Transition state affinity jump chromatography. A double selection method for isolating catalytically active enzymes and other molecules.
A double selection method for isolating active enzyme molecules, using substrate analog affinity chromatography and elution with transition state analogs, is described. To demonstrate the principle, a mixture containing native chymotrypsin and [3H]deoxychymotrypsin, in which the active site serine had been converted to [3H]alanine, was applied to a column containing immobilized D-tryptophan methyl ester. Both forms of chymotrypsin were retained. Catalytically active enzyme was selectively desorbed with the peptide aldehyde chymostatin, leaving catalytically inactive deoxychymotrypsin bound to the substrate analog affinity column. This affinity technique may afford a simple and general method for separating enzymes and other catalysts according to their molecular turnover numbers.
D000409:Alanine; D002846:Chromatography, Affinity / Q000379:methods*; D002918:Chymotrypsin / Q000302:isolation & purification* / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism; D012694:Serine; D014316:Tritium
D004800:Enzymes, Immobilized; D014316:Tritium; D012694:Serine; D002918:Chymotrypsin; D000409:Alanine