Comparison of ultraviolet induced photo-kinetics for lens-derived and recombinant beta-crystallins.
OBJECTIVE
The photobiology of purified recombinant crystallins has not been studied. Here we examine photo-induced aggregation of purified recombinant mouse betaA3-crystallin (rbetaA3) and compare it with that of betaL-crystallins isolated from bovine lenses.


METHODS
rbetaA3-Crystallin was expressed in baculovirus-infected Sf9 cells and purified by ion-exchange and gel-filtration chromatography. Protein solutions (pH 7.4) were irradiated at room temperature using a 308 nm excimer laser and light scattering was registered by attenuation of an unabsorbed beam of red light (670 nm).


RESULTS
Irradiation of bovine alpha-crystallin, betaL-crystallin, rbetaA3-crystallin and gammaB-crystallin resulted in formation of insoluble aggregates with subsequent light scattering. Different slopes and threshold energies were observed for light scattering by each of these species. Sensitivity to ultraviolet irradiation induced light scattering as determined from threshold energies varied, with gamma-crystallins showing the greatest sensitivity, the betaL- and rbetaA3-crystallins showing an intermediate sensitivity and alpha-crystallins much less sensitive. Low doses (100 J/cm2) resulted in irreversible formation of water soluble oligomers but no insoluble aggregates as indicated by changes in light transmission. The photo-behavior of rbA3 was similar to mixed betaL-crystallin and different from that of alpha- and gamma-crystallins.


CONCLUSIONS
Ultraviolet induced sensitivity of purified recombinant crystallins reflects that of mixed crystallin populations and should provide an indication of the pathogenicity of specific crystallin sequence changes associated with lens aging and hereditary cataract.
Ostrovsky
M A
MA
Institute of Biochemical Physics, Moscow, Russia. ostrovsky@sky.chph.ras.ru
Sergeev
Y V
YV
Atkinson
D B
DB
Soustov
L V
LV
Hejtmancik
J F
JF
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003459:Crystallins / Q000235:genetics / Q000302:isolation & purification / Q000528:radiation effects*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D007700:Kinetics; D007908:Lens, Crystalline / Q000737:chemistry / Q000528:radiation effects; D051379:Mice; D018462:Photobiology; D011489:Protein Denaturation; D011994:Recombinant Proteins; D012542:Scattering, Radiation; D014162:Transfection; D014466:Ultraviolet Rays; D038205:beta-Crystallin A Chain
C051374:Cryba1 protein, mouse; D003459:Crystallins; D011994:Recombinant Proteins; D038205:beta-Crystallin A Chain