Abstract
Purpose :
This study provides evidence for the role of intralenticular adenosine triphosphate (ATP) as an endogenous hydrotrope for prevention of Protein Aggregation (PA) known to be an etiology of age-related cataractogenesis.
Methods :
Calculation of ATP concentration and spectral line widths (indicators of atomic mobility) were determined in both H2O and 98% D2O buffers in intact (ex vivo) lens (N=12) among multiple species using a 200 MHz NMR spectrometer for phosphorus-31 detection. The line widths of each resonance multiplet of the three phosphorus atoms of ATP were determined during a time-course [baseline (0 hr) to 3 hr].
Results :
Intralenticular concentration of ATP [2.4 ±0.15 millimolar (mM)] far exceeds the micromolar (μM) amounts required for intracellular metabolism. The field strength of the spectrometer permitted resolution of the spectral line width in the α-group phosphate (11.6 ±0.5 Hz) relative to the γ-group phosphate (18.8 ±0.5 Hz) of ATP (P <0.01). When incubated in D2O buffer, the line widths of the phosphates progressively narrow with time such that after 3 hr the line width of the α-group phosphate was 9.7 ±0.5 Hz and the γ-group phosphate was 8.9 ±0.5 Hz which indicates the direct interaction of the hydrogen atoms of water with the phosphate groups. With time, the γ-group phosphate line width narrows significantly more than the α-group phosphate line width (P <0.01) indicating that the γ-group phosphate is interacting with the interstitial water, whereas the α-group is closer to the protein surface and less free to interact.
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Conclusions :
1. The large mM concentration of ATP in the metabolically quiescent lens is 3 orders of magnitude greater than what is believed to be required for all the known functions of ATP (metabolic or otherwise) combined. 2. Incubating in control H2O buffer, the different signal widths of the α-and γ-phosphate signals indicate that the ATP is acting as a hydrotrope with the adenine moiety bound to and shielding the protein surfaces and the γ-phosphate extending into the interstitial water between neighboring α-crystallin protein molecules. 3. With incubation in D2O buffer, the γ-group phosphate line width narrows significantly more than observed in the α-group phosphate. This data supports the hypothesis that ATP functions as a hydrotrope preventing cataractogensis secondary to PA. Prevention of PA is essential for maintaining lens transparency.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.