CHARACTERIZATION OF A SPECTROPHOTOMETRIC ASSAY FOR JUVENILE-HORMONE ESTERASE

Two surrogate substrates, methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-hexylthioacetothioate (HEXTAT) were utilized to compare a ne w spectrophotometric assay with the standard radiochemical partition a ssay used to quantify juvenile hormone esterase (JHE) activity, The su rrogate substrates were made with one common factor being a thiol este r moiety substituting for the ester moiety found in juvenile hormones (JHs) and a thioether replacing the 2,3-olefin of the SHs, As a result , nucleophilic attack by the serine residue of JHE at the carbonyl fun ctional group results in a hydrolytic reaction and release of methanet hiol. In the presence of Ellman's Reagent (DTNB) methanethiol will cle ave the disulfide bond of DTNB resulting in a chromophore detectable a t 405 nm, Methyl 1-hexylthioacetothioate and its oxygen ester analogue , methyl-1-hexylthioacetate, were compared for JHE activity, Statistic al analysis of the slopes indicated a very small but significant diffe rence between the hydrolytic rates for the thiol ester and oxygen este r. However, the data indicate that thiol esters can replace oxygen est ers to quantify hydrolytic activity by the JHEs examined. Results gath ered from different preparations of JHE including tissue culture media from a baculovirus expression system, affinity- and DEAE-purified enz yme, as well as insect hemolymph indicate an excellent correlation bet ween the two assays. Isoelectric focusing of pure and crude JHE prepar ations resulted in coinciding peaks of hydrolytic activity when using the standard partition assay and the spectrophotometric assay, with no other peaks of activity found in the crude preparations with either s ubstrate. Several esterase bands were found at different isoelectric p oints when gels were stained with alpha-naphthyl acetate. Substrates w ere further characterized by monitoring specific activity of SHE colle cted from hemolymph of larvae of Manduca sexta and Heliothis virescens .