Binding Analysis of 1α- and 17α-Dihydrotestosterone Derivatives to Homodimeric Sex Hormone-Binding Globulin

Jochen Metzger, Andreas Schnitzbauer, Manuela Meyer, Monika Söder, Claude Y. Cuilleron, Hagen Hauptmann, Erasmus Huber, Peter B. Luppa

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26 Scopus citations


Binding studies of the interaction of immobilized 1α- and 17α-aminoalkyl derivatives of 5α-dihydrotestosterone (DHT) with purified N-deglycosylated homodimeric human sex hormone-binding globulin (SHBG) were performed using a surface plasmon resonance biosensor. These 1α- and 17α-derivatives with spacers of appropriate lengths between the amine function and the steroid ring skeleton enabled privileged, sterically undisturbed, interactions of either the 17- or 3-characteristic functional groups of DHT with SHBG. The association constants (Ka1) for the binding of these immobilized DHT derivatives to the first binding site of SHBG, determined by SPR measurements, were 0.16 × 107 M-1 for 17α-aminopropyl-17β-hydroxy-5α-androstan-3-one (1), 1.64 × 107 M-1 for 17α-aminocaproyl-17β -hydroxy-5α-androstan-3-one (2), and 1.2 × 108 M -1 for 1α-aminohexyl-17β-hydroxy-5α-androstan-3-one (3). These values were compared with global Ka data for the corresponding nonimmobilized DHT derivatives from equilibrium measurements using competitions with a tritiated testosterone tracer: the Ka values were 1.25 × 107 M-1 for 1, 1.50 × 107 M-1 for 2, and 140 × 107 M -1 for 3, confirming a remarkably high binding affinity of this latter compound for SHBG. A global fitting analysis of the biosensor data revealed that the interaction of the three immobilized steroids with SHBG was best described by a kinetic model assuming two structurally independent binding sites. This hypothesis of a bivalent binding model was also directly suggested by a dual fluorescent signal observed by the flow cytometry analysis of SHBG immobilized as a hybrid complex binding simultaneously two 1α-aminohexyl DHT ligands, one formed by 3, covalently coupled to phycoerythrin-labeled latex microspheres, and the other by the same DHT derivative, coupled to a fluorescein derivative (4).

Original languageEnglish
Pages (from-to)13735-13745
Number of pages11
Issue number46
StatePublished - 25 Nov 2003
Externally publishedYes


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