Hannam C, Lange G L, Mellors A
Department of Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
Anal Biochem. 1998 May 1;258(2):246-50. doi: 10.1006/abio.1998.2631.
Radiolabeling permits the detection of trace amounts of zwitterionic detergent remaining in extracted hydrophobic or membrane proteins. To develop a sensitive and specific assay for its presence, the commonly used zwitterionic membrane protein detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) was synthesized in a tritiated form. Synthesis via 7-ketodeoxycholic acid gave [7-3H]Chaps in 53% yield with a specific activity of 0.85 mCi/mmol. A novel solvent extraction system for cholic acid obviated the need for chromatographic isolation of this intermediate. The protocol can be readily modified to yield [7-3H]Chaps of higher specific activity. [7-3H]Chaps was used to monitor the efficiency of various strategies for detergent removal from concentrated bacterial culture supernatants containing 0.2% (w/v) Chaps. Dialysis removed 95% of Chaps and the addition of detergent-affinity beads to the dialysis buffer resulted in 97% removal of Chaps. Gel-filtration chromatography removed 99.9% of Chaps, while a detergent-affinity bead chromatography column removed 99.99%. Overall, gel-filtration chromatography was the most convenient and economical method for the one-step removal of the zwittergent from complex biological mixtures.
放射性标记能够检测出提取的疏水蛋白或膜蛋白中残留的痕量两性离子去污剂。为开发一种针对其存在的灵敏且特异的检测方法，常用的两性离子膜蛋白去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（CHAPS）被合成了氚标记形式。通过7-酮去氧胆酸合成得到[7-³H]CHAPS，产率为53%，比活度为0.85 mCi/mmol。一种用于胆酸的新型溶剂萃取系统避免了对该中间体进行色谱分离的需求。该方案可轻松修改以获得更高比活度的[7-³H]CHAPS。[7-³H]CHAPS被用于监测从含有0.2%（w/v）CHAPS的浓缩细菌培养上清液中去除去污剂的各种策略的效率。透析去除了95%的CHAPS，向透析缓冲液中添加去污剂亲和珠导致97%的CHAPS被去除。凝胶过滤色谱去除了99.9%的CHAPS，而去污剂亲和珠色谱柱去除了99.99%。总体而言，凝胶过滤色谱是从复杂生物混合物中一步去除两性离子去污剂最方便且经济的方法。
