I want to study the structure of cross-linked enzyme aggregates (CLEAs) obtained with different precipitating agents. I do not known, if circular dichroism is the most suitable technique. So, any suggestion will be appreciated :)
Thank you for your answers.
Carlos
I guess in your case if your enzyme has any secondary structure before aggregation then you can quantify this with different precipitating agents. You can consider enzyme alone as the reference and see how the signature CD changes with different precipitating agents. However it might not be the best way to look at the structure of cross-linked enzyme aggregates. I guess you can use DLS, or SAXS to know about the order of aggregates or so.
Hi Carlos. Short answer, No.
Protein crosslinking mostly happens between charged surface amino acids, not the internal ones that can form distinct 2' structures.
Like Anil said, probably the best you can do is to know the order of the aggregates.
The problem I see with CD is that you will have light scattering by aggregated particles. which will complicate your date interpretation.
If you want more info on the structural behavior of your enzyme in the native state and the aggregated state, FTIR could help you: this can be done in solid state and can provide you with detailed secondary structure information from the comparison of the native anzyre and the different aggragted samples. Also the combination with AFM (Atomic FOrce Microscopy) or TEM (Transmission electron microscopy) can be powerfull to analyze the aggregated species (AFM and TEM can reveal the heterogeneity in your aggregated sample: If it is shown to be monodisperse, then certainly SAXS can provide additional detailed information.
Kris Pauwels is right. Light scattering of aggregated particles, of the size of the wavelength you are doing your spectra does impact the CD spectral amplitude. Seen from a different perspective: CD spectroscopy is quickly done, using little sample and may be used to test and identify aggregation. Several ways of inspecting your samples should include serial dilutions, as well as detector - sample distance adaptation. In combination with the FTIR, AFM, TEM and SAXs you will be able to describe and understand your sample best.
Well there are many techniques to understand aggregation rate and ratio. You can use Dynamic Light scattering (DLS) , Resonance light scattering (RLS) using flourescence technique, Circular Dichroism (CD), ALso in some cases a fibril formation of protein structure can also be seen under Environmental Scanning elcetron microscope (E-SEM). It depends what we are looking for and how we want to present data. CD would help us to know structural changes intensely well DLS with it would help to understand aggregation formation. SO both technique can be used in accordance with it we can also use Zetapotential studies to give verification of system unstability. FTIR would also give an answer similar to CD. AFM will also show aggregation provided aggregates formation is bigger. i think choose a proper technique relevant to ur system and data relevance.
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