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Int. J. Mol. Sci. 2013, 14(2), 3011-3025; doi:10.3390/ijms14023011
Article

2-(2-Hydroxy-5-nitrobenzylidene)-1,3-indanedione versus Fluorescein Isothiocyanate in Interaction with Anti-hFABP Immunoglobulin G1: Fluorescence Quenching, Secondary Structure Alteration and Binding Sites Localization

1,2,* , 2,3
, 3,4
 and 3,*
Received: 17 October 2012; in revised form: 11 January 2013 / Accepted: 15 January 2013 / Published: 31 January 2013
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Abstract: The first step in determining whether a fluorescent dye can be used for antibody labeling consists in collecting data on its physical interaction with the latter. In the present study, the interaction between the 2-(2-hydroxy-5-nitrobenzylidene)-1,3-indanedione (HNBID) dye and the IgG1 monoclonal mouse antibody anti-human heart fatty acid binding protein (anti-hFABP) has been investigated by fluorescence and circular dichroism spectroscopies and complementary structural results were obtained by molecular modeling. We have determined the parameters characterizing this interaction, namely the quenching and binding constants, classes of binding sites, and excited state lifetimes, and we have predicted the localization of HNBID within the Fc region of anti-hFABP. The key glycosidic and amino acid residues in anti-hFABP interacting with HNBID have also been identified. A similar systematic study was undertaken for the well-known fluorescein isothiocyanate fluorophore, for comparison purposes. Our results recommend HNBID as a valuable alternative to fluorescein isothiocyanate for use as a fluorescent probe for IgG1 antibodies.
Keywords: indane-1,3-dione derivative; fluorescein isothiocyanate; immunoglobulin G1; fluorescence spectroscopy; circular dichroism; molecular modeling indane-1,3-dione derivative; fluorescein isothiocyanate; immunoglobulin G1; fluorescence spectroscopy; circular dichroism; molecular modeling
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Stan, D.; Mihailescu, C.-M.; Savin, M.; Matei, I. 2-(2-Hydroxy-5-nitrobenzylidene)-1,3-indanedione versus Fluorescein Isothiocyanate in Interaction with Anti-hFABP Immunoglobulin G1: Fluorescence Quenching, Secondary Structure Alteration and Binding Sites Localization. Int. J. Mol. Sci. 2013, 14, 3011-3025.

AMA Style

Stan D, Mihailescu C-M, Savin M, Matei I. 2-(2-Hydroxy-5-nitrobenzylidene)-1,3-indanedione versus Fluorescein Isothiocyanate in Interaction with Anti-hFABP Immunoglobulin G1: Fluorescence Quenching, Secondary Structure Alteration and Binding Sites Localization. International Journal of Molecular Sciences. 2013; 14(2):3011-3025.

Chicago/Turabian Style

Stan, Dana; Mihailescu, Carmen-Marinela; Savin, Mihaela; Matei, Iulia. 2013. "2-(2-Hydroxy-5-nitrobenzylidene)-1,3-indanedione versus Fluorescein Isothiocyanate in Interaction with Anti-hFABP Immunoglobulin G1: Fluorescence Quenching, Secondary Structure Alteration and Binding Sites Localization." Int. J. Mol. Sci. 14, no. 2: 3011-3025.



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