This work used a combination of in vitro and cell culture experiments to study the effect of four biologically interesting porphyrins () and UVA exposure on lens α-crystallin. Coomassie staining showed that neither UVA radiation alone nor addition of porphyrin alone results in α-crystallin polymerization (). Increasing photosensitization, however, by either increased UVA exposure in the presence of a porphyrin or increased porphyrin concentration in the presence of UVA radiation, does result in more extensive protein polymerization. The NFK westerns show that NFK accumulation parallels protein polymerization, which also increases with increasing photosensitization. Of the porphyrins used, TPPS was the most potent photosensitizer in vitro, causing both more extensive polymerization and NFK accumulation at the lowest concentration of porphyrin and shortest duration of UVA exposure.
To differentiate between the contributions of Type I (radical) and Type II (1
) reactions to the protein modifications observed in we used the 1
quencher sodium azide and the radical scavenger/spin trap DMPO (). Azide reduced cross-linking of α-crystallin more effectively than DMPO, indicating that Type II reactions were primarily responsible for increased polymerization of the protein. Decrease in NFK accumulation roughly paralleled the differences in cross-linking, again suggesting that 1
was responsible for tryptophan residue conversion to NFK. The anti-DMPO western blots, however, showed that Type I reactions also play a role in porphyrin-mediated photosensitization of α-crystallin. Radicals are trapped by DMPO in all photosensitized samples, and, in the samples irradiated in the presence of Uro, radical trapping/scavenging reduces both cross-linking and NFK accumulation. In addition to Type I radical-mediated events there is also evidence for the production of radicals from 1
generated protein peroxides which can, in the presence of metals or UV radiation, decompose to radicals (16
). This UV radiation catalyzed decomposition of protein peroxides to radicals could explain the decrease in DMPO radical adduct production seen in photosensitized samples containing both DMPO and azide (, bottom panel).
Absorbance () and fluorescence () spectra were obtained to assess binding between protein and porphyrin. The absorbance and fluorescence maxima of both TPPS and THPP are shifted in the presence of α-crystallin, indicated binding between porphyrin and protein. Ce6 appears to participate in a less robust interaction with α-crystallin, as the spectra are altered in amplitude but show a less pronounced shift in wavelength. Uro, however, appears to have no interaction with α-crystallin as neither spectrum is altered in the presence of the protein ( and ) (11
). Porphyrin interaction with α-crystallin influences the extent of 1
-mediated polymerization seen in our in vitro
experiments ( and ). The porphyrins that interact the most strongly with the protein (TPPS and THPP) are also more resistant to azide quenching.
Recently, Medinas et al (29
) described the characterization of a tryptophan dimer resulting from the carbonate-dependent peroxidase activity of human SOD1 (30
). Interestingly, NFK formation and this novel mode of cross-linking and appear mutually exclusive. When they used reaction conditions focused on optimizing hSOD1 dimer yield Medinas et al (29
) detected no NFK in the dimer. Additionally, our original publication describing the anti-NFK antibody (25
) showed that while increasing H2
concentration up to 10 mM increased NFK accumulation in SOD1, dimer formation was maximal at 0.5 to 1.0 mM H2
. It is unlikely, therefore, that the protein polymers seen in are due to tryptophan dimerization. There is, however, evidence that dityrosine formation occurs in crystallin proteins via a radical-mediated process (32
). And, in addition to the 8 tyrosine residues found in the two subunits of α-crystallin, there are also a total of 16 histidine residues which can form His-His and His-Lys crosslinks (16
The confocal microscopy analysis () shows that in HLE cells the extent of porphyrin photosensitization is further influenced by their differential subcellular localization. In this experiment, while all four porphyins promote accumulation of NFK, substantial NFK accumulation that colocalizes with α-crystallin is only present in Ce6- and THHP-photosensitized cells. Thus, while TPPS promotes abundant oxidation of tryptophan to NFK in vitro
, in HLE cells, where the acidic TPPS accumulates in lysosomes (33
), there is little interaction between the porphyrin and the protein and therefore less photosensitized damage. THPP and Ce6, on the other hand, localize in HLE cells in the same compartment as α-crystallin, enabling physical interactions between protein and porphyrin, allowing opportunity for extensive NFK accumulation in α-crystallin protein molecules. The capacity of each porphyrin to photosensitize α-crystallin is a result of a combination of factors, including absorption of radiant energy, 1
quantum yield, and ability to bind to α-crystallin and/or to co-localize with α-crystallin in HLE cells.
Previous studies have examined photosensitization of α-crystallin by porphyrins and other biologically relevant substances (11
). Porphyrins (TPPS, Uro, hematoporphyin) and other photosensitizers such as hypericin, an ingredient of the herb St. John’s wort used to treat depression, and xanthurenic acid, a metabolite of tryptophan, all facilitated polymerization of α-crystallin under UVA irradiation and also promoted the oxidation of specific amino acid residues such as histidine, methionine, tyrosine and tryptophan. In this work we demonstrated that the porphyrin-mediated photooxidation of α-crystallin that leads to protein polymerization also leads to accumulation of NFK in monomers, dimers and higher molecular weight species. Additionally, we were able to detect the oxidation of cellular tryptophan residues to NFK in human lens epithelial cells, and to visualize the colocalization of NFK with α-crystallin in photosensitized cells.
The absorption spectrum of the human lens changes with age. The adult human lens absorbs most light and radiation between wavelengths of 295 to 400 nm due to the presence of 3-OH kynurenine and its glucoside, (36
) while the aged human lens absorbs longer wavelengths of light into the blue visible region due to a change in lenticular chromophores (20
). Photosensitizers which absorb wavelengths of light above 400-450 can damage both the lens and retina. Because NFK is itself a 1
-generating photosensitizer, accumulation of NFK in lens proteins serves to exacerbate the potential for light-mediated damage. Furthermore, the long-lived triplet state of a photosensitizer can react with reducing substrates to produce radicals in type I reactions (18
), and these radicals are also capable of altering amino acid side chains as well as the protein backbone.
Although the other eye lens crystallins (beta- and gamma-crystallins) represent a smaller percentage of the total protein of the lens, they are also susceptible to oxidation of tryptophan residues to NFK (38
). Because α-crystallin is a chaperone protein in the lens and other tissues, these observations may have implications that extend beyond eye functioning and health to areas related to protein folding and stress physiology. Indeed, there has been growing interest in the effect of post-translational modifications on α-crystallin efficiency as a chaperone (39
). Abraham et al (39
) observe an inverse correlation between glycation of lysine residues of and chaperone efficiency. The availability and accessibility of NFK immunological detection will facilitate the unraveling of the potential effect of tryptophan residue oxidation to NFK on chaperone activity.