420 IX. HEMATIN ENZYMES, H 



3.2. Horse-radish Peroxidase 



3.2.1. Isolation. Methods for the isolation of peroxidase from 

 horse-radish have been developed by Bach and Chodat, Willstatter, 

 Kuhn, Elliot, Keilin, and Theorell {109,668,670,1502,1616,3772,3093, 

 309 It). Fractional precipitation with ammonium sulfate and alcohol, 

 purification by dialysis and filtration, adsorption to alumina A, 

 precipitation by tannin or picric acid, and electrophoresis have been 

 applied. By electrophoresis at pH 7.5 {2772) a paraperoxidase migrat- 

 ing toward the cathode can be separated from peroxidase which 

 migrates anodically. The former was first called peroxidase I, the 

 latter, peroxidase II. Peroxidase I has now been recognized to be an 

 alteration product of peroxidase (peroxidase II) (2786) and is called 

 paraperoxidase (Theorell, 2778). While the isoelectric point of per- 

 oxidase is 7.2, that of paraperoxidase is 10.45. Peroxidase crystallizes 

 from ammonium sulfate solutions of 48-62% saturation, while para- 

 peroxidase requires more than 58%. Paraperoxidase is precipitated 

 by acid at pH 4.5. It is not always found and is rather unstable. 

 Enzymically it is almost as active as peroxidase. 



3.2.2. Absorption Spectra and Magnetochemical Properties 

 of Peroxidase and Its Compounds. An absorption spectrum with 

 four bands in the visible part of the spectrum was observed by Keilin 

 and Mann (1502) for horse-radish peroxidase in neutral solution: (I) 

 645, (II) 583, (III) 548, and (IV) 498 m/z. At pH 10 only bands (II) 

 and (III) are seen. According to Theorell (2772) bands (II) and (III) 

 of the neutral peroxidase solution are not bands of peroxidase, but of 

 paraperoxidase, and only bands (I) (640 mju) and IV are those of 

 peroxidase. Chance found e^M = 12 (424)- 



The Soret band lies at 400-402 niju (e,„M = 78) according to 

 Theorell (2772) and Itoh (1391), at 410 niM (tn.M = 125) according to 

 Chance (4-24), and at 415-420 m/x according to Kuhn and co-workers 

 (1616). 



By means of dithionite, jieroxidase is reduced to ferroperoxidase 

 with an absorption band at 558 m/x and a weaker band at 594 m/x 

 (1502). This combines with carbon monoxide. 



Table IV gives the absorption spectra of compounds of peroxidase 

 together with the number of free electrons found magnetochemically 

 (Theorell, 2775). 



Peroxidase forms three different compounds with hydrogen peroxide. 

 I, observed by Theorell, is green and in the absence of hydrogen 



