RAPID PURIFICATION AND N-TERMINAL AMINO-ACID-SEQUENCE OF A PHOTOAFFINITY-LABELED JUVENILE-HORMONE BINDING-PROTEIN FROM AN ARCTIID MOTH LARVA, PLATYPREPIA-VIRGINALIS

A novel two-step procedure has been developed for the purification of juvenile hormone binding proteins (JHBP) from caterpillars. Crude hemolymph was photoaffinity labeled with [H-3]EHDA, a JH II analog. After removal of excess ligand, 40 ml of buffer-diluted hemolymph containing over 200 mg protein was submitted to preparative isoelectric focusing (IEF) using a Rotofor device. After removal of ampholytes by dialysis, the H-3-labeled fractions were purified to > 95% homogeneity by anion-exchange HPLC. Over 1000-fold purification could be achieved in a few days on a scale which provides 100-1000-mu-g of purified JHBP. Proteins thus obtained can be used for proteolytic digestion or can be sequenced after electroblotting from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel onto a polyvinylidene fluoride (PVDF) membrane. This protocol is illustrated for the purification and N-terminal amino acid sequencing of a hemolymph JHBP from an arctiid wooly bear caterpillar, Platyprepia virginalis.