In this work we have developed and validated an immunological method for detection of proteins containing tryptophan residues oxidized to NFK. The hapten used to produce the antiserum retains the distinctive cleaved indole ring of NFK, but lacks the α-amine (), allowing us to use carbodiimide chemistry for ovalbumin conjugation without formation of hapten oligomers that can impede conjugation of hapten to carrier. The extra methylene group was added to facilitate the exposure of the characteristic oxidized, cleaved indole ring for production of NFK recognizing antibodies. Our previous work (unpublished) made us aware that production of antiserum to NFK and kynurenine was not achievable by the simple expedient of conjugating either NFK or kynurenine to a carrier protein.
Western blots with two commercially available antisera confirmed these observations. Although neither was promoted as a detection agent for NFK or kynurenine, it seemed prudent to test them. Not surprisingly, neither could detect NFK in photooxidized myoglobin (data not shown). A recent paper [28
] describes an antibody produced by blocking the α-amine group of tryptophan, oxidizing it with 1
and then conjugating the resulting product to a carrier protein. This antiserum recognized photooxidized protein, but was used at a dilution of 1:50, a concentration likely to prohibit its wide-spread application due to non-specific binding in complex protein mixtures. Also, no experiments were presented to discriminate between recognition of oxidized tryptophan and general recognition of photooxidized protein.
In contrast, we have shown that our anti-NFK antiserum is both highly specific to tryptophan residues with oxidized, cleaved indole rings, and highly sensitive. Myoglobin and hSOD1 samples modified so the tryptophan residues can no longer form NFK concomitantly lose cross-reactivity ( and ). Control samples of myoglobin, hSOD1 and tryptophan free bSOD1 all exhibit little or no anti-NFK immunoreactivity ( and ). The competition ELISA determined that our antiserum does not detect tryptophan residues with intact indole rings. We also saw very little competition from kynurenine, suggesting that our antiserum specifically recognizes NFK and NFK-like antigens.
Not surprisingly, competition ELISA also shows that recognition of the actual hapten occurs at concentrations 100-fold lower than that of NFK itself. These data, however, do not adequately reflect the actual affinity of our antiserum to proteins containing NFK. The myoglobin used in our experiments contains two tryptophan residues out of a total of 154 amino acid residues, representing just 1.3% of the total protein. hSOD1, a protein of comparable mw to myoglobin, contains but a single tryptophan, yet NFK detection in both proteins occurs at levels unquestionably above background cross-reactivity.
If the two tryptophan residues of the myoglobin were completely oxidized to NFK, the 2.5 μg protein sample used in both the western blot and ELISA of would result in the production of a total of 285 pmoles of NFK. The samples oxidized for only 2 minutes contain only a fraction of the NFK in the 60 min sample (ca. 20% by ELISA analysis), yet our antiserum definitively detected NFK above background levels. This suggests that we can detect ≤ 57 pmoles of NFK within 2.5 μg of total protein.
Because mass spectrometry is a widely accepted technique for NFK identification and quantification, we also used it as an independent measure of NFK content in photooxidized myoglobin (). A subset of the myoglobin samples used for the ELISA and western analyses in were desalted, lyophilized, resuspended, digested with trypsin, and then subjected to LC/MS. The data resulting from this analysis closely corroborate our ELISA and western data, showing increased accumulation of NFK with increased photooxidation. Our immunological assay for NFK, however, is not only as sensitive as mass spectrometry, but is more amenable to analysis of a greater number of samples in a shorter time with fewer steps.
Zhang et al. [13
] used mass spectrometry to detect the conversion of the single tryptophan residue of hSOD1 to NFK and kynurenine. Our work confirms that of Zhang et al. [12
], but was achieved with the much simpler techniques of immunochemistry. The higher throughput technology of immunochemistry should allow a more detailed dissection of the conditions that produce oxidation of tryptophan to NFK in a much shorter time. It is also feasible that the anti-NFK antiserum can be used for immunoprecipitation of proteins containing NFK residues, facilitating mass spectrometric and other analyses.
Additionally, immunological detection of NFK uses a relatively low level of processing and technology, which in turn reduces the possibilities for producing artifacts. The validity and utility of a number of oxidative stress assays have come into question due to the potential for creating artifacts during sample processing and analyses [29
]. Proteins can be recovered from cells and organisms with a minimum of manipulation, and the reducing conditions employed for SDS-PAGE do not facilitate tryptophan oxidation or alteration of the end product. This is clear from the myoglobin photooxidation experiment () in which the ELISA and western results are unambiguously equivalent.
The milk oxidation experiments () validate the use of this antiserum for detection of tryptophan oxidation products in protein mixtures. In conjunction with the simple purification step of acid precipitation, we derived a tentative identification for the oxidized protein(s). These westerns also illustrate a caveat important in all immunochemistry. The milk protein westerns cannot differentiate between endogenous pre-existing tryptophan oxidation in the samples and non-specific background. ELISA analysis of these samples without western data would likely lead to the conclusion that the background cross-reactivity in milk is too high to allow detection of specific protein bands containing NFK. In cases where the background cross-reactivity is low enough, however, ELISA allows quantitative corroboration of western data.
The experiments presented here provide compelling evidence that we have developed a practical and efficient approach for detection of proteins with tryptophan residues oxidized to NFK which can be used with both single proteins as well as protein mixtures. Use of this antiserum could prove to be a valuable and versatile tool applicable not only to in vitro analysis of oxidized proteins but potentially to analysis of cells, organs and organisms and should provide a straightforward, facile and expedient method for initial assays of oxidative protein modifications and, possibly, organismal oxidative stress. Western analysis could be used to identify single protein bands exhibiting cross-reactivity, which would greatly facilitate any further analysis using MS techniques, in some cases circumventing the need for large-scale proteomic mass spectrometric experiments using methodology not readily accessible to all. The low amount of background found in these experiments also suggests that our anti-NFK antiserum may be very useful in immunohistological study of cells undergoing oxidative and/or radical stress.