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closeMajor concern about the fluorescence spectra
Posted by mvdw on 05 May 2014 at 11:11 GMT
I have a major concern about the fluorescence spectra of renin and angiotensin-converting enzyme reported in this study (Figure 2). In my opinion the spectra indicate a problem with the wavelength calibration of the instrument, or a mistake in the preparation of the graphs. The authors report that both proteins give a very sharp fluorescence at 327/328 nm, but the peak at this wavelength is remarkably symmetric, and there is no signal (flat baseline) from about 300-315 nm. I have never ever seen fluorescence from any protein, or Trp and Tyr alone, that does not show any signal in the region 300-315 nm, nor have I seen such highly symmetric peaks for Trp or Tyr-based fluorescence. I believe the authors will not be able to find any similar spectra for ACE or renin from other groups either.
In addition, the spectra also contain another peak with a maximum around 370 (ACE) and 365 (renin) nm, which is not discussed at all. The band shape and width of this second peak actually fit well with what one may expect for (primarily)Trp fluorescence, but with a much larger red shift that expected.
The most obvious explanation is that the first, shapr peak is the Rayleigh scattering peak, which explains its high symmetry and absence of any signal between 300-315 nm, whereas the second peak is the actual fluorescence of the ACE. The first peak may also be the Raman scattering peak, but for various reasons I have more doubts about this.
I strongly urge the authors to repeat their measurements and include a fluorophore whose spectrum is very well known, for example Trp or its derivatives.
RE: Major concern about the fluorescence spectra
raluko replied to mvdw on 17 May 2014 at 00:44 GMT
Thanks for your comments on our recently published paper in PLoS One. Please find below our response.
1. Symmetry of the FI peak. Please see Fig. 2A in Yan et al. paper (Food Chemistry, 2013, 141, 3766–3773) and you will see that it is quite similar to our data in terms of symmetry of the first FI peak.
2. Lack of FI between 300-315 nm. There is nothing really odd about this data because it simply indicates partial quenching of tryptophan fluorescence, especially those residues buried deep in the hydrophobic pocket. The raw data show that it is not that there is complete absence of FI at the some of the lower wavelengths but due to the small values (<10), it appears as a zero in relation to scale of the plotted data (maximum of 100-200). So if we had plotted the figure on a scale of may be 40-50, the peak start at lower wavelengths would have been more obvious. Moreover, if the problem is with the instrument, then the FI values should be the same for all the samples at the 300-315 nm range. Looking at the raw emission fluorescence data, addition of peptides actually increased FI when compared to ACE alone within the 300-315 nm range but this was not obvious in the plotted figure because of the large scale (wide differences between minimum and maximum values). Therefore, it showed clearly that some of the trp residues were in a shielded or quenched environment prior to addition of the peptides; the latter probably caused protein unfolding and exposure of the previously shielded trp residues. In Fig. 11b of a previous paper (Schmid, 1989. Spectra methods of characterizing protein conformation and conformational changes. In "Protein Structure: a practical approach", Edited by TE Creighton, pp. 251-281) the bold line showed that the start of FI was longer than 300 nm. Also the initial FI was about ‘12’ in Fig. 11b, broken line but it was also flat and the peak did not start until about 317 nm. So really, our data is not uncommon.
3. As the raw data show, it is not that the actual data is flat in the 300-315 nm range, it is the scale of the Y-axis (wide differences between minimum and maximum FI values) that makes it to appear flat. And as shown in Fig 11b of the Schmid paper, some parts of the initial FI spectrum can indeed be flat.
4. As indicated above, there is no problem with the instrument otherwise the values in the presence and absence of peptides would have been the same in the 300-315 nm range. As the raw data show, the values are different suggesting that the instrument was able to pick up structural changes within the protein
5. Second peak. There was really no significant differences in the FI maximum of the different samples, hence we did not see the need to focus on the second peak, which is obviously tryptophan residues in a more hydrophilic environment than those detected at lower emission wavelengths (first peak).
Thanks.
RE: RE: Major concern about the fluorescence spectra
mvdw replied to raluko on 21 May 2014 at 09:20 GMT
The authors continue to maintain there is nothing odd about their spectra. I argue here with additional evidence that this is problematic.
First, the authors were kind enough to send me the data for ACE, for which I would like to thank them. The spectra as reported in the paper fit those in the excel sheet sent to me. However, that same sheet also shows a truly flat baseline from 300 to 310 nm (increase of at most 0.04), and this is the same for all spectra. Also, the increase in signal from the baseline does not pass a value of 1, a mere 0.5% of the maximum signal, until above 315 nm. The only difference upon addition of the peptides is a vertical shift that is not correlated to the concentration added. My contention that the spectrum is flat from 300-315 thus holds.
Second, the authors argue that Fig 11b in Schmid's book chapter supports their contention that a flat baseline in this spectral region is not abnormal. However, to make this argument the authors compare only to the spectrum obtained in 6 M guanidine, i.e., a fully unfolded protein. A comparison to the spectra in Fig 11a, which shows a folded protein, shows a quite different picture. Moreover, as the authors themselves note, the signal in the spectrum in 11b starts at 12; as it increase only to 25 at the peak maximum, and does decrease to a signal lower than 10 at 400 nm (scale goes only to 400), the authors cannot just claim the initial part of the spectra is a "baseline".
Third, the spectra in the current paper do not correspond to prior published spectra of these same proteins. In two earlier papers with one of the current authors, and which used the same two proteins and the same instrument, the spectra only show a single peak around 330 nm (renin) and 335 (ACE) (Deng et al, Pharm Biol 50 (2012) 401-406 and Yuan et al, Int J Biol Macromol 41 (2007) 274-280).
The authors could argue that those two papers performed their experiment at 37 degrees and that the ACE was excited at 295 nm, and that those differences could explain the different fluorescence spectra obtained. However, there are other reports with spectra for human renin, and those are at odds with the current paper.
For example, Epps et al (J Biol Chem 262 (1987) 10570-10573) show the spectrum of human renin upon excitation at 295 nm and at room temperature. This yields a single peak with a broad maximum around 320-330 nm, which makes this spectrum much closer to those reported in Yuan et al and Deng et al, even though the latter spectra were obtained by excitation at 280 nm and at 37 degrees. A spectrum shown in Morelock et al (J Med Chem 38 (1995) 1751-1761) obtained upon excitation at 285 nm and at 37 degrees also shows a single peak with a maximum around 325 nm.
For rabbit lung ACE I have unfortunately not been able to find any trustworthy sources of intrinsic fluorescence spectra, but they may well be out there, and if the authors have such references that confirm their own current measurements they are very welcome to give those references here.
My final comment relates to the supposed absence of a signal below 315 nm to the presence of highly quenched tryptophans. As reported by Epps et al (J Biol Chem 262 (1987) 10570-10573) human renin has three Trp residues, and despite selectively exciting the Trp residues they see a fluorescence already at 300 nm (spectrum cut off below this wavelength). That is, Epps et al do not find the presence of any fraction of a very deeply buried Trp that is completely quenched. Moreover, they show that complete unfolding in 6 M guanidine results in a peak shift to 353 nm, more than 10 nm lower than the peak maximum of the second peak the authors observed, and which they assigned to a Trp residue in a more hydrophilic environment. In other words, even if the human renin sample used in the current study had a Trp residue that was fully solvent-exposed, there should not be a peak maximum at such high wavelengths, unless the instrument used has a rather large wavelength-dependency.
In short, I would like to urge the authors again to check their instrument calibration or even go back to the raw data (not the Excel sheet) to check whether there is an issue with the x-axis of the figure. If I move the whole spectrum by about 45 nm to the left, placing the first peak as the Rayleigh scatter at 280 nm, the second peak is in a position that actually reasonably fits the spectra of renin and ACE reported in the earlier studies.
RE: RE: RE: Major concern about the fluorescence spectra
mvdw replied to mvdw on 08 Jan 2018 at 12:39 GMT
Prof. Aluko has now published a new paper containing fluorescence spectra of ACE and renin (https://doi.org/10.1016/j...). In this new paper, the ACE and renin spectra fit that of the prior papers that I mentioned, but are at odds with the current paper. This discrepancy needs to be explained.