J. Magee et al., PROPERTIES AND SIGNIFICANCE OF A RIBOFLAVIN-BINDING HEXAMERIN IN THE HEMOLYMPH OF HYALOPHORA-CECROPIA, Archives of insect biochemistry and physiology, 25(2), 1994, pp. 137-157
A riboflavin-binding hexamerin isolated from pupal hemolymph of Hyalop
hora cecropia has a native M(r) of 510,000, subunit M(r) of 85,000, an
d a 5% carbohydrate content. An intrachain cross-link was confirmed in
protease limit digests. Ellman titration confirmed the presence of a
sulfhydryl group, which is needed for this linkage. Though CU2+ is kno
wn to promote the linkage, heavy metals were not detected in the isola
te. Heat denaturation released ligand with the absorbency, fluorescenc
e spectra, and chromatographic behavior of riboflavin. Binding resulte
d in substantial quenching of the fluorescence of both the isoalloxazi
ne in riboflavin and of aromatic groups in the apoprotein. Kinetic ana
lysis indicated a K-D of 2.5x (-7) M for riboflavin, 1.3 x 10(-7) M fo
r lumiflavin, and greater than 1 x 10(-6) M for 10 FMN and FAD. Over f
our moles of flavin were bound per mole of hexamerin. The amount of ri
boflavin in pupal hemolymph is sufficient to occupy only 2-3 of these
sites. Riboflavin is also associated with lipophorin and vitellogenin,
but the molar ratios after protein isolation were low. On a standard
laboratory diet, riboflavin is in great excess, but most of it is appa
rently excreted before the apoprotein first appears in the hemolymph,
just before wandering. The concentration of riboflavin-binding hexamer
in rises to 15-30 mg/ml in pupae; relative to other hexamerins, very l
ittle is stored in the fat body. All of the apoprotein and 75% of ribo
flavin disappear from the hemolymph during adult development. An amoun
t of flavin at least equal to that stored in pupal hemolymph is transf
erred to the eggs formed during this period. (C) 1994 Wiley-Liss, Inc.