Isolation and partial characterization of chromoprotein from the larval hemolymph of the Japanese oak silkworm (Antheraea yamamai).

A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31,000 or 35,000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22,000 or 25,000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31,076 and 21,500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects.