A high-affinity juvenile hormone binding protein was purified from the hemolymph of the tobacco hornworm, Manduca sexta, employing ammonium sulfate precipitation and affinity and sizeseparation chromatography. The naturally occurring enantiomer of juvenile hormone III (107?) was converted to juvenile hormone III acid and then covalently attached to aminohexyl-Sepharose 4B. Hemolymph from early fifth stadium (60 h postecdysis) larvae was used as the source of hJHBP. The yield of hJHBP was approximately 25% of the starting material, with 3.5 mg of highly purified, biologically active hJHBP recovered from 100 mL of hemolymph. Binding parameters were examined using equilibrium dialysis and highly purified, enantiomerically correct juvenile hormone I and II and racemic JH III. The equilibrium dissociation constants for juvenile hormone I and II were approximately 6 X 10-10 M at 4 °C, while racemic juvenile hormone III displayed an equilibrium dissociation constant of 1.9 X 10~9M. At25 °C the equilibrium dissociation constant for juvenile hormone I was 1.6 X 10-9 M. Half-times of dissociation were also determined for the three homologs.

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