Purification And Biochemical Characterization Of A Fibrinolytic Protein From Black Ants

Ant which has a big variety, a large number of widely distribution and abundant resources,is one of the world’s largest and most prosperous insects,and it has a longhistory,according to the statistics it exists about 100 million years on the Earth. It has manyfeatures such as strong vitality, long life, nutrient-rich. Contains high protein,many essential amino acids, trace elements and minerals. Therefore,the ant not only has high food and nutritional value,but also has a very unique health and medicinal value.,Nowadays,more and more people like it. With the continuous development of social and economic, the edible and medicinal values of the ant also constantly are being developed and very broad application prospects.This was the first time to isolate and purify a Fibrinolytic Protein by ammonium sulfate fractionation, DEAE-52 ion-exchange and SephacrylS-l OOHR gel-filtration chromatography from the black ants, and then determined its fibrinolytic activity by fibrinplate, determined molecular weight by SDS-PAGE gel-electrophoresis analyze, determined the Protein concentr ationby Braford, measured the sugar content by anthrone sulfuric acid method,.The results showed that the protein salting optimal saturation concentration was 30% ?90%,and it had a strong fibrinolytic activity, the enzyme activity was 101.173 5U/ml,after Calculating,The protein from black ants was equivalent to 67.4552 urokinase international standards for energy units,the active protein showed an molecular weight of about 25 kD, the protein concentration was 2.8041mg/ml,the sugar content was 8.1714%.The study showed that different temperature,pH value have different effects o on the black ants fibrinolyticprotease activity. The results showed that the enzyme optimum temperature was 45。C,that meaned this protein had apoor heat resistance,it had the highest activity at pH3.0,but this enzyme was stable atpH from 4.0 to 8.0.In addition,this experiment also studied the impacts of a series of physical and chemical factors on the purpose protease activity.Experiment showed that:(1) different metal ions had different impacts on this fibrinolytic protein,and the degrees was varying.Na、K+ had little effect on its activity, had activation apparentlgnificantly inhibited byZii2+.Ba2+.Mn2+.Ca2+(2)differeiit inhibitors, surfactants and denatured agents affected the black ants fibrinolytic protein activity in different degrees.The erythromycin almost had no effect on fibrinolytic activity,while less affected by heparin;Penicillin had a weak role in promoting its activity,while PMSF has a strong inhibitory effect on its activity;However,p-mercaptoethanol,Tween-20,DFP,SDS were the strong inhibition of this enzyme activity;(3)the effects of ultrasound and UV on black ants fibrinolytic activity were different-Under the effect of ultrasound,with the extension of the ultrasonic frequency and processing time increasing, the black ants fibrinolytic enzyme activity significantly reduced.Since the same power,the longer time it was,the greater by inhibiting,the activity decreased more obvious;the same time,the greater power it was,the inhibition effect was more obvious.Under radiation,the enzyme activity decreased more significantly with the growing period.