Isolation, Purification And Study Of Some Properties Of Pyrophosphatase From Chicken Breast And Its Relationship With Protein Functionality

Three parts were included in this paper: (1) Purification of pyrophosphatase from chicken breast. (2) Study of pyrophosphatase properties, including the optimum pH,temperature,substrates specifity and the effects of metal ion on enzyme activity. (3) The connection between pyrophoaphatase and protein functionality.The pyrophosphatase has been isolated and purified from chicken breast by means of homogenation, 0.6M NaCl extractions, ammonium sulfate precipitation, and ion-exchange chromatography on DEAE cellulose column. Protein precipitated within 45-65% saturation of ammonium sulfate precipitation possessed the highest activity, resulting in a 5.31-fold increase in purity of the enzyme. Approximately 141.5-fold purification was achieved after ion-exchange chromatography on DEAE cellulose column.The method for detecting pyrophosphatase activity was established. This enzyme catalyzed the hydrolysis of sodium pyrophosphate but not that of other phosphate esters, ATP,HMP and STPP were not hydrolyzed by pyrophosphatase. Only Mg²⁺ was required for activity and stability and Ca²⁺ has no activating effect with PPi being the substrate of pyrophosphatase, its optimum pH and temperature is 7.4 and 40℃, respectively. In the presence of Mg²⁺, the activating effect of 0.1mM and 1mM EDTA-Na2 can be observed in the experiment, there is about 33.8% activating effect at 0.1mM EDTA-Na₂ and 28.4% at lmM. Higher concentration of EDTA-Na₂ can inhibit pyrophosphatase activity. Both of NaF and KIO₃ can inhibit pyrophosphatase activity.The relationship between pyrophosphatase and protein functionality was investigated. The influence of enzyme activation and inhibitor on protein solubility and gel water holding capacity (WHC) was studied. Mg²⁺ had no significant effect on protein solubility, and Ca²⁺ can significantly decrease protein solubility. Effects of NaF and KIO₃ on protein solubility and gel WHC were also studied. From 2h to 6h the protein solubility treated with NaF was higher than that of treated with KIO₃. 20mM and 30mM NaF can significantly increase protein solubility. At 12h 1mM,5mM,10mM KIO₃ can significantly decrease protein solubility and 20mM,30mM KIO₃ can increase solubility compared with the comparison. PP+ Mg²⁺,TPP+Mg²⁺,TPP,PP can significantly increase protein solubility compared to the control.