Synthesis of a radiolabeled zwitterionic detergent and its use in protein purification.
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.
Hannam
C
C
Department of Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
Lange
G L
GL
Mellors
A
A
D002793:Cholic Acids / Q000138:chemical synthesis* / Q000737:chemistry; D002850:Chromatography, Gel; D003902:Detergents / Q000138:chemical synthesis* / Q000737:chemistry; D015394:Molecular Structure; D011506:Proteins / Q000302:isolation & purification*
D002793:Cholic Acids; D003902:Detergents; D011506:Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate