Purification Of Luffin-B From The Seeds Of Luffa Cylindrica And The Studies Of Properties

Luffin-B, which was purified from seeds of Luffa cylindrica, is one of the single -chain ribosome-inactivating proteins of the highest activity so far investigated. But the obtain rate of luffin-B purified by previous methods was no less than 0.01%. I changed some procedure based on previous methods:1. The crude extract of Luffin-B first was purified by ice acetum (pH4.2) precipitation in order to get rid of many acidity proteins , followed by protein precipitation with 50%- 85% saturation ammonium sulfate; 2. After the crude extract was chromatographed on a CM-52 ion exchange chromatography column and Sephadex G-75 gel filtration, Superdex 75 , the high performance gel filtration had been used to purify Luffin-B from the seed extract secondly. 42.92 mg Luffin-B can be obtained from 100 g seeds of Luffa cylindrical. The obtain rate is be close to 0.043% ,which is much higher than that of the luffin by previous methods. This has prepared much for the furthest studies about Luffin-B.The purified Luffin-B could be demonstrated as a single band on SDS-PAGE, estimated to be 28 KDa as was previously reported . It can depurinate RNA in rat ribosome and produce the diagnostic R-fragment after rRNA were treated with acid aniline, which proved that Luffin-B is a typical ribosome-inactivating protein of rRNA N-glycosidase activity. It inhibits the protein synthesis of rabbit reticulocyte lysate strongly with a concentration causing 50% inhibition (IC50) of 2.1×10-11 M, much lower than that of trichosanthin(TCS), which is 2.8×10-10 M.So, it is promising that Luffin-Bthan that of trichosanthin(TCS), which is 2.8×10-10 M. So, it is promising that Luffin-B may be used as an effect toxin moiety of immunotoxins.