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closeModified Stern-volmer to analyse binding
Posted by mvdw on 28 Apr 2011 at 09:58 GMT
Therefore, a modified Stern-Volmer plot was used to analyze the quenching
http://plosone.org/article/info:doi/10.1371/journal.pone.0016810#article1.body1.sec3.sec9.p1
Authors and readers are advised that the Modified Stern-Volmer equation has been derived for collisional quenching. Here, the authors claim it is relevant for binding-related quencing. This is problematic for many reasons, most importantly the fact that the "fraction of accessible fluorophores" is then no longer necessarily a "fraction of accessible fluorophores". Rather, it is an indication, when done properly, of how much the fluorescence may be quenched upon full ligation of the protein. However, in such a case it may well be that one Trp residue is quenched by 10-15%, another by 40%, a third by 90%, while a fourth increases with 30%. The resulting quenching thus does not necessarily reflect how many Trp residues are involved in the binding.
Note also there are various other potentially confounding factors, such as the inner-filter effect, and the fact that the Modified Stern-Volmer equation is heavily biased towards the low ligand concentrations, where the added ligand concentration is likely much different from the free ligand concentration. This will also affect the f(a) calculated from the equation.
In short, I do not recommend to use this equation for binding-related quenching.
RE: Modified Stern-volmer to analyse binding
DNRAO replied to mvdw on 10 May 2012 at 04:52 GMT
Tryptophan fluorescence is sensitive to changes in solvent accessibility and the positions of polar residues. Protein conformational changes usually alter the observed fluorescence. The proximity of Trp residues in the region of the protein that contacts any ligand (AdoMet in this case) permits one to use fluorescence to study the conformational changes which occur upon ligand binding and therefore in principle one could use this to determine ligand dissociation constants.
The fluorescence intensities used to determine Kd values for AdoMet was corrected for the inner-filter effect since the adenine ring has a absorption maximum at 259 nm in aqueous solution which is close to 280 nm, the excitation wavelength.
The intensities were corrected using the following equation (Lakowicz, 1983),
Fc = F(10εCd/2)
where Fc is the corrected intensity, F is the observed intensity, c is the extinction coefficient of the quencher at the excitation wavelength, C is the concentration of quencher, and d is the path length. Extinction coefficient of 1887 for AdoMet was calculated at pH 8.0 and 280 nm using the Beer-Lambert law.
Since the adenine moiety can acts as a collisional quencher (Lakowicz, 1983), quenching could occur from random collisional quenching at high AdoMet concentration in addition to static quenching due to binding of AdoMet. This was taken care by carrying out the same control experiments that Maegley et al (1992) performed. There was no significant change in fluorescence intensity at 80 µM AdoMet. We agree that the resulting quenching thus does not necessarily reflect how many Trp residues are involved in AdoMet binding.
Earlier, Maegley et al (1992) showed that AdoMet binds to EcoRI MTase using fluorescence quenching. EcoRI MTase contains two tryptophan residues and authors had to use modified Stern-Volmer relationship to calculate Ksv. However, mutation of one of the tryptophan residue showed that Ksv can be calculated using Stern-Volmer relationship. This study also compared dissociation constants determined using other techniques.
Modified Stern-Volmer analyses was necessary in our case as Stern-Volmer plot showed a downward curvature indicating that Tryptophan residues were not equally accessible to the quencher (AdoMet).
After thorough analysis of the Maegley et al (1992) article, we resorted to use fluorescence quenching technique to calculated Ksv in our studies.
References
1. Lakowicz, J.R. (1983). Principles of Fluorescence Spectroscopy, p.259, Plenum Press, New York.
2. Maegley KA, Gonzalez L Jr, Smith DW, Reich NO. Cofactor and DNA interactions in EcoRI DNA methyltransferase. Fluorescence spectroscopy and phenylalanine replacement for tryptophan 183. J Biol Chem. 1992 Sep 15;267(26):18527-32. PubMed PMID: 1526989.
RE: RE: Modified Stern-volmer to analyse binding
mvdw replied to DNRAO on 06 Jun 2012 at 08:29 GMT
I appreciate the response of the authors, and it is good to see the authors indeed corrected for the inner-filter effect. I do strongly recommend to mention this, because so many others do not make this correction.
I do want to point out that Maegly et al had an experimental set-up that is more appropriate when using the Modified Stern-Volmer equation to determine the binding constant, compared to the experimental set-up used in the present article. Maegly et al used a ligand concentration range that was much higher than that of the protein. The lowest ligand concentration in their study is almost a factor 100 higher than that of the protein. This makes it possible to assume that the free and added ligand concentration are essentially the same.
In contrast, the current paper uses ligand concentrations of 0.5-6.25 µM, with a protein concentration of 2 µM. This causes significant problems in the Modified Stern-Volmer equation. Take, for example, the lowest ligand concentration: since it is lower than the protein concentration, any binding of the ligand to the protein will lower the free ligand concentration. Suppose the dissociation constant is indeed 2 µM, the same concentration of the protein. In that case a simple quadratic equation shows the the free ligand concentration upon adding 0.5 µM ligand is in reality about 0.27 µM. This is almost a factor two lower, and will thus create an error of almost a factor two in the calculated dissociation constant when using the Modified Stern-Volmer equation.
Re-reading the paper on this section, I also have become quite skeptical about the claim that the intercept value of 1.85 is indicative that more than one class of fluorophores is involved in the binding. What the intercept value of the Modified Stern-Volmer plot tells us in the case of a binding process is how much of the fluorescence is quenched upon full ligation compared to the unligated protein (if the analysis is performed properly). It tells us no more and no less.