Purification and Characterization of Prophenoloxidase from the Hemolymph of Coleopteran Insect, Holotrichia diomphalia Larvae

Prophenoloxidase (pro-PO), a precursor of phenol oxidase (PO), was purified from the hemolymph of coleopteran Holotrichia diomphalia larvae. The enzyme was purified to apparent homogeneity, in six steps of chromatography, using Sephadex G-100, CM-52, Dextran-sulfate Sepharose CL-6B, Phenyl Sepharose CL-4B, Sephacryl S-200, and a Mono-Q column. The preparation exhibited a single band on SDS-PAGE. The proenzyme had a molecular weight of 158 kDa, as estimated by gel filtration. On SDS-PAGE under reducing conditions, it gave a 79 kDa band, indicating that it forms a dimer of the 79 kDa protein. On the other hand, the purified pro-PO gave two well-separated peaks, named proPO-1 and pro-PO-2, on reverse-phase HPLC. Amino acid compositions of both proteins were indistinguishable, thereby suggesting the presence of an allelic variant or an isoprotein. On dextran-sulfate Sepharose CL-6B chromatography, a fraction containing prophenoloxidase activating enzyme(s) (PPAE fraction), free from pro-PO, was also separated. In reconstitution experiments, the activation of purified pro-PO by PPAE fraction was observed in the presence of 5 mM Ca²⁺, with a specific limited proteolysis. The NH₂-terminal sequence of generated PO was determined to be NH₂-Phe-Gly-Glu-Asp-Asp-. The activated PO oxidized o-diphenols but did not oxidize mono-phenol and p-diphenol substrates. The purified pro-PO was not activated by trypsin, α-chymotrypsin, and SDS.