The association of acrylamide with proteins. The interpretation of fluorescence quenching experiments

Topics in Fluorescence Spectroscopy

“…Blatt et al (114) have recently emphasized this point, in comparison with quenching studies with micelles (see Section 2.4.1). If there is a high local quencher concentration, then the apparent values (obtained with reference to the bulk quencher concentration) will give an overestimate of the kinetic exposure of the fluorophore.…”

“…(52,84) For larger quenchers, such as acrylamide, there has been some question regarding the mechanism of quenching of internal fluorophores. (114) Here I will comment on these various alternative interpretations. (73,110) Some have argued that, instead of the quencher diffusing inward, a segmental unfolding of the protein occurs to increase the exposure of the fluorophore to the aqueous phase and hence to the quencher.…”

Interfacial Effects on the Conformation of Amyloid-Beta Peptide

“…The increased openness of ABP conformation at the sub-interfacial layer would allow for interaction between quenching agent and other side chains, partitioning the quenching agent and thus resulting in a biphasic SternVolmer curve (Fig. 3) [26]. This type of behavior is not seen with bulk ABP, suggesting that these partitioning sites are not accessible and thus consistent with bulk ABP being more ordered.…”

Application of Fluorescence Spectroscopy for Determining the Structure and Function of Proteins

“…Iodide is the most common of these and was employed in Lehrer's original accessibility studies (Lehrer, 1971). In the last decade, however, acrylamide has gained favor in such studies because being a neutral quencher, its quenching rate constant (k q ) is not affected by the surface charge of the protein (Eftink and Ghiron, 1981;Blatt et al, 1986;Calhoun et al, 1986). Finally, of all the quenchers only oxygen is able to diffuse into the interior of a protein and quench-buried fluorophores (Lakowicz and Weber, 1973a,b;Gratton et al, 1984b;Jameson et al, 1984b).…”