Ribosome-inactivating proteins (RIPs) are RNA N-glycosidase enzymes, which are isolated from seeds of many plants. RIPs remove a site-specifically single adenine residue (A 4324 in the case of rat liver ribosome) from the highly conserved sarcin/ricin loop of the 28S rRNA, thus arresting protein synthesis.1,2) RIPs are conventionally classified into three types: type I, II and III based on the conformation of their subunits.2) Type I RIPs are single-chained proteins with enzymatic activity and type II RIPs consist of a catalytic chain linked to a lectin binding chain. Type III RIPs, which are identified mostly in maize and barely, consist of an N-terminal active chain linked to an unrelated C-terminal domain with unknown function.Almost plants of the Cucurbitaceae family are found to contain several type I RIPs in the same species such as Trichosanthin and TAP-29 from Trichosanthes kirilowii, 3,4) Luffin-a and Luffin-b from Luffa cylindrica, 5,6) Bryodin I and Bryodin II from Bryonia dioica, 7) a-Momorcharin and bMomorcharin from Momordica charantia. 8) Type I RIPs are highly basic proteins with pI values of 9-10 and display molecular weight of 26-30 kDa. The proteins from the Cucurbitaceae plants exhibit an array of biological activities including anti-HIV-1, 4,9) immunomodulatory, 10) antiproliferative, 10) deoxyribonuclease 11) and ribonuclease.12) It has recently been described that Momordica cochinchinensis consisted of two isoforms of type I RIPs: namely Momorcochin from root tubers and Momorcochin-S from seeds with molecular mass of approximately 32 kDa and 30 kDa, respectively. 13,14) The proteins are highly basic and have anti-tumor activities. In the present investigation, we reported a novel type I RIPs named as Cochinin B, which was isolated from the seeds of M. cochinchinensis. Cochinin B possessed all typical properties of type I RIPs with a broad range of potent anti-tumor activities. MATERIALS AND METHODSPurification of Cochinin B Ripe seeds of M. cochinchinensis (collected from Nakornpathom, Thailand during April-November 2002) were decorticated in a mortar and a pestle. Endosperm was separated and mixed in ice-cooled 10 mM sodium phosphate buffer pH 6.3 containing 150 mM NaCl and 5 mM EDTA. The mixture was homogenized by a blender and the homogenized sample was filtered through double cheesecloth and centrifuged at 15000 rpm for 30 min at 4°C. After filtering through filter paper, the clear solution was added with 30-60% saturation of ammonium sulfate, stirred for 1 h, and centrifuged at 8000 rpm for 15 min at 4°C. The precipitate was dissolved and dialyzed against 10 mM sodium phosphate buffer pH 6.3 and then applied to a fast protein liquid chromatography (FPLC) system with a SP Sepharose, cation-exchange column (1 cmϫ10 cm), which had previously been equilibrated with the same buffer. The column was initially eluted with the same buffer to remove unadsorbed proteins and subsequently with a linear gradient of 0-1.0 M NaCl in 10 mM sodium phosphate buffer pH 6.3. Fractions that contained 28 k...