Binding behavior of subphthalocyanine-tagged testosterone with human serum albumin at the n-hexane/water interface

Subphthalocyaninatoboron(III) was applied for the first time as a novel marker-tag of testosterone (Subpc-test) for the binding analysis with human serum albumin (HSA) at a liquid/liquid interface. The binding interaction of Subpc-test with HSA at the n-hexane/water interface was studied by UV-visible absorption and circular dichroism (CD) spectroscopy combined with a centrifugal liquid membrane cell at different pHs of aqueous solutions. Complementary studies by a high-speed stirring experiment and an interfacial tension measurement were also performed to characterize the interfacial adsorptivity of Subpc-test and HSA molecules, respectively. The n-hexane solution of Subpc-test showed no optical chirality, but the contact with the aqueous solution of HSA induced its optical chirality, clearly suggesting the formation of Subpc-test/HSA complexes at the n-hexane/water interface. Furthermore, pH profiles of CD signals showed that the interaction between Subpc-test and HSA was very sensitive to the neutral-to-base transition (N-B transition). Interfacial formation of the Subpc-test/HSA complex was studied in the presence of the site-selective ligand, furosemide (site I) or cefaclor (site II). The experimental results suggested that Subpc-test is bound to site I of the HSA molecule at the n-hexane/water interface. This technique using subphthalocyanine as a tag molecule and the liquid/liquid interface as a two-dimensional nanoreaction field will be useful for the evaluation of the interaction between drugs or hormones and proteins.