Program Information
Evaluating Proteomic CEST NMR Response in Radiation-Sensitive Metabolic Pathways
R Hachadorian*, S Goerke , M Zaiss , K Klika , M Schnoelzer , P Kunz , P Bachert , J Seco , German Cancer Research Center (DKFZ), Heidelberg, Baden-Wuerttemberg
Presentations
WE-F-205-6 (Wednesday, August 2, 2017) 1:45 PM - 3:45 PM Room: 205
Purpose: (1) To further understand how irradiation damage effects the proteome, and how these changes are reflected in a CEST (chemical exchange saturation transfer) NMR spectrum. (2) To link the observed protein damage to an existing cell model in development, which focuses on the mechanisms behind free radical formation.
Methods: Two iron-containing metalloproteins (Hemoglobin and Aconitase) and one non-metalloprotein (BSA) were analyzed using NMR and fluorescence spectroscopy after successive high doses of gamma radiation between 0.5kGy-4kGy (Siemens Linear Accelerator) in vitro [2]. The NMR Spectrometer (Bruker, 14.1 Tesla) was used to monitor changes in the CEST amide mobile-proton peak (Δω=+3.6ppm) and the relayed nuclear Overhauser effect (rNOE) signal (Δω =-3.5ppm) in the AREX spectrum [1]. In future work, use of carbon ion irradiation (HIT DKFZ) will allow us to compare the observed signal changes when the radiative biological effectiveness is increased.
Results: When high doses (kGy range) of gamma irradiation were administered to protein BSA, increase in signal was observed at the two aforementioned peaks (p=0.066, 0.067, two-tailed unpaired, not quite significant), which mimics the observed response when BSA is denatured by heat shock (p<0.0001) [1]. Analysis suggests this could be a result of protein cross-linking. The signal was compared to the decrease in the fluorescent signal, for which the correlation was significant (p<0.0001).
Conclusion: Non-metallic proteins are highly-radiation resistant macromolecules, yet changes are observable in the CEST NMR spectrum between 0.5kGy and 4kGy. These increase in signal contradicts expectations that dipolar coupling interactions would decrease with denaturation. Further, a preliminary result shows an increase in cell damage due to radiation in the presence of iron-II, thus it is estimated that the irradiation of these iron-containing metalloproteins will damage the protein structure in a more relevant and feasible, in vivo dose range.
Funding Support, Disclosures, and Conflict of Interest: Funding provided by the DKFZ (German Cancer Research Center) - Medical Physics in Radio-Oncology.
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