASE 



427 



tion (FeOH) ;=^ (Fe)"^ + 0H~ . This supports covalent linkage of one of 

 the carboxyUc acid groups of the hematin to a protein group. 



The combination of the hematin with apoenzyme is slow and, on treatment 

 of the enzyme with normal hydrochloric acid, slow changes of absorption 



Fig. 3. Forms of horse-radish peroxidase, according to Theorell and Paul {2789). 



were found by spectrophotometric measurements. This speaks in favour of 

 an ester linkage, broken slowly at Jow pH values. 



Theorells arguments have a number of weak points and the results of the 

 two papers do not appear to have been correlated satisfactorily. Thus, in 

 the first paper (before the discovery of the formation of the hydroxyl com- 

 pound at jsHo), a difference of one equivalent between ferriperoxidase and 

 ferroperoxidase between pH 8 and 9.5 was found, which now remains unex- 

 plained. No clear evidence for a change of one equivalent at 7>>H 5 was 

 found, but the results of this differential titration were not reliable below 

 pYl 1.5. It is difficult to see why a difference of three equivalents should be 

 expected if the pK value of the acidic hematin-linked protein group is above 

 o (cf. point 4 above). 



Part of the reaction (Fe)+ + OH^ -> (FeOH) will be titrated in the pH 

 range of 5.5 to 8 (cf. point 1)\ since, however, 'i.5 equivalents are titrated, 2 

 may still be normal histidine imidazoles. 



Theorell showed that the titrated apoenzyme could be reunited with 

 hematin to fully active enzyme after neutralization. This excludes, however, 

 only irreversible, not reversible, denaturation. Perhaps the large differences 

 found at low pH values {cf. Table VI) are due to reversible denaturation of 

 the apoenzyme which does not occur as long as hematin remains combined 

 with the apoenzyme by ester linkage. 



Another difficulty, stressed by Theorell himself, is the structure ascribed 

 to alkaline peroxidase (compound IV of Fig. 3). This compound is magneto- 

 chemically (covalent linkage) and spectroscopically very different from 

 alkaline hematin; the linkage of the hematin carboxyl to protein cannot 

 explain this difference, since there is no resonance between the porphyrin-iron 

 system and this carboxylic acid group. It must not be forgotten, however, 



