Quenching bovine serum albumin

Papadopoulou A, Green RJ, Frazier RA. 2005. Interaction of flavonoids with bovine serum albumin A fluorescence quenching study. J Agric Food Chem 53 158-163. 

Flanagan, S. D. and Yost, B. (1984) Calmodulin-binding proteins visualization by 125I-Calmodulin overlay on blots quenched with Tween 20 or bovine serum albumin and poly(ethylene oxide). Anal. Biochem. 140, 510-519. 

Soares, S. Mateus, N. de Freitas, V. 2007. Interaction of different polyphenols with bovine serum albumin (B SA) and human salivary 3-amylase (HSA) by fluoreseenee quenching. J. Agric. Food Chem. 55 6726-6735. 

If the sample is fixed with aldehyde fixatives it should be quenched with a solution of 0.1 M glycine that binds to free aldehyde groups. Non-specific binding of antibodies should be blocked using 1-2% BSA (Bovine Serum Albumin) in PBS with or without 5 pgm/mL of normal IgG from the same species as the secondary antibody. 

In this work, the solutions of human serum albumin (HSA) (>96%, Sigma) and of bovine serum albumin (BSA) (>98%, MP Biomedicals) in a phosphate buffer (0.01 M, pH 7.4) have been used. The proteins concentrations were lO- (absorption spectra measurement) and 10- M (fluorescence measurement at the nanosecond laser fluorimeter). All of the experiments were performed at a temperature of 25 1 °C. The structure and biological functions of HSA and BSA can be found in (Peters, 1996). Tryptophan, tyrosine, and phenylalanine (with relative contents of 1 18 31 in HSA and 2 20 27 in BSA) are the absorption groups in these proteins (as in many other natural proteins). The tyrosine fluorescence in HSA and BSA (as in many other natural proteins) is quenched due to the effect of adjacent peptide bonds, polar groups (such as CO, NH2), and other factors, and phenylalanine has a low fluorescence quantum yield (0.03) (Permyakov, 1992). Therefore, the fluorescence signal in these proteins is determined mainly by tryptophan groups. In that case the fluorescence, registered in nonlinear and kinetic laser fluorimetry measurements, correspond to tryptophan residues (this fact will be used in Section 6.1). 

Weber, G., Daniel, E. Cooperative effects in binding by bovine serum albumin. II. The binding of l-anilino-8-naphthalenesulfonate. Polarization of the ligand fluorescence and quenching of protein fluorescence. Biochemistry 5, 1900-1907 (1966)... 

FIA of anionics without a phase separation may be based on the quenching effect of surfactants on the fluorescence intensity of 8-anilino-l-naphthalenesulfonic acid coupled with bovine serum albumin (83). Another method of avoiding a phase separation is to use a surfactant-selective electrode. Since interference is a problem, the surfactant-selective electrode approach is best coupled with online concentration with a reversed-phase column. Such a system has been demonstrated for trace analysis of sodium dodecylsulfate (84). 

Ellipticine and several derivatives were able to displace 7,12-dimethyl-benz[a]anthracene from binding to bovine and human serum albumin using the fluorescence-quenching technique (192,193). The site of binding of ellipticines appears to be on the hydrophobic regions of the enzyme. A fast kinetic experimental technique, namely, temperature-jump spectroscopy, has been developed in order to study the interaction of elliptinium, and other molecules, with biological macromolecules (194). 

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