Fig. 2. Final step of purification of S. haematocheir OHSS. (A) RP-HPLC analysis of the proteins. The active fractions eluted by molecular-sieve chromatography (gel filtration) were concentrated, and the proteins were eluted with a linear gradient of 8%–52% acetonitrile containing 0.1% HCl over 80 min (flow rate at 0.6 ml min^–1). Numbers (1–6) indicate peaks of protein. (B) OHSS biological assay of fractions eluted by FPLC (gel filtration) and RP-HPLC. Bioassay carried out for 1.5 h with two ovigerous setal segments per fraction (see Saigusa and Iwasaki, 1999). C, control assay in distilled water. Values are means ± S.D. (C) Protein analysis by SDS-PAGE. The polyacrylamide gel was stained with Coomassie Brilliant Blue. The marker proteins were ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa; Amersham). Two bands (25 and 22 kDa) appeared in fractions 3–5, while a single band (25 kDa) appeared in fraction 6.