FIGURE 2.

SFP uses multiple substrates but newly collected plasma contains only Tyr. A, melanization rate of SFP. Plasma was prepared in GSH (5 mm final) to block melanization and then washed three times on a 30-kDa filter to prepare SFP. SFP (20 μl) was diluted in PBS (80 μl) along with substrate (0.5 mm final) and monitored for melanin formation at A₄₇₀. B, fractionation of substrates in plasma by HPLC. Plasma from a fifth instar was boiled for 1 min, cooled on ice, and centrifuged to remove precipitate, and the supernatant was separated by HPLC. The dark line indicates the timing of when peaks from plasma eluted, whereas the light line indicates the timing when peaks corresponding to the tyrosine, DOPA, and dopamine standards eluted. Collected fractions (0.5 ml) were lyophilized, resuspended in PBS, and tested for melanizing activity using B. mori SFP. The active fraction at 21 min co-eluted with a tyrosine standard. The tyramine standard also eluted at 21 min (not shown; see “Results”). Inset, absorbance spectrum of the 21 min peak with and without a co-injected tyrosine standard.