Çiğdem BİLEN, Nahit GENÇER, Oktay ARSLAN

İnek Sütünden Alkalen Fosfataz Enziminin Saflaştırılması ve Bazı Veteriner İlaçların Enzim Aktivitesi Üzerine İnhibisyon Etkilerinin İncelenmesi

Bu çalışmada, fosfat metabolizmasında önemli role sahip olan Alkalen Fosfatazın ALP , sütte pastörizasyon belirteci olarak kullanılması gerekçesiyle sütte bulunan izoformu biyokimyasal yöntemlerle saflaştırılmış ve bazı veteriner ilaçların enzim aktivitesi üzerine etkileri araştırılmıştır. Enzim, çeşitli organik çözücülerle muamele edilerek, amonyum sülfat çöktürme aralıkları belirlenmiş ve elde edilen çökelek diyaliz edilerek, Sephadex G-100 jel filtrasyon kolonuna tatbik edilmiştir. Saflaştırılan enzim SDS poliakrilamid jel elektroforezine uygulanarak, elektroforezde gözlenen kazein fraksiyonları dışında, yaklaşık 85 kDa ve 185-190 kDa molekül ağırlığına sahip bandlar elde edilirken; doğal jel elektroforezinde, yaklaşık 170-190 kDa aralığında tek band gözlenmiştir. Enzim, pastörizasyon sıcaklığına gelindiğinde inaktive olacağından enzim aktivitesinin gözlenmemesi sütün pastörize olduğunu gösterir. Fakat, veteriner ilaçlar tarafından enzimin inaktivasyonu söz konusu olduğunda, pastörizasyon sıcaklığına ulaşılmadan enzim inhibe olacağından doğru bir pastörizasyon gerçekleştirilemeyecektir. Bu sebeple çalışmamızda, bazı ilaçların furosemide, atropine sulfate, toldimfos sodium and levamisol/ - - tetramisol HCI enzim aktivitesi üzerine inhibisyon etkileri incelenmiştir. Atropin sülfat enzim aktivitesini etkilemez iken, enzim aktivitesi üzerinde inhibisyon etki gösteren tetramisol, furosemid ve toldimfos sodyuma ait IC50 değerleri sırasıyla 45.48 μM, 2.19 ve 126.315 mM olarak hesaplanmıştır. ALP’nin bilinen inhibitörü olan Levamisol ile karşılaştırıldığında, enzim aktivitesini azaltan ilaçların zayıf inhibitör etki gösterdiği belirlenmiştir.

Anahtar Kelimeler:

Alkalen fosfataz, saflaştırma, inhibisyon, veteriner ilaçlar

Purification of Alkaline Phosphatase from Bovine Milk and Investigation of Inhibitory Effects Of Some Veterinary Drugs on Enzyme Activity

In this study, Alkaline Phosphatase EC 3.1.3.1, ALP which has a critical role in phosphate metabolism was purified via bi- ochemical methods and the effects of some veterinary drugs on purified milk ALP were determined on account of the fact that using as a pasteurization indicator in milk. Enzyme was homogenized with different organic solvents, ammonium sulfate precipitation, dialysis and following Sephadex G-100 gel filtration chromatography. The enzyme product yielded bands, app- roximately 85 and 185-190 kDa on SDS-PAGE except casein fractions, while a single band, approximately 170-190 kDa interval. ALP is the most important enzyme in dairy industry as the check for the presence of residual ALP activity in milk is a good indication of proper pasteurization. Heat treatment of milk for adequate pasteurization, which kills pathogenic microorga- nisms, give rise to inactivate ALP. It seems as if milk is pasteurized before not to reach pasteurization temperature, in case of decreasing ALP activity notably by the veterinary drugs. Therefore, in vitro inhibitory effects of some drugs furosemide, atropine sulfate, toldimfos sodium and levamisol/synonim: - - tetramisol HCI were investigated in this study. While atropine sulfate didin’t effect the enzyme activity, the IC50 values of drugs which have inhibitory effect were obtained 45.48 μM, 2.19 and 126.315 mM for tetramisol, furosemide and toldimfos sodium, respectively. When compared to Levamisol, which is a po- tent inhibitor of the enzyme, the inhibitory effects of the drugs on the enzyme activity are weak.

Keywords:

Alkaline phosphatase, purification, Inhibition, veterinary drugs,

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