FIGURE 6.

Collection of plasma in PTU enhances formation of the high mass complex in fraction 1. A, PTU blocks formation of most PO-containing complexes in plasma. PTU (10 μm to 1 mm) was added to newly collected plasma and then subjected to SDS-PAGE and immunoblot analysis as described in the legend to Fig. 5B. Numerous PO-containing high mass bands are visible in the plasma sample with no PTU, whereas most of these bands are absent in the sample with 1 mm PTU. B, plasma prepared with and without PTU (1 mm) was separated by gel filtration chromatography as outlined in the legend to Fig. 4. Fraction 1 was then added to a 4–20% SDS-polyacrylamide gel under reducing conditions, followed by immunoblotting using α-PPO1/2. The PO-containing high mass complex (arrow) is clearly more visible in fraction 1 from plasma where PTU was added than in fraction 1 from plasma without PTU. In contrast, the abundance of monomeric PPO and PO is similar. Mass markers are indicated to the left. C, melanization activity of fraction 1 from plasma samples with or without PTU (1 mm) using DA or Tyr as the substrate at a final concentration of 0.5 mm. Melanization activity was measured by monitoring dopachrome or dopaminechrome formation at A₄₇₀.