The synthesis of biotinyl-hexaglycyl-NEDA (abbreviation:BGN), a biotinyl derivative of propranolol, is described. This bifunctional molecule binds with high affinity to the biotin-binding protein, avidin. The duck erythrocyte was used as a model 0-receptor system. Formation of an avidin-BGN-0-receptor complex was demonstrated in intact erythrocytes, in erythrocyte ghosts, and in the digitonin-solubilized 0-receptor. The avidin-BGN complex will be used for localization and purification of the 0-receptor.
Key words: avidin-biotin complex, (3-adrenergic antagonist, (3-adrenergic receptor, adenylate cyclasc, duck erythrocytes, biotinyl derivatives of hormones
The distribution of hormone receptors 011 cell surfaces may provide important information on the mechanism by which hormones function. To this end, biotinyl derivatives of hormones and other ligands are potentially useful tools in receptor research. Avidin, a protein isolated from egg white, binds with very high affinity to biotin and biotin derivatives [ l ] . Avidin can be labeled with fluorescein [2] and ferritin [3] for use in receptor localization. In addition, avidin-Sepharose columns may be prepared [4] for affinity chromatography of receptors bound t o biotinyl ligands. While this work was under way, the syntheses of biotinyl derivatives of insulin were reported [ S , 61. This paper describes the synthesis and characterization of a biotinyl derivative of the 0-adrenergic antagonist propranolol. This compound will be utilized for the localization and purification of the 0-receptor.
MATERIALS AND METHODS
Ma ter ia Is
Avidin, (+)-biotin, L-isoproterenol-D-bitartrate, 4-hydroxyazobenzene-2'-carboxylic acid, N-hydroxysuccinimide, and hexaglycine were obtained from the Sigma Chemical Co. 1,l '-Carbonyldiimidazole was obtained from Aldrich Chemical Co. ["C] -Biotin and