Two different biliverdin-binding proteins, designated BBP-I and BBP-II, were purified from the larval hemolymph of the
Eri-silkworm, Samia cynthia ricini. These proteins were readily isolated from the hemolymph of fifth instar larvae using
two chromatographic steps, hydrophobic interaction chromatography and ion exchange chromatography. Both BBPs were
easily separated by Q-Sepharose HP column chromatography. BBP-I has an apparent molecular weight of 24 kDa, as
determined by gel-filtration and sodium dodecyl sulfate–polyacrylamide gel electrophoresis SDS–PAGE.. Native BBP-II
had a molecular weight of 48 kDa estimated by gel-filtration. SDS–PAGE revealed a single band with a molecular weight
of 26 kDa. Moreover, the molecular weights of BBP-I and BBP-II were determined to be 20,468 and 22,708 by
MALDI-TOFrMS matrix-assisted laser desorption ionization-time of flightrmass spectrometry., respectively. On this
basis, BBP-I and BBP-II molecules are assumed to be a monomer and a dimer, respectively. The blue color of BBPs
collected from the hemolymph is attributed to the presence of biliverdin IXg , which is non-covalently and stoichiometri-
cally bound to the apoprotein, based on absorbance maxima at 359 and 695 nm in methanol:HCl 95:5, vrv.. One molecule
of BBP-I contains one molecule of biliverdin IXg , whereas BBP-II contains two molecules of biliverdin IXg . The amino
acid compositions of BBP-I and BBP-II are different, although the N-terminal sequences of both BBPs have a 48% identity.
These BBPs were found in the hemolymph of fourth and fifth instar larvae. The newly molted fifth instar larvae had the
highest concentration of BBP-I in the hemolymph. This gradually decreased during larval development. In contrast to
BBP-I, the level of BBP-II was low, and increased slightly at the same developmental stage in S. cynthia ricini larvae.
q1998 Elsevier Science B.V.