title: Fluorescence spectroscopic studies on tryptophan at the saccharide-binding site of castor bean hemagglutinin.
abstract: The environment of tryptophan in castor bean hemagglutinin (CBH) was analyzed by fluorescence spectroscopy with regard to saccharide binding. Upon binding of specific saccharides, the fluorescence maximum of 333 nm of CBH shifted to a wavelength 2 nm shorter, owing to the change in the environment of tryptophan at the saccharide-binding site. By analyzing the change in the fluorescence intensity at 320 nm as a function of concentration of saccharides, the association constants for binding of saccharides to CBH were determined. The results suggest that the saccharide-binding site on each B-chain is actually composed of a subsite with which the saccharide residue linked to galactopyranoside at the non-reducing end can interact, and another site which recognizes the galactopyranoside moiety. Quenching data indicated that five out of 22 tryptophans in CBH are surface-localized and are available for quenching with both KI and acrylamide, and three other tryptophans are buried and are available only to acrylamide. Binding of raffinose to CBH decreased by 2 the number of tryptophan residues accessible to quenchers in the CBH molecule. We speculate that raffinose binds to CBH in such a manner as to shield the tryptophan located at the subsite from quenching by KI and acrylamide. The results also suggest that the tryptophan residue at the saccharide-binding site on each B-chain is localized near the surface, and present in the positively charged environment.
authors.affiliation: Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Yamasaki
authors.forename: N
authors.initials: N
authors.lastname: Absar
authors.forename: G
authors.initials: G
authors.lastname: Funatsu
mesh_terms: D000178:Acrylamides; D002352:Carrier Proteins; D011956:Receptors, Cell Surface; D012276:Ricin; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan
chemical_list: D000178:Acrylamides; D002352:Carrier Proteins; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins; D014364:Tryptophan; D012276:Ricin