Separation And Purification Of The Superoxide Dismutase From Allium Sativum Plasma

Allium Sativum contains a quantity of Superoxide Dismutase(SOD) having the function of removing the superoxide free radical (O2-).so it can prevent O2- from harming the organism effectively. In this research three methods are used to separate and purify SOD from Alliun Sativum cell plasma , which is thermodenaturation, precipitation of Polyethylene glycol(PEG) and the column chromatography of DEAE-cellulose. Enzymes activity is measured with improved Pyragallol autoxidation, meanwhile the stabilities of enzyme towards temperature and pH, the type of enzyme and absorption spectrum in ultraviolet region are studied. So the results of study are following.1.There is something unknown to interfere SOD activity in the crude extract of Allium Sativum cell plasma. This material is unstable to heat, but SOD is opposite. So the primary method of purification is based on the different heat stability among SOD, interfering material, heteroprotein. When the crude extract is keeping in 60℃ for 20 min ,it can reduce the total protein concentration from 24mg/ml to 15.8mg/ml, and disactivate the interfering material from Allium Sativum cell plasma. The enzyme protein can be obtained which specific activity is 15.9U/mg.2.Polyethylene glycol 6000(PEG) is a kind of water soluble and non-ionic polymer.lt can make protein to precipitate and has a better alternative to precipitation of protein. For example, the concentration of PEG within 16%-28%, the SOD activity of supernatant fluid is steady; while it raises to 28%, the activity begins to reduce; At the point of 40%, the SOD activity of supernatant fluid becomes zero. It indicates that SOD is precipitated completely. The SOD activity can reach 129.3U/mg by utilizing gradient elution of PEG precipitation.3.SOD precipitated with PEG is separated effectively from heteroprotein by the column chromatography of DEAE-cellulose(DE-52) and eluted gradualy by pH7.8 0.05mol/L Phosphoric acid(PB) and 1.0mol/L Nacl. The best column chromatography conditions: the flow rate is 1.2ml/min, the rate of diameter to height is 1:10,the added quantity is 6 ml;The time is 33 minute and the elution volume is 40ml when SOD is completely eluted, and the time is 66 minute and the volume is 80 ml when the most heteroprotein are eluted. So the column chromatography of DEAE-cellulose (DE-52) is high effective and differential. Finally, SOD which specific activity is 3145.5U/mg can be obtained.4.SOD has a good stability to heat and pH. The enzymatic activity iscomparatively stable below 60℃, for example the exact being kept in 60℃ for 30 minute, the enzymatic activity only reduces 19.1%;however the temperature raised higher than 70℃, the activity reduces obviously, such as reducing 56.6% on the condition of 70℃ for 30 minute and 70.8% on the condition of 80℃ for 30 minute. The enzymatic activity is stable within pH 4-9; Once exceeding this range , it reduces deeply. For example, the rest activity is 21% on the condition of pH 2 and 49.1% of pH 10. The activity is nothing on the condition of pH 12.5.The SOD from Allium Sativum cell plasma is sensitive to H2O2 and cyanide, so it indicates this type of SOD is copper/zinc-SOD.The absorption apex exhibits at 258nm. it demonstrates the enzyme contains few Tyrosine and Tryptophan.6.The product weighted 15.6mg can be obtained from 500g Allium Sativum thought three methods .The specific activity of purified SOD is 3145.5U/mg, purification times is 197.8 and yield is 39.2% .