Why is my protein Raman spectrum changing dramatically with each measurement?

[evidenso]

Raman spectroscopy of protein please help

Hey any Raman experts out there?
Im doing Raman spectroscopy of a protein called c-reactive protein (in aquous solution) using 532nm 10mW and the protein placed in a nmr tube. I can nicely get a spectrum of benzene and cyclohexane, but when I am doing Raman on the protein, the spectrum chanches dramatical for every measurement (flourescence like). how can this be? and how can i test if I have destroyed the protein?

MB

 

[chemisttree]

Is there a high fluorescence background in the sample? Does this change (lower) each time you analyze the same sample or within the same sample during long scans? If so, you could be photobleaching the sample.

If the sample changes over time regardless of the time spent in the spectrometer, you could be looking at degredation, aggregation or secondary structural changes.

To test for 'destruction' you must have a test to determine the titre of the sample in mind. If the titer changes (lowered) after the raman measurement vs. handling in the same manner minus the actual raman measurement, you have degraded the sample. I don't know of any other way to determine whether or not the protein has been degraded.

 

[evidenso]

okay thanks. I then think I am photobleaching the sample, is this a process which can be reversed?

 

[chemisttree]

Probably not, in proteins the photobleaching is likely an irreversible process. It is probably a free radical oxidation of the tyrosine residues. If the spot size is small relative to the sample, you will see fluorescence recover mainly as fresh sample diffuses back into the lightpath. If the spot size is large relative to the sample size (5mm NMR tube?) there won't be as much 'recovery'.

 

[Mike H]

FRAP (fluorescent recovery after photobleaching), as described by chemistree, is typically used to probe cell membranes and single cells (and interactions within membranes and cells) but not individual proteins, at least from the literature I've skimmed on the topic.

I'm not familiar with C-reactive protein, but there should be some sort of assay that you (or a collaborator) use as part of the purification and characterization process to make sure that the sample you're working with is functional/of adequate quality. In both of the biophysical spectroscopy labs I've worked in, we'd always check a sample by the assay (spectrophotometric, ligand/substrate binding, enzymatic activity, or whatever), do our experiments, and then redo the assay to make sure it hadn't completely died on us.

 

[Ygggdrasil]

Sounds to me like you're doing something similar to a fluorescence correlation spectroscopy measurement (basically, you focus a laser beam in a solution containing a fluorescent species. The fluorescence signal fluctuates because of molecules diffusing in and out of the focal volume of the laser. The autocorrelation of the fluctuations can tell you information about the diffusion of the fluorescent molecules in the solution). Is the protein solution relatively dilute?

 

[evidenso]

Probably not, in proteins the photobleaching is likely an irreversible process. It is probably a free radical oxidation of the tyrosine residues. If the spot size is small relative to the sample, you will see fluorescence recover mainly as fresh sample diffuses back into the lightpath. If the spot size is large relative to the sample size (5mm NMR tube?) there won't be as much 'recovery'.

ok thanks. is oxidation the only way of photobleaching? and why is it the aromatic aminos which are affected? are they the true flourephores?

 

[Mike H]

ok thanks. is oxidation the only way of photobleaching? and why is it the aromatic aminos which are affected? are they the true flourephores?

In a very general way, photobleaching occurs whenever the fluorophore(s) becomes unable to fluoresce due to photochemical damage and/or covalent modifications. Oxidation of aromatic residues is a pretty safe sort of explanation, but if there are other fluorophores in the protein (bound cofactors, metal centers, and the like) they could be the primary source of the fluorescence signal and, accordingly, the reason for photobleaching. They could be undergoing some sort of photochemical rearrangement or decomposition after some period of time.

 

[evidenso]

In a very general way, photobleaching occurs whenever the fluorophore(s) becomes unable to fluoresce due to photochemical damage and/or covalent modifications. Oxidation of aromatic residues is a pretty safe sort of explanation, but if there are other fluorophores in the protein (bound cofactors, metal centers, and the like) they could be the primary source of the fluorescence signal and, accordingly, the reason for photobleaching. They could be undergoing some sort of photochemical rearrangement or decomposition after some period of time.

ok thanks, that helped a lot. But why have some molecules flouresence an some not. Ofcause the photon energy must correspond to an excited electronic state. But is this the only demand, and does aromatic compounds emit flourescence because they fulfill this demand?

 

[Mike H]

ok thanks, that helped a lot. But why have some molecules flouresence an some not. Ofcause the photon energy must correspond to an excited electronic state. But is this the only demand, and does aromatic compounds emit flourescence because they fulfill this demand?

Some molecules are capable of dissipating the absorbed energy before fluorescence would occur. Those that do don't fluoresce. Molecular rigidity plays a role - if a molecule is flexible, it has more options to dissipate away the absorbed energy, but if a molecule is more rigid (like most conjugated/aromatic compounds) it has fewer degrees of freedom to work with in dissipating the energy.

I would suggest checking out Lakowicz's book on fluorescence spectroscopy from your nearest library for answers to pretty much all of your questions. If you're encountering any sort of wonky issue with fluorescence in biophysical spectroscopy - desired or not - it's pretty much a standard reference to be aware of in one's research.