NADH photo-oxidation is enhanced by a partially purified lambda-crystallin fraction from rabbit lens.
OBJECTIVE
In the rabbit lens, high levels of reduced nicotinamide adenine dinucleotide (NADH) can function as a near-ultraviolet light (near-UV) filter, an effect apparently achieved by specific nucleotide binding to lambda-crystallin. The present investigation asks whether lambda-crystallin enhances NADH photo-oxidation by superoxide radicals produced via a photosensitization reaction of near-UV with NADH.


METHODS
Lambda-crystallin was partially purified from rabbit lens soluble fraction by a two-step gel filtration and affinity column chromatography procedure. NADH solutions with or without partially purified lambda-crystallin were subjected to near-UV irradiation or exposed to superoxide generated enzymatically by the xanthine/xanthine oxidase system. NADH oxidation was determined by assaying the decrease of absorbance at 340 nm.


RESULTS
When irradiated with near-UV, free NADH was oxidized very little in the absence of lambda-crystallin. In contrast, NADH photo-oxidation was rapidly initiated in the presence of partially purified lambda-crystallin. This lambda-crystallin-enhanced NADH photo-oxidation was totally inhibited by adding superoxide dismutase. We also found that lambda-crystallin largely increased NADH oxidation by a superoxide that is generated enzymatically. These results indicate that NADH bound to lambda-crystallin rapidly reacts with superoxides. The reactivity of bound NADH with superoxide was almost equivalent to that of ascorbic acid. However, lambda-crystallin-enhanced NADH oxidation exceeded the superoxide levels generated by NADH photosensitization and xanthine/xanthine oxidase.


CONCLUSIONS
We conclude that NADH binding to lambda-crystallin enhances NADH photo-oxidation through a radical chain reaction mechanism that is initiated by superoxides generated by NADH photosensitization and propagated by another superoxide from the molecule oxygen. High concentrations of NADH bound to lambda-crystallin may be beneficial to the rabbit lens in scavenging the low amounts of superoxide produced by near-UV absorption, since oxygen tension in the lens is very low. We also discuss the function of near-UV-filtering and the anti-photo-oxidation systems in other vertebrate lenses.
Bando
Masayasu
M
Department of Ophthalmology, Tokai University School of Medicine, Isehara, Japan.
Oka
Mikako
M
Kawai
Kenji
K
Obazawa
Hajime
H
Takehana
Makoto
M
D000818:Animals; D003459:Crystallins / Q000302:isolation & purification* / Q000378:metabolism*; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000737:chemistry*; D009243:NAD / Q000378:metabolism* / Q000528:radiation effects*; D010084:Oxidation-Reduction; D011817:Rabbits; D013481:Superoxides / Q000378:metabolism; D014466:Ultraviolet Rays; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
D003459:Crystallins; D009243:NAD; D013481:Superoxides; D019820:Xanthine; D014969:Xanthine Oxidase