Abstract
Purpose: :
to demonstrate the autofluorescense emission of a small heat shock protein (Alpha crystallin) as part of the autofluorescense emission from lipofuscin of the RPE at 488 nm of excitation.
Methods: :
1) Human alpha crystallin were purified from sonicated human lens fibers by using fast performance liquid chromatography and detected through western blotting and mass spectrometry. Also we used commercial available alfa crystallin.2) Alfa crystallin autofluorescence in solution with excitation wavelength of 488 nm was recorded in a spectrofluorimeter.3) Aggregates of alfa crystallin was identified by immunoblots of fractions belonging to ultracentrifugated discontinuous sucrose gradients in an chemical denaturant agent, in these fractions was measured the presence of autofluorescence, as well.4) The presence of alfa crystallin in lipofuscin was demonstrated like we showed in "Alpha Crystallin In Lipofuscin From Human Retinal Pigment Epithelium. 2011 Annual Meeting on Visionary Genomics. ARVO 2011 Annual Meeting"5) The autofluorescence of lipofuscin from RPE cells was detected by epifluorescence and confocal fluorescence microscopy of specimens from human cells and ARPE-19 cells.
Results: :
Alpha crystallin shows autofluorescence at 488 nm of excitation, has increasing light scattering and autofluorescence in higher concentration of the protein, wich are dismunished with denaturant agent in the buffer media.
Conclusions: :
Alpha crystallin has autofluorescence with 488 nm of excitation wavelength that depends on oligomerization state of the protein and could be related to the lipofucsin autofluorescence in between others fluorophores in lipofuscin.
Keywords: crystallins • ipofuscin • retinal pigment epithelium