Purification and characterization of GPI-anchored proteins. (A) Delipidated SDS extracts from bloodstream form trypanosomes containing [³H]-labeled VSG before (▪) and after (□) treatment with GPI-PLD were applied to an octyl-Sepharose column (2 mL bed volume) previously equilibrated in 100 mmol/L ammonium acetate containing 5% (vol/vol) 1-propanol. The column was washed with 5 mL of the same buffer (fractions 1 through 10) followed by 5 mL of 100 mmol/L ammonium acetate containing 20% (vol/vol) 1-propanol (fractions 11 through 20), and was eluted with a linear gradient of 20% to 40% (vol/vol) 1-propanol in 100 mmol/L ammonium acetate (fractions 21 through 40). Fractions of 0.5 mL were collected and aliquots were counted for radioactivity. Intact VSG eluted at 31% 1-propanol. After treatment with GPI-PLD, VSG eluted in the column flow-through. Intact procyclin (P) and AChE (A) eluted at 25% and 35% 1-propanol, respectively (as indicated by the arrows), whereas GPI-PLD–treated procyclin and AChE eluted in the same fractions as GPI-PLD–treated VSG. The actual 1-propanol concentration in an individual fraction was determined by measuring the refractive index. (B) Purified radiolabeled VSG and procyclin were treated with large amounts of Pronase to completely digest the protein portions of the molecules followed by repurification by octyl-Sepharose chromatography. Control untreated VSG (lane a) and procyclin (lane c) and the corresponding Pronase reaction products (lanes b and d, respectively) were analyzed by SDS-PAGE and autoradiography.