Purification and characterization of an isoform of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii

Isolation of the crustacean hyperglycemic hormone (CHH) from the eyestalk o f the giant freshwater prawn Macrobrachium rosenbergii was performed using 5,000 ground eyestalks extracted in methanol-acetic acid-water (90:1:9). Af ter the extract was partially purified using C18 cartridges, it was further purified by eight steps of RP-HPLC using four kinds of columns: C18, C8, c yano and phenyl, and three solvent systems: acetonitrile (ACN)/trifluoroace tic acid, ACN/heptafluoroacetic acid and ACN/triethylammonium acetate. The bioassay of CHH during purification was done by injection of eluate fractio ns into eyestalk-ablated prawns and determination of the ability of the fra ctions to elevate glucose in the haemolymph. A complete amino acid sequence analysis was performed on one isoform of CHH (Mar-CHH-1), consisting of 71 residues. The sequence of Mar-CHH-1 shows considerable similarity (45-68%) to CHHs reported in other crustaceans. It is expected that there might be more than one isoform of CHH in M. rosenbergii. J. Exp. Zool. 284:217-224, 1999. (C) 1999 Wiley-Liss, Inc.