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K. Sharma, S. Yellamaraju; Interaction of Prodan With Alpha B-Crystallin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2034. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the interaction of neutral fluorescence probe, prodan (6-propionyl-2-(N,N-dimethyl)-aminonaphthalene) with αB-crystallin.
Human αB-crystallin was expressed in E.coli BL21(DE3)pLyS cells and purified by ion-exchange and gel filtration chromatography. The interaction of the purified protein with prodan was measured using excitation at 380 nm and EM at 400-600 nm. The number of prodan binding site(s) per subunit was estimated by Scatchard plot whereas the effect of prodan interaction on chaperone-like activity was determined using citrate synthase and ADH aggregation assays
Prodan fluorescence was partially quenched when it interacted with αB-crystallin. Excitation of αB-crystallin mixed with prodan at 295 nm resulted in a decrease in Trp emission at 343 nm and an increase in fluorescence at 506 nm, emission maximum for protein bound prodan, suggesting an energy transfer from Trp in αB-crystallin to protein bound prodan. Scatchard analysis showed that one mole of prodan binds to each subunit of αB-crystallin. Partial unfolding of αB-crystallin with 2M urea lead to an increase in prodan fluorescence whereas exposure of αB-crystallin to 50oC resulted in decreased prodan binding suggesting that the two denaturing conditions have differing effect on αB-crystallin. Prior binding of prodan to αB-crystallin decreased its chaperone-like activity toward denaturing ADH and citrate synthase.
Prodan interacts with αB-crystallin at or near the chaperone site. The prodan interacting hydrophobic site(s) exposed during urea and heat denaturation of αB-crystallin are different.
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