Abstract
A derivative of methoxypolyethylene glycol (mPEG2) was covalently attached to ricin D, and some properties of the resulting conjugate (niPEG2-ricin D) were studied. The conjugate was prepared by modification of ricin D with the TV-hydroxysuccinimide ester of mPEG2 [Yamasaki et al., Agric. Biol. Chem., 52, 2125 (1989)], and purified by DEAE-cellulose chromatography, followed by gel filtration on a Sephadex G-75 column. Analytical data indicated that in the conjugate, 9 mol of mPEG2 with an average molecular weight of 10, 000 were covalently attached to the ricin D molecule. The cytoagglutinating activity of mPEG2-ricin D for Sarcoma 180 ascites tumor cells was 11% of that of native ricin D. While ricin D was insolubilized in frozen acidic solution at - 20°C accompanied by the loss of the cytoagglutinating activity irrespective of the coexistence of free mPEG2, the conjugate was not insolubilized in frozen acidic solution and its cytoagglutinating activity was completely retained. An acetylated derivative of ricin D in which 7 amino groups/mol were modified with acetic anhydride was insolubilized in frozen solution similar to native ricin D. The insusceptibility of mPEG2-ricin D to freeze-insolubilization may be due to the covalent attachment of the amphipathic copolymer, mPEG2, to the amino groups of ricin D, but not due to the decrease in the positive charge by modification of amino groups.
