Abstract
Fluorescence spectroscopy has long been used to characterize the equilibrium binding of retinoids to proteins (1). Changes in steady-state fluorescence are monitored as the protein is titrated with aliquots of retinoid. The resultant binding curve can be analyzed yielding information about the stoichiometry and affinity of retinoid binding. There are several advantages to this approach. For one, this is a true equilibrium technique. Binding is monitored directly, with no need to physically separate bound from free ligand. A second advantage is the high sensitivity of fluorescence. Titrations can be routinely performed on as little as 2.5 mL of 10−6-10−7 M CRABP. Another advantage is that no alteration of the retinoid or protein is necessary. The fluorescence signals are intrinsic to the native protein and retinoid.
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References
Cogan, U, Kopelman, M., Mokady, S, and Shinitzky, M. (1976) Binding affinities of retinol and related compounds to retinol binding binding proteins. Eur J Biochem. 65, 71–78.
Stryer, L. (1978) Fluorescence energy transfer as a spectroscopic ruler Ann Rev Biochem. 47, 819–846.
Birdsall, B., King, R. W., Wheeler, M. R., Lewis Jr., C. A., Goode, S. R., Dunlap, R. B., and Roberts, G. C. K. (1983) Correction for light absorption in fluorescence studies of protein-ligand interactions Anal. Biochem. 132, 353–361.
Norris, A W., Cheng, L, Giguère, V., Rosenberger, M, and Li, E. (1994) Measurement of subnanomolar retinoic acid binding affinities for cellular retinoic acid binding proteins by fluorometric titration Biochim. Biophys. Acta 1209, 10–18
Cheng, L, Norris, A. W., Tate, B. F., Rosenberger, M., Grippo, J. F, and Li, E. (1994) Characterization of the ligand binding domain of human retinoid X receptor α expressed in Escherichia coli. J. Biol. Chem. 269, 18,662–18,667
Glatz, J F. C., and Veerkamp, J. H. (1983) Removal of fatty acids from serum albumin by Lipidex 1000 chromatography J Biochem. Biophys. Meth. 8, 57–61.
Lowe, J B., Sacchettini, J C., Laposata, M., McQuillan, J J., and Gordon, J I. (1987) Expression of rat intestinal fatty acid-binding protein in Escherichia coli J Biol Chem 262, 5931–5937
Lohman, T. M., and Mascotti, D. P. (1992) Nonspecific ligand-DNA equlibrium binding parameters determined by fluorescence methods. Methods Enzymol 212, 424–458.
Szuts, E. Z., and Harosi, F. I. (1991) Solubility of retinoids in water. Arch Biochem. Biophys 287, 297–304
Halfman, C. J., and Nishida, T (1972) Method for measuring the binding of small molecules to proteins from binding-induced alterations of physical-chemical properties. Biochemistry 11, 3493–3498.
Straume, M., and Johnson, M. L (1992) Analysis of residuals criteria for determining goodness-of-fit Methods Enzymol. 210, 87–105
Johnson, M L., and Frasier, S G (1985) Nonlinear least-squares analysis Methods Enzymol. 117, 301–342.
Lohman, T. M, Chao, K., Green, J. M., Sage, S, and Runyon, G T (1989) Largescale purification and characterization of the Escherichia coli rep gene product J Biol. Chem. 264, 10,139–10,147.
Gill, S C and von Hlppel, P H (1989) Calculation of protein extinction coefficients from amino acid sequence data Anal. Biochem 182, 319–326
Fiorella, P D, Giguère, V, and Napoli, J L (1993) Expression of cellular retinoic acid-binding protein (type II) in Escherichia coli J Biol Chem 268, 21,545–21
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© 1998 Humana Press Inc, Totowa, NJ
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Norris, A.W., Li, E. (1998). Fluorometric Titration of the CRABPs. In: Redfern, C.P.F. (eds) Retinoid Protocols. Methods in Molecular Biology, vol 89. Humana Press. https://doi.org/10.1385/0-89603-438-0:123
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DOI: https://doi.org/10.1385/0-89603-438-0:123
Publisher Name: Humana Press
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