Preparation of a photoactivatable fluorescent derivative of lactose and its application to photoaffinity labeling of a conger eel lectin.
A photoactivatable heterobifunctional fluorescent reagent, 1-azido-5-naphthalene sulfonyl (ANS) hydrazide, was synthesized and characterized. ANS-hydrazide reacted with lactose to form a photoactivatable hydrazone. The derivative (ANS-lactose) had the same binding affinity for a conger eel lectin as lactose judging from the hemagglutinating-inhibition assay with rabbit erythrocytes. ANS-lactose was used for photoaffinity labeling of a conger eel lectin. The photolabeled lectin was digested with chymotrypsin to isolate photolabeled peptides by reversed-phase HPLC by monitoring fluorescence. A major labeled peptide was located at positions 31-45 in the lectin by amino acid analysis and N-terminal sequencing. The identified segment was close to the highly conserved region throughout animal beta-galactoside-binding lectins.
Muramoto
K
K
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
Yamauchi
F
F
Kamiya
H
H
D000345:Affinity Labels / Q000138:chemical synthesis*; D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000494:pharmacology; D004524:Eels; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000276:immunology; D037161:Galectins; D006384:Hemagglutination / Q000187:drug effects; D006385:Hemagglutination Inhibition Tests; D006835:Hydrazones / Q000138:chemical synthesis* / Q000302:isolation & purification; D007785:Lactose / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification; D037102:Lectins / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011817:Rabbits
C088617:1-azido-5-naphthalenesulfonylhydrazide-lactose; D000345:Affinity Labels; D002241:Carbohydrates; D037161:Galectins; D006835:Hydrazones; D037102:Lectins; D010446:Peptide Fragments; C074516:congerin protein, Conger myriaster; D007785:Lactose