토끼 erythrocyte lysate으로부터 hemoglobin을 $(NH_4)_2SO_4$ 분별침전, CM-Sephadex 크로마토그래피, hydroxyapatite 크로마토그래피, Sephadex G-l00 겔 크로마토그래피 방법을 이용하여 순수 정제하였다. 정제한 토끼 hemoglobin은 peroxidase 활성을 나타냈으며 guaiacol과 o-dianisidine을 기질로 사용하였을 경우 식물 peroxidase보다 10배 가량 낮은$K_m$값을 나타냈다. 여러 phenol 화합물들을 기질로 사용하였을 때 토끼 hemoglobin은 caffeic acid, chlorogenic acid, indole-3-acetic acid를 빠르게 산화하였으며, disubstituted phenol 화합물과 methoxylated phenol 화합물들이 rabbit hemoglobin에 대하여 친화력이 큰 것으로 나타났다. Chemical modification 실험결과 토끼 hemoglobin은 iodoacetamide와 N-ethylmaleimide에 의하여 활성이 억제되지 않지만 p-chloromercuribenzoate에 의해서는 활성이 현저히 억제되었다. 또한 식물 peroxidase와는 달리 hemoglobin의 peroxidase 활성은 diethylpyrocarbonate에 의하여 급격히 불활성화 되었다.
Rabbit hemoglobin was purified by using ammonium sulfate fractionation, CM-Sephadex chromatography, hydroxyapatite chromatography and Sephadex G-100 gel filtration. The purified hemoglobin oxidized various synthetic and naturally occurring phenolic compounds in the presence of $H_2O_2$. The substrate specificity studies indicate that rabbit hemoglobin has about 10 times lower $K_m$ values for guaiacol (0.39 mM) and o-dianisidine (0.09 mM) when compared to those of plant peroxidases. Among natural substrates tested, rabbit hemoglobin catalyzes the oxidation of caffeic acid, chlorogenic acid and indole-3-acetic acid quite rapidly compare to other naturally occurring phenolic compounds such as scopoletin, esculetin, ferulic acid and p-coumaric acid. In general, phenolic compounds having methoxy group appears to have high affinity to rabbit hemoglobin as revealed by their very low $K_m$ values. Chemical modification studies indicate that p-chloromercuribenzoate inactivates the peroxidase activity of rabbit hemoglobin, whereas N-ethylmaleimide or iodoacetamide does not. Furthermore, the peroxidase activity of rabbit hemoglobin is rapidly inactivated by diethylpyrocarbonate, a histidyl selective reagent, unlike plant peroxidases.
