Purification and Characterization of Multiple-Charged Forms of Permethrin Carboxylesterase(s) from the Hemolymph of Resistant Colorado Potato Beetle

Abstract

Permethrin carboxylesterases (e.g., pI4.2–4.8 CbEs) have been purified from the hemolymph of a permethrin-resistant strain of Colorado potato beetle,Leptinotarsa decemlineata, through several chromatographic procedures in order to determine those proteins most responsible for the DEF-synergized aspect of resistance. The pI4.8 CbE is a 46- to 48-kDa monomeric protein whereas the pI4.5 CbE appears to be a 57- to 59-kDa dimeric protein. Both the pI4.8 CbE and the pI4.5 CbE are, in fact, composed of groups of multiple-charged forms. All pI4.5–4.8 CbEs are glycoproteins but the charge heterogeneity is not associated withN-glycan moieties. A consistent tendency of increasing affinities for the pI4.2–4.8 CbEs as the hydrophobicity of naphthyl substrate increased implicates that the catalytic sites of all the pICbEs have hydrophobic properties. The hydrophobic catalytic site, however, is separate entity from the domain determining overall surface hydrophobicity of CbE as determined by hydrophobic interaction chromatography of the purified pI4.5–4.8 CbEs. Overall, pI4.8 CbE possessed the highest affinity and catalytic efficiency for all three naphthyl substrates tested and for permethrin. The hydrolysis rates for permethrin, however, were extremely low compared to those of the naphthyl substrates. Nevertheless, the over production and the high affinity toward hydrophobic substrates of the permethrin CbE in the hemolymph in the permethrin-resistant strain of Colorado potato beetle is likely to result in efficient hydrolysis of permethrin. In general, the PI4.8 and 4.5 CbEs share a number of similarities in their biochemical properties and functional role in permethrin resistance despite their distinct molecular properties. The availability of highly purified and characterized forms of permethrin CbE will allow the development of efficient immunochemical and DNA-based diagnostics for the monitoring of permethrin resistance in field populations of beetles.