An assay for lectin activity using microtiter plate with chemically immobilized carbohydrates.
A simple microtiter plate assay for lectins or carbohydrate-binding proteins was developed. The method utilizes carbohydrates immobilized in the wells of the microtiter plate containing primary amino groups on their surface. After incubation of the lectins, bound proteins are measured by the protein assay using the colloidal gold solution. When the binding of Ricinus communis agglutinin, concanavalin A, and wheat germ agglutinin was measured using the microtiter plate wells coated with lactose, mannose, or N-acetylglucosamine, binding of the lectins according to their known specificity was observed. Inhibition experiments with various carbohydrates also demonstrated that the specificity of lectins for different carbohydrates could be determined quantitatively. Since there is no need for modification of the lectins, such as biotinylation or conjugation with marker enzymes, the carbohydrate-binding ability of intact proteins can be easily determined by this method. When gel filtration fractions from the extract of the marine invertebrate Cucumaria echinata were subjected to this assay, different carbohydrate-binding activities were observed with different elution profiles, suggesting that this assay could also be widely applicable for the simultaneous detection of lectins from various sources.
Hatakeyama
T
T
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Fukuoka, 812-81, Japan.
Murakami
K
K
Miyamoto
Y
Y
Yamasaki
N
N
D000818:Animals; D001826:Body Fluids / Q000737:chemistry; D050260:Carbohydrate Metabolism; D002241:Carbohydrates; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D005069:Evaluation Studies as Topic; D017979:Gold Colloid; D037102:Lectins / Q000032:analysis* / Q000378:metabolism; D008722:Methods; D012616:Sea Cucumbers
D002241:Carbohydrates; D002352:Carrier Proteins; D017979:Gold Colloid; D037102:Lectins