A tetrodotoxin-binding protein in the hemolymph of shore crab Hemigrapsus sanguineus: purification and properties.
The shore crab Hemigrapsus sanguineus hemolymph contains soluble proteins that bind tetrodotoxin (TTX) and are responsible for high resistance of the crab to TTX. The TTX-binding protein was purified from the hemolymph by ultrafiltration, lectin affinity chromatography and gel filtration HPLC. The purified protein gave only one band in native-polyacrylamide gel electrophoresis (PAGE), confirming its homogeneity. Its molecular weight was estimated to be about 400k by gel filtration HPLC, while it was estimated to be about 82k under non-reducing conditions and about 72 and 82k under reducing conditions by SDS-PAGE, indicating that the TTX-binding protein was composed of at least two distinct subunits. The TTX-binding protein was an acidic glycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% amino sugar. The protein selectively bound to TTX, with a neutralizing ability of 6.7 mouse unit TTX/mg protein, but not to paralytic shellfish poisoning toxins. However, its neutralizing activity was almost lost by treatments with enzymes (protease XIV, thermolysin, trypsin, amyloglucosidase and alpha-amylase) and denaturing agents (1% SDS, 1% dithiothreitol, 8 M urea and 6 M guanidine hydrochloride), suggesting the involvement of both proteinaceous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein.
Nagashima
Yuji
Y
Department of Food Science and Technology, Tokyo University of Fisheries, Konan, Minato, Tokyo 108-8477, Japan. yujicd@tokyo-u-fish.ac.jp
Yamamoto
Kazuhiko
K
Shimakura
Kuniyoshi
K
Shiomi
Kazuo
K
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000992:Antitoxins / Q000097:blood* / Q000302:isolation & purification; D003386:Brachyura / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000737:chemistry*; D008970:Molecular Weight; D015222:Sodium Channels / Q000097:blood* / Q000302:isolation & purification; D013779:Tetrodotoxin / Q000037:antagonists & inhibitors / Q000378:metabolism
D000596:Amino Acids; D000992:Antitoxins; D015222:Sodium Channels; C023790:tetrodotoxin-binding protein; D013779:Tetrodotoxin