422 IX. HEMATIN ENZYMES, II 



from the enzyme and Chance {If25) showed the inhibition to be 

 reversible and competitiv'e. The enzyme combines with one mole 

 of cyanide (K = 4 X 10"^ M). Whereas the affinities for cyanide 

 and hydrogen peroxide are of the same order, the rate of combination 

 with cyanide is about 100 times slower than that with hydrogen 

 peroxide (c/. Section 3.2.4.). Fluoride, azide, hydroxylamine, and 

 hydrazine (1482,1502) are also strong inhibitors, although in some- 

 what higher concentration, (10"-^ M), while carbon monoxide has no 

 inhibitory effect (671,2777). Balls and Hale (127), measuring the 

 disappearance of hydrogen peroxide iodometrically, claimed that 

 peroxidase is inactivated by sulfhydryl compounds which act on it in 

 the absence of peroxide, while aniline, phloroglucinol, resorcinol, and 

 toluidines inhibit the enzyme in the presence of peroxide. Randall 

 {220Jf) has recently shown, however, that the method used by Ball 

 and Hale cannot be applied in the presence of sulfhydryl compounds, 

 and that the latter do not really inhibit peroxidase. They are them- 

 selves oxidized as substrates of the hydrogen peroxide -peroxidase 

 system. 



3.2.4. Kinetics and Estimation. The enzyme has a double speci- 

 ficity, which we shall call hydrogen donor and hydrogen acceptor 

 specificity, whereas some authors speak of hydrogen peroxide as the 

 substrate and of the hydrogen donors as "acceptors." Peroxidase 

 combines with hydrogen peroxide through the hematin iron and with 

 the hydrogen donor through a group in the protein molecule (1862). 

 It can oxidize a variety of hydrogen donors, of which polyphenols 

 (particularly pyrogallol), leuco dyes such as leucomalachite green, 

 and ascorbic acid are used frequently. The pH optimum varies with 

 the hydrogen donor used (127). As hydrogen acceptors hydrogen 

 peroxide and (less effectively) monoalkylperoxides are active, but not 

 dialkylperoxides (3075). Catalase does not inhibit peroxidase effec- 

 tively (1375). Horse-radish peroxidase can also catalyze the oxidation 

 of p-aminobenzoic acid by hydrogen peroxide (1759). The reaction 

 is inhibited by sulfanilamide, but has no correlation with the anti- 

 bacterial activity of the latter substance. 



For quantitative estimations, the purpurogailiu method of Willstatter is 

 commonly used. The "Puipurogallin Zahl" (PZ) is defined as tlie number 

 of milhgrams of purpurogalhn, an orange pigment, produced per milHgram 

 of enzyme preparation (dry weight) in five minutes at '20° C. when I'i.o mg. 

 of hydrogen peroxide and 1 .^.l g. of pyrogallol in .500 cc. of water are employed. 

 It now appears that this method was not fortunately chosen. Several authors 



