ECE2014 Poster Presentations Female reproduction (54 abstracts)
Inverse relationship between hSHBG affinity for testosterone and hSHBG concentration revealed by surface plasmon resonance
Laurence Heinrich-Balard 1, , Wael Zeinyeh 1, , Henri Déchaud 1, , Pascaline Rivory 1, , Amandine Roux 1, , Catherine Grenot 1 , Claude Yves Cuilleron 1 , Michel Pugeat 1, & Richard Cohen 1,
1Université de Lyon, Lyon, France; 2MATEIS UMR-CNRS 5510, ISPB Faculté de Pharmacie, F-69373 Lyon, France; 3Hospices Civils de Lyon, Groupement Hospitalier Est, Centre de Biologie Est, Laboratoire d’Hormonologie, Bron, France; 4INSERM U1060 CarMen Institute, Faculté de Médecine Lyon-sud-BP12, Lyon, France; 5Hospices Civils de Lyon, Laboratoire de Biochimie et de Biologie Moléculaire, Lyon, France.
Introduction: Testosterone circulates in the blood mainly bound to albumin and human sex-hormone-binding globulin (hSHBG). It is generally admitted that only the protein unbound fraction of testosterone (free hormone hypothesis) is biologically active. From our routine SHBG binding assay, we observed that SHBG concentration may have an influence on its affinity for testosterone (personal data). To further explore this assumption we used surface plasmon resonance (SPR, Biacore) technic.
Methods: The immobilization of testosterone derivative on the sensorchip CM5 through an oligoethylene glycol linker introduced at the position C-4 was performed by a standard amine coupling. For different plasmas with hSHBG concentrations ranged from 4.4 to 680 nmol/l, an estimation of the equilibrium affinity constant of hSHBG for T (KaSHBG) was obtained by a kinetic analysis from a set of sensorgrams at different analyte concentrations associated with a curve-fitting of these data.
Results: When the total hSHBG concentration was used to perform the fit to the Langmuir 1/1 model or to the heterogeneous ligand model, the measured KaSHBG decreased from 1.0×1010 to 1.7×108 l/mol or from 2.0×1010 to 2.2×107 l/mol respectively, when the total hSHBG plasma concentration increased from 4.4 to 680 nmol/l. This inverse relationship between KaSHBG and concentration was significant for both models: r=0.88, 0.72 and 0.66 for KA, KA1, and KA2 providing respective P values 1.3×10−12, 6.9×10−7 and 1.1×10−5 for Langmuir and heterogeneous ligand model.
Comments: These unexpected results suggested that allosteric effects may modulate the hSHBG binding affinity for testosterone. These findings challenged the paradigm which assumes that each SHBG dimer binds two testosterone molecules with similar binding affinity, and questioned the validity of current recommendations for calculating free testosterone for evaluating androgen disorder in humans.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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