Sarımsak filizinden asit fosfatazın kısmi saflaştırılması ve özelliklerinin incelenmesi

Abstract

Asit fosfataz (ortofosforik monoester fosfohidrolaz; EC 3.1.3.2.) çeşitli fosfat esterlerini hidrolizini katalizleyen pH optimumu 6'nın altında olan enzimdir. Asit fosfataz tabiatta çok rastlanan bir enzim olup ve genelde seçici değildir. Bu çalışmanın amacı sarımsak filizinden asit fosfatazın izolazyonu, saflaştırılması ve özelliklerinin incelenmesi olarak belirlenmiştir. Sarımsak filizi asit fosfataz (EC 3.1.3.2.) inert komponentlerin çöktürülmesi, amonyum sülfat ile fraksiyonlama ve DEAE-seluloz kolon kromotografisi ile kısmi saflaştınlmıştır. Saflaşma başlangıç supernatanta göre 40 kat olmuştur. Enzim aktivitesi ve büyüme hızı arasındaki ilişki incelenmiştir. Amonyum sülfat konsantrasyonu, pH, sıcaklık, asetat tamponunun iyonik şiddeti ve stabilite profilleri saflaştınlmış sarımsak filizi asit fosfatazı için incelenmiştir. Ham homojenatın spesifik aktivitesi 4.2 (U/mg protein) olarak bulunmuştur. Saflaştırmanın son kademesinde elde edilen enzimin spesifik aktivitesi ise 168 (U/mg protein) olarak tespit edilmiştir.The biological role of plant and animal acid phosphatase remains largely unknown. Acid phosphatases ( orthophosphoric monoester phosphohydrolases; EC 3.1.3.2) are enzymes that catalyse the hydrolysis of a variety of phosphate esters and appear to exhibit pH optima below 6. The enzymes are widly found in nature and are usually rather nonspecific. Acid phosphatases are apperently ubiquitous in nature, having already been identified in numerous organisims and tissues. In general, the acid phosphatases accure in very small quantities, are unstable in diluted solutions, and are subject to surface denaturation in the pure state. These properties, and their tendency to occure in multiple forms, often make the isolation of acid phosphatases difficult. Studies on pure acid phosphatases have so far focused mainly on those of clinical significance from animal sources. Perhaps because of the difficulty of obtaining the pure enzymes, the function of the nonspecific acid phosphatases are poorly defined. Among possible functions of acid phosphatases might be the role of providing in organic phosphate for metabolic excretory and some secretory purposes. Acid phosphatases may have a role the mammalian reproductive process. A possible role of human prostatic acid phosphatase may be in the dephosphorylation of esters to liberate fructose, which serves as an energy source for spermatozoa, or in the formation of choline from choline phosphate. In plants, acid phosphatase activity in seeds often increases greatly during germination, indicating a possible role in phosphate mobilation. Garlic acid phosphatase is a metalloprotein as a monomer with a molecular weight 56000-58000. Interestingly, sun flower seed acid phosphatase consists of two, polypeptide chains, each of which is simular in size to the one that is present in the wheat germ enzyme. The effect of eddit metal ions and the metalloenzyme charecter of acid phosphatases has been of continiual interest in literature. The metal ions are bound quite firmly to the protein and was inactivated by dialysis. The isoelectric point of the enzyme is 5.2 and optimum pH is between 4.5-6.5. The objective of this study is to obtain pure acid phosphatases than produced by prior art by operating under conditions that improve the final product. The study features;. The use of a mild non-ionic detergent,. 40-80 % saturation with (NH4)2S04,. Maintaining at low temperature to remove impurity. The use of chromatografic columns to concentrate the acid phosphatase and remove non-acid phosphatase proteins with lower or higher molecular weights.