AFFINITY PURIFICATION AND BINDING ANALYSIS OF THE HEMOLYMPH JUVENILE-HORMONE BINDING-PROTEIN FROM MANDUCA-SEXTA

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 size-separation chromatography. The naturally occurring enantiomer of juvenile hormone III (10R) was converted to juvenile hormone III acid and then covalently attached to aminohexyl-Sepharose 4B. Hemolymph from early fifth stadium (60 h pos tecdysis) larvae was used as the source of hJHBP. The yield of hJHBP w as approximately 25% of the starting material, with 3.5 mg of highly p urified, biologically active hJHBP recovered from 100 mL of hemolymph. Binding parameters were examined using equilibrium dialysis and highl y purified, enantiomerically correct juvenile hormone I and II and rac emic JH III. The equilibrium dissociation constants for juvenile hormo ne I and II were approximately 6 x 10(-10) M at 4-degrees-C, while rac emic juvenile hormone Ill displayed an equilibrium dissociation consta nt of 1.9 x 10(-9) M. At 25-degrees-C the equilibrium dissociation con stant for juvenile hormone I was 1.6 x 10(-9) M. Half-times of dissoci ation were also determined for the three homologs. The half-time of di ssociation was 30 s for juvenile hormone I, 20 s for juvenile hormone II, and 13 s for juvenile hormone III at either 4 or 25-degrees-C. Usi ng the new equilibrium dissociation constants, we calculate that bette r than 99% of the circulating juvenile hormone titer may be bound to t his hemolymph protein.