MILK PEROXIDASE . 429 



contained 1% hematin. After removal of some less active hematin compound 

 the absorption band was at 640 m/i- This peroxidase appears to be closely 

 related to horse-radish peroxidase. 



The peroxidase of Asclepias si/riaca L. (milkweed) was studied by Sumner 

 and Gjessing {2701). The enzyme works with a much higher optimal 

 hydrogen peroxide concentration than horse-radish or turnip peroxidase and 

 is rapidly destroyed by peroxide in the absence of pyrogallol, as well as by 

 pyrogallol in the absence of peroxide. For the estimation of such peroxidases 

 the purpurogallin method must therefore be modified. 



3.4. Milk Peroxidase 



The peroxidase in milk was discovered by Arnold in 1881 {81) and 

 studied by Elliot (667), who separated it from caseinogen by frac- 

 tional ammonium sulfate precipitation. More recently it has been 

 purified further and studied thoroughly by Theorell and collaborators 



{2786,2789,2790). 



Impurities were removed by heating for fifteen minutes at 70° C, followed 

 by basic lead acetate precipitation and precipitation at pH 5.9 by acetone. 

 By electrophoresis at pH 5.9 the enzyme w^as separated from an accompany- 

 ing red iron-containing substance. The enzyme moved cathodically, its 

 isoelectric point being at pH 7.7. Further purification was effected by 

 isoelectric precipitation with ammonium sulfate. It was thus obtained in 

 thin, indistinctly crystalline leaflets. The yield is low (^2% of the total 

 enzyme present), and the preparation does not work during certain seasons. 



Its molecular weight was found to be 92,700 and the frictional 

 ratio, 1.18 (ratio of axes in prolate ellipsoid would be 4.1 for the 

 nonhydrated, less than 4 for the hydrated, enzyme). The pure 

 enzyme with one hematin group per mole would be expected to con- 

 tain 0.06% iron, while the best preparations gave 0.076%. 



The absorption spectra of lactoperoxidase and its compounds {cf. 

 Table VII) show that it is not a protohematin compound. The 

 brown-green ferriperoxidase is reduced by dithionite to the green 

 ferroperoxidase. 



Theorell classes this enzyme with the leucocyte peroxidase as a 

 "verdoperoxidase," but it differs in its properties as much from the 

 green leucocyte peroxidase as from horse-radish peroxidase. It 

 appears possible that the hematin group is monoazahematin {cf. 

 Chapter V, Section 8.2.). 



With a small excess of hydrogen peroxide the enzyme gives a reddish 

 hydrogen peroxide compound, which decomposes to yield the free enzyme, 

 while a larger excess (10 moles of peroxide per mole of enzyme) produces a 

 brow^nish compound. Combination with fluoride decreases the absorption 



