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Time-Resolved
Spectroscopic Measurements of Anaerobically Modified  -Crystallin
and Aged Human Lens Protein
Ervin, Lisa1,
Dillon, James2 and Gaillard, Elizabeth1
Northern Illinois University, DeKalb, IL 601151
Columbia University, New York, NY 100322
Abstract-
3-Hydroxykynurenine (3-HK) and 3-hydroxykynurenine glucoside (3-HKG)
have been found to covalently attach to lens protein resulting in protein
yellowing. Irradiating -crystallin
in the presence of 3-HK under anaerobic conditions results in spectroscopic
changes similar to those seen in the primate lens. The yellow chromophores
of the modified protein may accelerate damage to the lens through photosensitization,
however the mechanism is not known. In this study, samples of 3-HK and
-crystallin
were bubbled with Ar and irradiated for 17 hours with light similar
to that which reaches the lens in vivo. The modified -crystallin
was isolated from the reaction mixture and analyzed by time-resolved
spectroscopy in solutions that were air saturated or bubbled with Ar
or O2. Excitation of the modified -crystallin
results in a broad transient absorption with maxima at approximately
350, 430, and 500 nm. The decay kinetics are best fit to a double exponential
model indicating that the spectra arise from at least two different
transient species or from a chromophore that resides in several different
environments that are non-interconverting on the time scale of the measurement.
The decay rate constants observed for the modified -crystallin
are all faster when compared to similar data for pure -crystallin.
The absorption at 350 nm appears to be more sensitive to O2
than the absorption at 430 nm and the bimolecular rate constant for
quenching by O2 is estimated to be 3 x 107 M-1s-1.
Data from the modified -crystallin
will also be compared to that from aged human lens protein. -Crystallin
modified in this model system may allow the photochemical mechanisms
of damage in the lens to be determined.
Keywords: lens
protein, 3-hydroxykynurenine, light damage
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