Biotinylinsulins as potential tools for receptor studies

Abstract

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 the N-hydroxysuccinimido ester of biotin to form NaB'-biotinylinsulin. The ability of this modified insulin to stimulate rat epididymal adipocytes was (mean i SD) 94 + 9.6% (PF 0.05) that of the control insulin. NaOB'-Biotinylinsulin displaced 4-hydroxyazobenzene-2'-carboxylic acid from avidin, demonstrating affinity for this protein. The formation of the Na2B'-biotinylinsulin-avidin complex was visualized by cellulose acetate electrophoresis at pH 4. Na6B'-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. The effectiveness of affinity columns for receptor studies is dependent on the highly specific complementarity between hormone and receptor, and consequently the binding region of the hormone should not be modified. The problem of attaching polyfunctional molecules such as insulin to solid supports without disturbing their receptor-binding potential represents a challenging problem which we address in this communication.Insulin was selectively acylated with biotin ( Fig. 1 upper) at the amino terminus of the B chain to form NaB -biotinylinsulin (Fig. 1 lower). This compound, named "biotinylinsulin" for simplicity's sake, was prepared with the expectation that it would exhibit affinity for insulin receptors and also toward the egg-white protein, avidin (for a review, see ref. 1). As a result of this bivalency, it provides the basis for the targeted preparation of selective insulin receptor affinity columns. Such columns result when the biotinylinsulin is added to a solid support containing covalently attached avidin. Fundamentally they are based on the high affinity of biotin for avidin (dissociation constant, KD, approximately 1015).The advantages of the method are: (i) biotin can be attached to the hormone in a targeted manner; (ii) the chemical manipulations are performed with the free hormone, and thus their effect on binding capacity and biological activity can be readily assessed by bioassay; (iii) the attachment of the modified hormone to the support involves a strong noncovalent bond that forms specifically and spontaneously on simple mixing of the Schleicher an...

Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.

“…Insulin We synthesized NaB'-biotinylinsulin (biotinylinsulin) (19) and found that its ability to stimulate lipogenesis in rat epididymal adipocytes was identical to that of insulin. Thus, biotinylation of insulin on the NaB' position does not interfere with receptor binding.…”

Fluorine-18 labeled insulin: a prosthetic group methodology for incorporation of a positron emitter into peptides and proteins

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Induction of antibodies to hyaluronic acid by immunization of rabbits with encapsulated streptococci.

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