Open AccessBiotinylinsulins as potential tools for receptor studies
Author(s) -
Klaus Hofmann,
Frances M. Finn,
Heinz-Jürgen Friesen,
Cornelia Diaconescu,
Helmut Zahn
Publication year - 1977
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.74.7.2697
Subject(s) - avidin , biotinylation , sepharose , biotin , chemistry , biochemistry , affinity chromatography , chromatography , enzyme
The preparation of affinity columns that contain insulin attached to Sepharose in a targeted manner by way of biotin-avidin noncovalent bonds is described. Insulin was acylated selectively at the amino terminus of the B chain with theN -hydroxysuccinimido ester of biotin to formN α,B1 -biotinylinsulin. The ability of this modified insulin to stimulate rat epididymal adipocytes was (mean ± SD) 94 ± 9.6% (P , 0.05) that of the control insulin.N α,B1 -Biotinylinsulin displaced 4-hydroxyazobenzene-2′-carboxylic acid from avidin, demonstrating affinity for this protein. The formation of theN α,B1 -biotinylinsulin-avidin complex was visualized by cellulose acetate electrophoresis at pH 4.N α,B1 -Biotinylinsulin combined with avidin attached to Sepharose to form affinity columns in which the hormone was attached to the support by strong noncovalent bonds. The determination of the loading of avidin-Sepharose columns with biotinylinsulin was greatly facilitated by the attached biotin which provided a marker whose concentration could be assessed accurately by titration with avidin. Biotinylinsulin attached to avidin-Sepharose beads retained the ability to stimulate rat epididymal adipocytes. The activity of several samples of these beads was about 15% that of free biotinylinsulin, based on the amount of biotinylinsulin anchored to the support. The advantages of biotinylated hormones for the targeted attachment of hormones to solid supports are discussed.