LETTER pubs.acs.org/jchemeduc
Fluorescence Quenching, Lifetimes, and Fluorophore Solvent Accessibility Lorenzo Stella* Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, 00133 Rome, Italy ABSTRACT: The influence of the excited-state lifetime of a fluorophore on its susceptibility to collisional quenching is discussed. In the case of cytochrome c, FRET to the heme moiety strongly reduces the tryptophan fluorescence lifetime. This should be taken into account to correctly evaluate the tryptophan solvent accessibility from acrylamide quenching experiments. KEYWORDS: Upper-Division Undergraduate, Physical Chemistry, Misconceptions/Discrepant Events, Fluorescence Spectroscopy, Proteins/Peptides appreciated the recent article titled “Quenching of Tryptophan Fluorescence in Unfolded Cytochrome c: A Biophysics Experiment for Physical Chemistry Students” by Kim and coworkers.1 This interesting paper describes an experiment to determine the solvent exposure of the single Trp residue in horse heart cytochrome c under different denaturing conditions by measuring its susceptibility to the water-soluble quencher, acrylamide. The authors suggest combining this experiment with the measurements of F€orster resonance energy transfer (FRET) between the Trp residue and the heme moiety of the protein, reported by them in a previous study,2 to provide a comprehensive set of biophysics experiments. I would like to point out that the presence of FRET in this protein actually introduces some complications in the analysis of solvent exposure, which students should be made aware of. In a quenching experiment, by measuring the decrease in fluorescence as a function of quencher concentration, a so-called SternVolmer constant Ksv is determined,1,3 which measures the susceptibility to the quencher of the fluorophore in its excited state. The authors directly equate this constant to the solvent exposure of the fluorophore (“A relatively large ...constant... indicates that the fluorophore is accessible to bulk solvent, whereas a small quenching constant suggests that the fluorophore is relatively inaccessible to solvent, or buried”). However, as shown in the literature,46 Ksv is the product of two terms:
I
Ksv ¼ kq τo
shorter lifetime. This significantly changes the data interpretation, and explains the authors’ observation that “the quenching constant found here for fully denatured cyt c are lower than those found for other denatured proteins”. The lifetime of NATA is 3.0 ns,5 and is similar to the 3.3 ns average lifetime of horse heart apocytochrome c.7 However, in the native holo-protein, the lifetime is reduced to just 0.01 ns by the FRET process to heme.8 The lifetime of the partially denatured holo-protein in 7 M urea can be estimated by the data reported by Kim and co-workers in Figure 3 of ref 2: under these conditions, the FRET efficiency with respect to NATA was about 0.77. This efficiency was determined from steady-state experiments, but it is also equal to:5 E ¼ 1 τðcyt c 7 M ureaÞ=τðNATAÞ
Therefore, the average lifetime of the partially denatured protein in 7 M urea is about 23% of that of NATA, that is, approximately 0.7 ns. The difference between this lifetime and that of NATA cannot be neglected in the analysis of the acrylamide quenching data. The ratio of the reported values of the SternVolmer acrylamide quenching constants for NATA and for the partially denatured protein is Ksv(NATA)/Ksv(cyt c 7 M urea) = 5.5. However, by taking into account the different lifetimes, we can calculate the ratio kq(NATA)/kq(cyt c 7 M urea), which is much more significant regarding the fluorophore’s solvent exposure. This ratio is just 1.3, indicating that the single Trp of cytochrome c partially denatured in 7 M urea is actually rather solvent-exposed, because its accessibility to acrylamide is not very different from that of NATA. It is also worth commenting in these terms on what the authors call the “screening effect” of urea. They observed that quenching of NATA in water is higher than in concentrated urea solutions. This denaturant significantly influences the solution viscosity, which increases approximately by a factor of 2 when going from water to 10 M urea.9 The term kq depends inversely on viscosity,5 but this is not the only effect, as the τ0 lifetime of NATA is also affected by this change: it increases by a factor of 1.52 when going from water to 10 M urea.10 These opposing effects contribute to the change in Ksv, and, from the values
ð1Þ
In eq 1, kq is the bimolecular quenching constant, which depends (in addition to other parameters) on the solvent accessibility of the quencher, and τ0 is the fluorophore’s excited state lifetime in the absence of the water-soluble quencher. The presence of τ0 in eq 1 is somewhat obvious: the longer the fluorophore remains excited, the higher is the probability that it will collide with a quencher. In the case of cytochrome c, the Trp lifetime is significantly reduced due to FRET to the heme, and this effect varies with denaturant concentration. Therefore, the different Ksv of the protein with respect to NATA (N-acetyl-L-tryptophanamide), which in the acrylamide quenching experiments was used as a reference for a completely solvent-exposed Trp, could be due to a lower solvent accessibility of the fluorophore, but also to its Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.
ð2Þ
Published: April 18, 2011 695
dx.doi.org/10.1021/ed101147s | J. Chem. Educ. 2011, 88, 695–696
Journal of Chemical Education
LETTER
reported above, a decrease by a factor of 1.32 can be predicted for the SternVolmer constant (when comparing water to a 10 M urea solution), which is very close to the 1.33 factor reported in ref 1. In this connection, it should be mentioned that in general the correction factor for the urea effects on viscosity and lifetime might be different for the NATA and protein data sets.10,11 In my opinion, this discussion of the effects of lifetimes on quenching would improve students’ understanding of the laboratory activity, and provide an occasion to teach several important aspects of emission spectroscopy.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT The author gratefully acknowledges support by the Italian Ministry of Education, University and Research (PRIN 2008). ’ REFERENCES (1) Schlamadinger, D. E.; Kats, D. I.; Kim, J. E. J. Chem. Educ. 2010, 87, 961–964. (2) Sanchez, K. M.; Schlamadinger, D. E.; Gable, J. E.; Kim, J. E. J. Chem. Educ. 2008, 85, 1253–1256. (3) Coutinho, A.; Prieto, M. J. Chem. Educ. 1993, 70, 425–428. (4) Legenza, M. W.; Marzacco, C. J. J. Chem. Educ. 1977, 54, 183–184. (5) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. (6) Eftink, M. R.; Ghiron, C. A. Anal. Biochem. 1981, 114, 199–227. (7) Vincent, M; Brochon, J. C.; Merola, F.; Jordi, W.; Gallay, J. Biochemistry 1988, 27, 8752–8761. (8) Das, T. K.; Mazumdar, S.; Mitra, S. Eur. J. Biochem. 1998, 254, 662–670. (9) Kawahara, K.; Tanford, C. J. Biol. Chem. 1966, 241, 3228–3232. (10) Eftink, M. R. Biophys. J. 1994, 66, 482–501. (11) Eftink, M. R.; Hagaman, K. A. Biophys. Chem. 1986, 25, 277–282.
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