Choma C T, Kaplan H
Department of Chemistry, University of Ottawa, Ontario, Canada.
J Invertebr Pathol. 1992 Jan;59(1):75-80. doi: 10.1016/0022-2011(92)90114-j.
The 16 cysteine residues of reduced protoxin from Bacillus thuringiensis subsp. kurstaki HD-73 can be quantitatively reacted with: (a) iodoacetic acid, to give carboxymethyl protoxin; (b) iodoacetamide, giving carbaminomethyl protoxin and (c) N-(beta-iodoethyl)trifluoroacetamide to give aminoethyl protoxin. The carboxymethyl derivative was found to be significantly more soluble at neutral pH values where both the native protoxin and the carbaminomethyl derivative exhibit low solubilities. At the alkaline pH values (pH 9.5-10.5) normally used to solubilize the crystal protein, the native protein was slightly more soluble than either the carboxymethyl or the carbaminomethyl derivatives. The aminoethyl derivative had an extremely low solubility at all pH values. Succinic anhydride reacted with only 35% of the lysine residues in both the carboxymethyl and the carbaminomethyl protoxin derivatives. Nonetheless, these succinylated protoxins exhibited significantly increased solubilities at neutral pH values. All the derivatives were found to retain full insecticidal activity toward spruce budworm (Choristeneura fufimerana) larvae. It is concluded that all the cysteine residues and modified lysine residues are on the surface of the protein and that derivatization does not alter the conformation of the solubilized protoxin.
苏云金芽孢杆菌库斯塔克亚种HD - 73还原原毒素的16个半胱氨酸残基可与以下物质进行定量反应：(a) 碘乙酸，生成羧甲基原毒素；(b) 碘乙酰胺，生成氨甲酰甲基原毒素；(c) N - (β - 碘乙基)三氟乙酰胺，生成氨乙基原毒素。发现羧甲基衍生物在中性pH值下的溶解度显著更高，而天然原毒素和氨甲酰甲基衍生物在该pH值下溶解度较低。在通常用于溶解晶体蛋白的碱性pH值（pH 9.5 - 10.5）下，天然蛋白的溶解度略高于羧甲基或氨甲酰甲基衍生物。氨乙基衍生物在所有pH值下的溶解度都极低。琥珀酸酐仅与羧甲基和氨甲酰甲基原毒素衍生物中35%的赖氨酸残基发生反应。尽管如此，这些琥珀酰化原毒素在中性pH值下的溶解度显著增加。所有衍生物对云杉芽虫（Choristeneura fufimerana）幼虫均保留了全部杀虫活性。得出的结论是，所有半胱氨酸残基和修饰的赖氨酸残基都在蛋白质表面，并且衍生化不会改变溶解的原毒素的构象。
