Pyrene maleimide as a probe of microenvironmental and dynamics properties of protein binding sites

S. Benci; S. Vaccari; G. Schianchi; Donata Locatelli; P. Vaghi; Giovanni F. Bottiroli

doi:10.1117/12.198721

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4 January 1995 Pyrene maleimide as a probe of microenvironmental and dynamics properties of protein binding sites

S. Benci; S. Vaccari; G. Schianchi; Donata Locatelli; P. Vaghi; Giovanni F. Bottiroli

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Proceedings Volume 2324, Optical Biopsy and Fluorescence Spectroscopy and Imaging; (1995) https://doi.org/10.1117/12.198721
Event: International Symposium on Biomedical Optics Europe '94, 1994, Lille, France

Abstract

N-(1-Pyrene)maleimide is highly fluorescent upon covalent binding with sulfhydryl and amino groups of the proteins. Multiexponential fluorescence decays were observed for the dye bound to different proteins even when a single binding site is involved. The lack of information about the fluorescence decay of free dye does not allow to define the variations of fluorescence parameter following the conjugation and their correlation with the binding properties of the fluorophore. In this work, a study of the fluorescence of the probe, free in solution, bound to different antibodies and to the antigen-antibody complex both in solution and in cell, has been performed. The experimental results showed that chemico-physical properties of the medium influence the fluorescence decay of the probe in both the free and bound forms, although to a different extent. The variations of fluorescence decay and anisotropy of the bound probe are related to the electronic characteristics of microenvironment and show an increased stabilization of the probe binding site with the increasing complexity of the substrate. The sensitivity of the fluorescence properties of the probe to the binding site environment opens interesting perspectives concerning the application of Py- maleimide fluorochromization to assess the degree of specificity of immunocytochemical labelling.

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S. Benci, S. Vaccari, G. Schianchi, Donata Locatelli, P. Vaghi, and Giovanni F. Bottiroli "Pyrene maleimide as a probe of microenvironmental and dynamics properties of protein binding sites", Proc. SPIE 2324, Optical Biopsy and Fluorescence Spectroscopy and Imaging, (4 January 1995); https://doi.org/10.1117/12.198721