title: Juvenile hormone esterase purified by affinity chromatography with 8-mercapto-1,1,1-trifluoro-2-octanone as a rationally designed ligand.
abstract: Trifluoromethyl ketones are potent inhibitors of a variety of serine hydrolases. Based on this chemistry improved affinity chromatography procedures were developed for juvenile hormone esterase from insects. New affinity gels were prepared by binding rationally designed ligands to epoxy-activated Sepharose. One ligand is 8-mercapto-1,1,1-trifluoro-2-octanone which has a methylene group replacing a sulfide sulfur beta to the carbonyl of the trifluoromethyl ketone of the previously reported ligand, 3-(4-mercaptobutylthio)-1,1,1-trifluoro-2-propanone. With many loading levels and esterases, the original gel bound enzymes too tightly, resulting in elution difficulties. This replacement of the sulfur beta to the ketone thought to interact with the catalytic serine decreases the binding capacity of the gel at similar loading by approximately 56% compared to the affinity gel with the thioether. However, elution of the enzyme from the column can be accomplished with less potent inhibitors such as 3-n-butylthio- or 3-n-pentylthio-1,1,1-trifluoro-2-propanone, which can easily be removed from the enzyme by dialysis in the presence of the detergent n-octyl beta-D-glucopyranoside. An alternative approach allowing elution with less potent inhibitors involved varying concentrations of the previous high-affinity ligand to optimize the concentration of ligand on the column. Low concentrations of the high-affinity ligand also allowed the use of less potent eluting agents. These two improved affinity chromatography systems have been successfully used to purify juvenile hormone esterase of Heliothis virescens to near homogeneity with a 30-90% recovery of recombinant esterase secreted into the cell media in a baculovirus expression system. The purity of the esterase after affinity chromatography with newly prepared gel was comparable to that produced using the original affinity system based on analyses by SDS-PAGE and isoelectric focusing. A library of affinity gels with ligands of different affinities used at several loading levels and a library of eluting inhibitors of varying potency facilitate the rational selection of conditions for the affinity purification of esterases.
authors.affiliation: Department of Entomology, University of California, Davis 95616.
authors.forename: T
authors.initials: T
authors.lastname: Shiotsuki
authors.forename: T L
authors.initials: TL
authors.lastname: Huang
authors.forename: T
authors.initials: T
authors.lastname: Uematsu
authors.forename: B C
authors.initials: BC
authors.lastname: Bonning
authors.forename: V K
authors.initials: VK
authors.lastname: Ward
authors.forename: B D
authors.initials: BD
authors.lastname: Hammock
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D007659:Ketones; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
chemical_list: C090160:8-mercapto-1,1,1-trifluoro-2-octanone; D007202:Indicators and Reagents; D007659:Ketones; D008024:Ligands; D008823:Micelles; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase