A biochromatographic approach is developed to measure for the first time thermodynamic data and magnesium (Mg(2+)) effect for the binding of testosterone (TT) to sex hormone-binding globulin (SHBG) in a wide temperature range. For this, the SHBG was immobilized on a chromatographic support. It was established that this novel SHBG column was stable during an extended period of time. The affinity of TT to SHBG is high and changes slightly with the Mg(2+) concentration because the number of Mg(2+) linked to binding is low. The determination of the testosterone retention with the steroid hormone at different Mg(2+) concentrations and temperatures demonstrated that the Mg(2+) binding heat effect associated with this Mg(2+) release or uptake during this binding was in magnitude around 17kJ/mol corresponding to the model describing the electrostatic attraction that occurs between the negatively charged non specific areas of SHBG and the positively charged of magnesium. At all the magnesium concentrations studied, the DeltaH values were negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface confirming strong TT-SHBG hydrogen bond networks. As well, the DeltaS values were all positive due to hydrophobic forces in the testosterone-SHBG complex formation. In addition our results suggest that adaptive conformational transitions contribute to the specific testosterone-SHBG complex formation. As well, in the biological Mg(2+) concentration domain, it was clearly demonstrated that there was an uncompetitive inhibition of Mg(2+) on TT-SHBG binding which led an enhancement of bioavailable TT. Our work indicated that our biochromatographic approach could soon become very attractive for study other SHBG-steroid (or phytoestrogen) binding.