I would like to say congratulations, but unfortunately I have to be mean to you about it: the fluorescence is disastrously wrong. Ever thought about how you could determine a binding constant by fluorescence that is a factor 10 (EG) and a factor 30 (ECG) higher than the value determined by ITC? Why did you or any of the reviewers not notice that, uhm, "slight discrepancy" at all?
You may want to check my paper in the Journal of Molecular Structure (volume 998, 2011, pp 144-150) to see what you (and with you at least a few hundred others - there's 360+ in my database and it's not complete) have likely done wrong. It starts with ignoring the inner-filter effect, and ends with using improper equations (and you most assuredly did not find that equation in Theodor Förster's book - your reference 26).
One way of checking whether I might be right is to measure the absorbance spectrum of BSA together with the ECG. You may need slightly higher concentrations to be able to measure it properly. If my suspicions are right, you will see an absorbance band at ca. 340-350 nm. That would be the explanation for that strange apparent double peak in figure 2B. Also check for absorbance increases as the excitation wavelength, 280 nm.
I'm not convinced about some of the other data either, e.g., the ITC analysis of especially the EC; and do you have any explanation why the CD spectra of BSA in figure 6A and B are so different (see in particular the magnitude on the y-axis)? The FTIR also does not fit with the CD: a 10% absolute loss of alpha-helix at the expense of beta-sheet should not give shifts to slightly higher wavenumbers, but to lower wavenumbers.
All I can say for now is: "another paper to add to my database"!
Contact me if you want to discuss, you can find the address on the paper cited earlier.
I would like to say congratulations, but unfortunately I have to be mean to you about it: the fluorescence is disastrously wrong. Ever thought about how you could determine a binding constant by fluorescence that is a factor 10 (EG) and a factor 30 (ECG) higher than the value determined by ITC? Why did you or any of the reviewers not notice that, uhm, "slight discrepancy" at all?
ReplyDeleteYou may want to check my paper in the Journal of Molecular Structure (volume 998, 2011, pp 144-150) to see what you (and with you at least a few hundred others - there's 360+ in my database and it's not complete) have likely done wrong. It starts with ignoring the inner-filter effect, and ends with using improper equations (and you most assuredly did not find that equation in Theodor Förster's book - your reference 26).
One way of checking whether I might be right is to measure the absorbance spectrum of BSA together with the ECG. You may need slightly higher concentrations to be able to measure it properly. If my suspicions are right, you will see an absorbance band at ca. 340-350 nm. That would be the explanation for that strange apparent double peak in figure 2B. Also check for absorbance increases as the excitation wavelength, 280 nm.
I'm not convinced about some of the other data either, e.g., the ITC analysis of especially the EC; and do you have any explanation why the CD spectra of BSA in figure 6A and B are so different (see in particular the magnitude on the y-axis)? The FTIR also does not fit with the CD: a 10% absolute loss of alpha-helix at the expense of beta-sheet should not give shifts to slightly higher wavenumbers, but to lower wavenumbers.
All I can say for now is: "another paper to add to my database"!
Contact me if you want to discuss, you can find the address on the paper cited earlier.