Purification and partial characterisation of catalase from chicken liver and the inhibition effects of some chemicals on the enzyme activity

Katalaz, kısmi indirgenmiş oksijen türleri ve H2O2 nin yükseltgen etkisinden dokuların korunmasında önemli rol oynar. Tavuk karaciğer katalazı, (NH4)2SO4 çöktürmesi, etonol-kloroform muamelesi, CM-Selükoz ve Sephadeks G-200 kromotografisi kullanılarak saflaştırıldı. Jel filitrasyon kromotografisi ile tavuk karaciğer katalazının molekül ağırlığı yaklaşık olarak 235000 Da bulundu. SDS-jel elektroforezi sonucu tavuk karaciğer katalazının molekül ağırlığı 57500 Da olarak bulundu ve 4 alt birimden oluştuğu tespit edildi. Spesifik aktivite 55765.5 U/mg olarak saptandı. Hem içeriğinin kabaca ölçülmesini sağlayan A407/A270 oranı 0.52 olarak bulundu. Enzim için Km değeri 100 mM olarak belirlendi. Enzimin 7.0-10.0 aralığında geniş bir pH optimumuna sahip olduğu görüldü. Tavuk karaciğer katalaz enziminin optimum sıcaklığı 25 °C olarak tespit edildi. Saflaştırılmış enzim aktivitesi KCN, NaN3, β-merkaptoetanol, DTT, iyodoasetamit tarafından inhibe edildi, fakat sodyum dietilditiyokarbamatın enzimi inhibe etmediği görüldü.

Anahtar Kelimeler:

tavuk, saflaştırma, karaciğer, katalaz, enzim aktivitesi, kromatografi, engelleme, moleküler ağırlık

Catalase plays a major role in the protection of tissues from oxydizing effects of H2O2 and partially reduced oxygen species. It was purified chicken liver by (NH4)2SO4 precipitation, ethanol-chloroform treatment, CM-cellulose and Sephadex G-200 chromatography. The molecular weight of the native chicken liver catalase was estimated by gel filtration to be approximately 235000 Da. SDS-gel electrophoresis results indicated that chicken liver catalase consists of four apparently identical sub-units, with a molecular weight of about 57500 Da. The specific activity was found to be 55765,3 U/mg. The optical spectrum of the purified enzyme shows a Soret band at 407 nm which is the characteristic for heme and there was apparent reduction by dithionite. The Km was found to be 100 mM. The maximal activity of the enzyme was observed between pH 7.0 and 10.0. The enzyme has a optimum reaction temperature at 25 °C. The activity of purified catalase was inhibited by azide, cyanide, β-mercaptoethanol, DTT, GSH and iodoacetamide.

Keywords:

fowls, purification, liver, catalase, enzyme activity, chromatography, inhibition, molecular weight,

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