Acrylamide quenching

Other corrections, besides those for static interactions, are important for certain quenchers. For example, acrylamide quenching is often used to help determine the relative solvent accessibility of aromatic residue side chains. In addition to a correction for static quenching,(60,66) acrylamide quenching data for tyrosine residues require both primary and secondary inner filter corrections since acrylamide absorbs both 280- and 305-nm light.(67)... 

P. C. Leavis, E. Gowell, and T. Tao, Fluorescence lifetime and acrylamide quenching studies of the interactions between troponin subunits, Biochemistry 23, 4156 1161 (1984). 

Fig. 10. Highly schematic representation of the orientation of several tryptophan-containing peptides with respect to calmodulin. (A) With tryptophan in position 1, the indole is located on the hydrophilic side of the helix and is exposed to solvent. Peptides with tryptophan on this face of the helix should exhibit emission maxima near that of indole in water ( 350 nm), a small anisotropy, and a high accessibility for acrylamide quenching. (B) In position 2, the tryptophan is partially exposed at the interface between the peptide and calmodulin. Peptides with a tryptophan in this location should have fluorescence properties that are intermediate between example A and C. (C) The tryptophan is on the hydrophobic side of the helix and is almost entirely buried. The emission maximum should be strongly blue-shifted, the anisotropy should be large, and the accessibility to acrylamide quenching low. Taken from O Neil et al. (1987).

Fig. 8. Stem-Volmer plot for acrylamide quenching of melittin monomer and tetramer. From M. Eftink, University of Mississippi, Chemistry Department, unpublished observations. The lifetimes are from [35J. The broken lines are the initial slopes, corresponding to the values on the figure. 

Once again melittin illustrates the effect of protein structure on the fluorescence emission. Acrylamide quenching data for melittin monomer and tetramer are shown in Fig. 8. Stem-Volmer plots are often used to present quenching data. The Stem-Volmer equation is... 

The quenching data for both the monomeric and tetrameric forms of melittin indicate the tryptophan residues are accessible to acrylamide with the accessibility being greater in the monomeric state. This conclusion is reached by comparison with acrylamide quenching data for NATA. At 25 °C in water the acrylamide... 

Figure 8.17 St -Vblmer plot for acrylamide quenching of cAMP receptor protein in the absence (o) and in the presence (a) of cAMP. Revised from Ref. 4l.

Figure S.23. Steiv-Vbliiier plots for acrylamide quenching of lAEDANS (o). OPH ( ), and lAEDANS and OPH (Ci) in SDS nicc a. For the mixtuie. die solid line rqueseaits die fit with calctdaled parameleis AT = 9.9 AT. iCj=0 Ar, /i = 0.69, and/j = 0.31 at 473 nm. TV ioncr pand shows die inadoals for this fit Revised from Ref. 49.

Figure 9.29. Effect of acrylamide quenching on the intradty decay of Y -base. The FD intensi decays in the ibseacc (o) and in the presence of O.SAf acrylamide are shown. The solid curves represent (he best ringJe-decay-time fits to data. The lower panels show the deviations from the bestsingle-decay-time fits. Revised atidr wintedfnHnRef. 105. Copyright 91988. with pennission from Elsevier Scienoe.

Figure 16.39. Dependence of Ure emission maxima (A), acrylamide quenching constants (B), and steady-state anisotropies (C) of the MLCK pepddes bound to calmodulin on the position of the tryptophan residue. Reprinted, with permission, from O Neil, K. T, Wolfe. H. R., Erickson-Viitanen, S. and DeGrado, W. F., Fluorescence properties of calmodulin-binding peptides reflect alpha-helical periodicity, Science 236 1454-1456, Copyright O 1987, American Association for the Ad-vancement of Stience.

Acrylamide quenching. 238. 239, 244-246. 250,251,252 absorption spectra. 249 bimolecular quenching constants. 252 covalent adduct formation, 257 intensity decays, 282,283 NATA, 285 jHx>teins, 463... 

In acrylamide quenching experiments it is important that X x — 290 nm. Acrylamide has an appreciable absorption at 285 nm. At the high concentrations of acrylamide that are normally used (0.01-0.5 m) a large proportion of excitation light at 285 nm will be absorbed by the acrylamide. This is an inner filter effect that will render the data and analysis invalid. 

Measurement of of a sample 59 Acrylamide quenching of protein fluorescence 65... 

MnA, manganese-adequate sample MnD, manganese-deficient sample K, Iodide quenching constant and Ka2, acrylamide quenching constant for exposed and partly-exposed fluorophores, respectively. 

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