Intact sea urchin spermatozoa were successfully biotinylated with NHS-LC-Biotin and the biotinylated spermatozoa retained the viability. Analysis of the membrane prepared from the biotinylated spermatozoa of the sea urchin Hemicentrotus pulcherrimus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that several proteins such as wheat-germ agglutinin (WGA)-binding protein (220 kDa), guanylyl cyclase (131 kDa), sperm-activating peptide-l (SAP-l)-crosslinking protein (71 kDa), GPI-anchored protein (63 kDa) and functionally unknown proteins (50 kDa and 30 kDa) were specifically biotinylated. Experiments using spermatozoa of sea urchins, Anthocidaris crassispina and Clypeasterjaponicus showed that several proteins similar to those of H. pulcherrimus spermatozoa were also labeled with NHS-LC-Biotin.

Fucose sulfate glycoconjugate (FSG) isolated from the jelly coat of H. pulcherrimus was mixed with solubilized biotinylated sperm membrane proteins of H. pulcherrimus, A. crassispina or C. japonicus and then subjected to gel filtration chromatography on a Sepharose 2B column, indicating that only two biotinylated H. pulcherrimus sperm proteins were coeluted with H. pulcherrimus FSG.