Characterization and Quantification of Oxidized High Mobility Group Box 1 Proteoforms Secreted from Hepatocytes by Toxic Levels of Acetaminophen.

The high mobility group box 1 (HMGB1), which is released during acute acetaminophen (APAP) overdose, is thought to mediate a subsequent immune response, particularly hepatic infiltration of macrophages. The redox behavior of HMGB1 and the proteoforms of HMGB1 present in oxidative environments has been the subject of a number of confusing and contradictory studies. Therefore, a stable isotope dilution two-dimensional nanoultrahigh-performance liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry method was developed in order to characterize and quantify oxidative modifications to the cysteine (Cys) residues (Cys-23, Cys-45, and Cys-106) that are present in HMGB1. Disulfide linkages were determined using carbamidoethyl derivatization before and after reduction as well as by direct analysis of disulfide cross-linked peptides. A stable isotope labeled form of HMGB1 was used as an internal standard to correct for sample to sample differences in immunoaffinity precipitation, derivatization, and electrospray ionization. Four discrete HMGB1 proteoforms were found to be released from a hepatocarcinoma cell model of APAP overdose after 24 h. Fully reduced HMGB1 with all three Cys-residues in their free thiol state accounted for 18% of the secreted HMGB1. The proteoform with disulfide between Cys-23 and Cys-45 accounted for 24% of the HMGB1. No evidence was obtained for a disulfide cross-link between Cys-106 and the other two Cys-residues. However, 45% of the HMGB1 formed a cross-link with unidentified intracellular proteins via an intermolecular disulfide bond, and 12% was present as the terminally oxidized cysteic acid. Surprisingly, there was no evidence for the formation of HMGB1 disulfides with GSH or other low molecular weight thiols. Secreted plasma HMGB1 Cys-23/Cys45 disulfide proteoform together with the Cys-106/protein disulfide proteoforms could potentially serve as early biomarkers of hepatoxicity after APAP overdose as well as biomarkers of drug-induced liver injury.

Simultaneous Quantification of Mitochondrial Mature Frataxin and Extra-Mitochondrial Frataxin Isoform E in Friedreich's Ataxia Blood.

Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by an intronic guanine-adenine-adenine (GAA) triplet expansion in the frataxin (FXN) gene, which leads to reduced expression of full-length frataxin (1-210) also known as isoform 1. Full-length frataxin has a mitochondrial targeting sequence, which facilitates its translocation into mitochondria where it is processed through cleavage at G41-L42 and K80-S81 by mitochondrial processing (MPP) to release mitochondrial mature frataxin (81-210). Alternative splicing of FXN also leads to expression of N-terminally acetylated extra-mitochondrial frataxin (76-210) named isoform E because it was discovered in erythrocytes. Frataxin isoforms are undetectable in serum or plasma, and originally whole blood could not be used as a biomarker in brief therapeutic trials because it is present in erythrocytes, which have a half-life of 115-days and so frataxin levels would remain unaltered. Therefore, an assay was developed for analyzing frataxin in platelets, which have a half-life of only 10-days. However, our discovery that isoform E is only present in erythrocytes, whereas, mature frataxin is present primarily in short-lived peripheral blood mononuclear cells (PBMCs), granulocytes, and platelets, meant that both proteins could be quantified in whole blood samples. We now report a quantitative assay for frataxin proteoforms in whole blood from healthy controls and FRDA patients. The assay is based on stable isotope dilution coupled with immunoprecipitation (IP) and two-dimensional-nano-ultrahigh performance liquid chromatography/parallel reaction monitoring/high resolution mass spectrometry (2D-nano-UHPLC-PRM/HRMS). The lower limit of quantification was 0.5 ng/mL for each proteoform and the assays had 100% sensitivity and specificity for discriminating between healthy controls (n = 11) and FRDA cases (N = 100 in year-1, N = 22 in year-2,3). The mean levels of mature frataxin in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 7.5 ± 1.5 ng/mL and 2.1 ± 1.2 ng/mL, respectively. The mean levels of isoform E in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 26.8 ± 4.1 ng/mL and 4.7 ± 3.3 ng/mL, respectively. The mean levels of total frataxin in whole blood from healthy controls and homozygous FRDA patients were significantly different (p < 0.0001) at 34.2 ± 4.3 ng/mL and 6.8 ± 4.0 ng/mL, respectively. The assay will make it possible to rigorously monitor the natural history of the disease and explore the potential role of isoform E in etiology of the disease. It will also facilitate the assessment of therapeutic interventions (including gene therapy approaches) that attempt to increase frataxin protein expression as a treatment for this devastating disease.

Extra-mitochondrial mouse frataxin and its implications for mouse models of Friedreich's ataxia.

Mature frataxin is essential for the assembly of iron-sulfur cluster proteins including a number of mitochondrial enzymes. Reduced levels of mature frataxin (81-20) in human subjects caused by the genetic disease Friedreich's ataxia results in decreased mitochondrial function, neurodegeneration, and cardiomyopathy. Numerous studies of mitochondrial dysfunction have been conducted using mouse models of frataxin deficiency. However, mouse frataxin that is reduced in these models, is assumed to be mature frataxin (78-207) by analogy with human mature frataxin (81-210). Using immunoaffinity purification coupled with liquid chromatography-high resolution tandem mass spectrometry, we have discovered that mature frataxin in mouse heart (77%), brain (86%), and liver (47%) is predominantly a 129-amino acid truncated mature frataxin (79-207) in which the N-terminal lysine residue has been lost. Mature mouse frataxin (78-207) only contributes 7-15% to the total frataxin protein present in mouse tissues. We have also found that truncated mature frataxin (79-207) is present primarily in the cytosol of mouse liver; whereas, frataxin (78-207) is primarily present in the mitochondria. These findings, which provide support for the role of extra-mitochondrial frataxin in the etiology of Friedreich's ataxia, also have important implications for studies of mitochondrial dysfunction conducted in mouse models of frataxin deficiency.

Voriconazole enhances UV-induced DNA damage by inhibiting catalase and promoting oxidative stress.

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer and is associated with cumulative UV exposure. Studies have shown that prolonged voriconazole use promotes cSCC formation; however, the biological mechanisms responsible for the increased incidence remain unclear. Here, we show that voriconazole directly increases oxidative stress in human keratinocytes and promotes UV-induced DNA damage as determined by comet assay, 8-oxoguanine immunofluorescence and mass spectrometry. Voriconazole treatment of human keratinocytes potentiates UV-induced apoptosis and activation of the p38 MAP kinase and 53BP1 UV stress response pathways. The p38 MAP kinase activation promoted by voriconazole exposure can be mitigated by pretreating keratinocytes with N-acetylcysteine. Voriconazole increases oxidative stress in keratinocytes by directly inhibiting catalase leading to lower intracellular NADPH levels and the triazole moieties in voriconazole are critical for inhibiting catalase. Furthermore, voriconazole is shown to promote UV-induced dysplasia in an in vivo model. Together, these data demonstrate that voriconazole potentiates oxidative stress in UV-irradiated keratinocytes through catalase inhibition. Use of antioxidants may mitigate the pro-oncogenic effects of voriconazole.

Evaluation of antibodies for western blot analysis of frataxin protein isoforms.

Frataxin is the protein that is down-regulated in Friedreich ataxia (FRDA), an autosomal recessive genetic disease caused by an intronic GAA repeat expansion in intron-1 of the FXN gene. The GAA repeats result in epigenetic silencing of the FXN gene and reduced expression of the cytosolic full-length frataxin (1-210) protein. Full length frataxin translocates to the mitochondria, leading to formation of mature frataxin (81-210) formed by cleavage of the mitochondrial targeting sequence at K-80 of the full-length protein. There are currently no approved treatments for FRDA, although experimental approaches involving up-regulation or replacement of mature frataxin protein through numerous approaches are being tested. Many of the pre-clinical studies of these experimental approaches are conducted in mouse and monkey models as well as in human cell lines. Consequently, well-validated antibodies are required for use in western blot analysis to determine whether levels of various forms of frataxin have been increased. Here we examined the specificity of five commercially available anti-frataxin antibodies and determined whether they detect mature frataxin in mouse heart tissue. Four protein standards of monkey, human, and mouse frataxin as well as mouse heart tissue were examined using polyacrylamide gel electrophoresis (PAGE) in combination with western blot analysis. One antibody failed to detect any of the frataxin standards or endogenous frataxin in mouse heart tissue. Three of the antibodies detected a protein in mouse heart tissue that ran slightly faster on PAGE (at 23.4 kDa) to that predicted for full-length frataxin (23.9 kDa). One antibody detected all four frataxin standards as well as endogenous mouse mature frataxin in mouse tissue. Significantly, this antibody, which will be useful for monitoring mature frataxin levels in monkey, human, and mouse tissues, did not detect a protein in mouse heart tissue at 23.4 kDa. Therefore, antibodies detecting the immunoreactive protein at 23.4 kDa could be misleading when testing for the up-regulation of frataxin in animal models.

Characterization of a new N-terminally acetylated extra-mitochondrial isoform of frataxin in human erythrocytes.

Frataxin is a highly conserved protein encoded by the frataxin (FXN) gene. The full-length 210-amino acid form of protein frataxin (1-210; isoform A) expressed in the cytosol of cells rapidly translocates to the mitochondria, where it is converted to the mature form (81-210) by mitochondrial processing peptidase. Mature frataxin (81-210) is a critically important protein because it facilitates the assembly of mitochondrial iron-sulfur cluster protein complexes such as aconitase, lipoate synthase, and succinate dehydrogenases. Decreased expression of frataxin protein is responsible for the devastating rare genetic disease of Friedreich's ataxia. The mitochondrial form of frataxin has long been thought to be present in erythrocytes even though paradoxically, erythrocytes lack mitochondria. We have discovered that erythrocyte frataxin is in fact a novel isoform of frataxin (isoform E) with 135-amino acids and an N-terminally acetylated methionine residue. There is three times as much isoform E in erythrocytes (20.9 ± 6.4 ng/mL) from the whole blood of healthy volunteers (n = 10) when compared with the mature mitochondrial frataxin present in other blood cells (7.1 ± 1.0 ng/mL). Isoform E lacks a mitochondrial targeting sequence and so is distributed to both cytosol and the nucleus when expressed in cultured cells. When extra-mitochondrial frataxin isoform E is expressed in HEK 293 cells, it is converted to a shorter isoform identical to the mature frataxin found in mitochondria, which raises the possibility that it is involved in disease etiology. The ability to specifically quantify extra-mitochondrial and mitochondrial isoforms of frataxin in whole blood will make it possible to readily follow the natural history of diseases such as Friedreich's ataxia and monitor the efficacy of therapeutic interventions.

Quantification of Serum High Mobility Group Box 1 by Liquid Chromatography/High-Resolution Mass Spectrometry: Implications for Its Role in Immunity, Inflammation, and Cancer.

High mobility group box 1 (HMGB1) is a non-histone chromosomal protein, which can be secreted through a variety of pathways and bind to pattern recognition receptors to release pro-inflammatory cytokines. Previous studies have suggested that HMGB1 is upregulated in numerous inflammatory diseases and that it could be a biomarker for such diseases. However, these studies used immunoassay-based methods to analyze serum HMGB1. Autoantibodies to HMGB1 in serum are found in healthy control subjects as well as in patients with different diseases. HMGB1 also binds to haptoglobin, a highly abundant plasma protein. This means that antibodies used in immunoassays must compete with binding of HMGB1 to endogenous serum HMGB1 autoantibodies and haptoglobin. To overcome these potential problems, we developed and validated a specific and sensitive assay based on stable isotope dilution and immunopurification to quantify HMGB1 in plasma and serum using two-dimensional nano-ultra-high-performance liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry. Using this assay, we found that serum HMGB1 in 24 healthy control subjects (6.0 ± 2.1 ng/mL) was above the mean concentration reported for 18 different diseases (5.4 ± 2.8 ng/mL) where the analyses were conducted with immunoassay methodology. In light of our finding, the role of HMGB1 in these diseases will have to be re-evaluated. The concentration of HMGB1 in citrated and EDTA-treated plasma from the same healthy control subjects was below the limit of detection of our assay (1 ng/mL), confirming that HMGB1 in serum arises when blood is allowed to clot. This means that future studies on the role of HMGB1 in vivo should be conducted on plasma rather than serum.

The CPT1a inhibitor, etomoxir induces severe oxidative stress at commonly used concentrations.

Etomoxir (ETO) is a widely used small-molecule inhibitor of fatty acid oxidation (FAO) through its irreversible inhibitory effects on the carnitine palmitoyl-transferase 1a (CPT1a). We used this compound to evaluate the role of fatty acid oxidation in rapidly proliferating T cells following costimulation through the CD28 receptor. We show that ETO has a moderate effect on T cell proliferation with no observable effect on memory differentiation, but a marked effect on oxidative metabolism. We show that this oxidative metabolism is primarily dependent upon glutamine rather than FAO. Using an shRNA approach to reduce CPT1a in T cells, we further demonstrate that the inhibition of oxidative metabolism in T cells by ETO is independent of its effects on FAO at concentrations exceeding 5 µM. Concentrations of ETO above 5 µM induce acute production of ROS with associated evidence of severe oxidative stress in proliferating T cells. In aggregate, these data indicate that ETO lacks specificity for CTP1a above 5 µM, and caution should be used when employing this compound for studies in cells due to its non-specific effects on oxidative metabolism and cellular redox.

An oxidized abasic lesion inhibits base excision repair leading to DNA strand breaks in a trinucleotide repeat tract.

Oxidative DNA damage and base excision repair (BER) play important roles in modulating trinucleotide repeat (TNR) instability that is associated with human neurodegenerative diseases and cancer. We have reported that BER of base lesions can lead to TNR instability. However, it is unknown if modifications of the sugar in an abasic lesion modulate TNR instability. In this study, we characterized the effects of the oxidized sugar, 5'-(2-phosphoryl-1,4-dioxobutane)(DOB) in CAG repeat tracts on the activities of key BER enzymes, as well as on repeat instability. We found that DOB crosslinked with DNA polymerase ß and inhibited its synthesis activity in CAG repeat tracts. Surprisingly, we found that DOB also formed crosslinks with DNA ligase I and inhibited its ligation activity, thereby reducing the efficiency of BER. This subsequently resulted in the accumulation of DNA strand breaks in a CAG repeat tract. Our study provides important new insights into the adverse effects of an oxidized abasic lesion on BER and suggests a potential alternate repair pathway through which an oxidized abasic lesion may modulate TNR instability.

Analysis of HETEs in human whole blood by chiral UHPLC-ECAPCI/HRMS.

The biosynthesis of eicosanoids occurs enzymatically via lipoxygenases, cyclooxygenases, and cytochrome P450, or through nonenzymatic free radical reactions. The enzymatic routes are highly enantiospecific. Chiral separation and high-sensitivity detection methods are required to differentiate and quantify enantioselective HETEs in complex biological fluids. We report here a targeted chiral lipidomics analysis of human blood using ultra-HPLC-electron capture (EC) atmospheric pressure chemical ionization/high-resolution MS. Monitoring the high-resolution ions formed by the fragmentation of pentafluorobenzyl derivatives of oxidized lipids during the dissociative EC, followed by in-trap fragmentation, increased sensitivity by an order of magnitude when compared with the unit resolution MS. The 12(S)-HETE, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid [12(S)-HHT], and 15(S)-HETE were the major hydroxylated nonesterified chiral lipids in serum. Stimulation of whole blood with zymosan and lipopolysaccharide (LPS) resulted in stimulus- and time-dependent effects. An acute exposure to zymosan induced ∼80% of the chiral plasma lipids, including 12(S)-HHT, 5(S)-HETE, 15(R)-HETE, and 15(S)-HETE, while a maximum response to LPS was achieved after a long-term stimulation. The reported method allows for a rapid quantification with high sensitivity and specificity of enantiospecific responses to in vitro stimulation or coagulation of human blood.

Liquid Chromatography-High Resolution Mass Spectrometry Analysis of Platelet Frataxin as a Protein Biomarker for the Rare Disease Friedreich's Ataxia.

Friedreich's ataxia (FA) is an autosomal recessive disease caused by an intronic GAA triplet expansion in the FXN gene, leading to reduced expression of the mitochondrial protein frataxin. FA is estimated to affect 1 in 50 000 with a mean age of death in the fourth decade of life. There are no approved treatments for FA, although experimental approaches, which involve up-regulation or replacement of frataxin protein, are being tested. Frataxin is undetectable in serum or plasma, and whole blood cannot be used because it is present in long-lived erythrocytes. Therefore, an assay was developed for analyzing frataxin in platelets, which have a half-life of 10 days. The assay is based on stable isotope dilution immunopurification two-dimensional nano-ultra high performance liquid chromatography/parallel reaction monitoring/mass spectrometry. The lower limit of quantification was 0.078 pg frataxin/µg protein, and the assay had 100% sensitivity and specificity for discriminating between controls and FA cases. The mean levels of control and FA platelet frataxin were 9.4 ± 2.6 and 2.4 ± 0.6 pg/µg protein, respectively. The assay should make it possible to rigorously monitor the effects of therapeutic interventions on frataxin expression in this devastating disease.

Abnormal polyamine metabolism is unique to the neuropathic forms of MPS: potential for biomarker development and insight into pathogenesis.

The mucopolysaccharidoses (MPS) are rare genetic disorders marked by severe somatic and neurological symptoms. Development of treatments for the neurological manifestations of MPS has been hindered by the lack of objective measures of central nervous system disease burden. Identification of biomarkers for central nervous system disease in MPS patients would facilitate the evaluation of new agents in clinical trials. High throughput metabolite screening of cerebrospinal fluid (CSF) samples from a canine model of MPS I revealed a marked elevation of the polyamine, spermine, in affected animals, and gene therapy studies demonstrated that reduction of CSF spermine reflects correction of brain lesions in these animals. In humans, CSF spermine was elevated in neuropathic subtypes of MPS (MPS I, II, IIIA, IIIB), but not in subtypes in which cognitive function is preserved (MPS IVA, VI). In MPS I patients, elevated CSF spermine was restricted to patients with genotypes associated with CNS disease and was reduced following hematopoietic stem cell transplantation, which is the only therapy currently capable of improving cognitive outcomes. Additional studies in cultured neurons from MPS I mice showed that elevated spermine was essential for the abnormal neurite overgrowth exhibited by MPS neurons. These findings offer new insights into the pathogenesis of CNS disease in MPS patients, and support the use of spermine as a new biomarker to facilitate the development of next generation therapeutics for MPS.

Comparison of statistical methods for detection of serum lipid biomarkers for mesothelioma and asbestos exposure.

AIM: We compared three statistical methods in selecting a panel of serum lipid biomarkers for mesothelioma and asbestos exposure. MATERIALS & METHODS: Serum samples from mesothelioma, asbestos-exposed subjects and controls (40 per group) were analyzed. Three variable selection methods were considered: top-ranked predictors from univariate model, stepwise and least absolute shrinkage and selection operator. Crossed-validated area under the receiver operating characteristic curve was used to compare the prediction performance. RESULTS: Lipids with high crossed-validated area under the curve were identified. Lipid with mass-to-charge ratio of 372.31 was selected by all three methods comparing mesothelioma versus control. Lipids with mass-to-charge ratio of 1464.80 and 329.21 were selected by two models for asbestos exposure versus control. CONCLUSION: Different methods selected a similar set of serum lipids. Combining candidate biomarkers can improve prediction.

Identification of proximal sites for unwound DNA substrate in Escherichia coli topoisomerase I with oxidative crosslinking.

Topoisomerases catalyze changes in DNA topology by directing the movement of DNA strands through consecutive cleavage-rejoining reactions of the DNA backbone. We describe the use of a phenylselenyl-modified thymidine incorporated into a specific position of a partially unwound DNA substrate in crosslinking studies of Escherichia coli topoisomerase I to gain new insights into its catalytic mechanism. Crosslinking of the phenylselenyl-modified thymidine to the topoisomerase protein was achieved by the addition of a mild oxidant. Following nuclease and trypsin digestion, lysine residues on topoisomerase I crosslinked to the modified thymidine were identified by mass spectrometry. The crosslinked sites may correspond to proximal sites for the unwound DNA strand as it interacts with enzyme in the different stages of the catalytic cycle.

The phospholipase A2 activity of peroxiredoxin 6 modulates NADPH oxidase 2 activation via lysophosphatidic acid receptor signaling in the pulmonary endothelium and alveolar macrophages.

Peroxiredoxin 6 (Prdx6) is essential for activation of NADPH oxidase type 2 (NOX2) in pulmonary microvascular endothelial cells (PMVECs), alveolar macrophages (AMs), and polymorphonuclear leukocytes. Angiotensin II and phorbol ester increased superoxide/H2O2 generation in PMVECs, AMs, and isolated lungs from wild-type (WT) mice, but had much less effect on cells or lungs from Prdx6-null or Prdx6-D140A-knock-in mice that lack the phospholipase A2 activity (PLA2) of Prdx6; addition of either lysophosphatidylcholine (LPC) or lysophosphatidic acid (LPA) to cells restored their oxidant generation. The generation of LPC by PMVECs required Prdx6-PLA2 We propose that Prdx6-PLA2 modulates NOX2 activation by generation of LPC that is converted to LPA by the lysophospholipase D activity of autotaxin (ATX/lysoPLD). Inhibition of lysoPLD with HA130 (cells,10 µM; lungs, 20 µM; IC50, 29 nM) decreased agonist-induced oxidant generation. LPA stimulates pathways regulated by small GTPases through binding to G-protein-coupled LPA receptors (LPARs). The LPAR blocker Ki16425 (cells, 10 µM; lungs, 25 µM; Ki, 0.34 µM) or cellular knockdown of LPAR type 1 decreased oxidant generation and blocked translocation of rac1 to plasma membrane. Thus, Prdx6-PLA2 modulates NOX2 activation through generation of LPC for conversion to LPA; binding of LPA to LPAR1 signals rac activation.-Vázquez-Medina, J. P., Dodia, C., Weng, L., Mesaros, C., Blair, I. A., Feinstein, S. I., Chatterjee, S., Fisher, A. B. The phospholipase A2 activity of peroxiredoxin 6 modulates NADPH oxidase 2 activation via lysophosphatidic acid receptor signaling in the pulmonary endothelium and alveolar macrophages.

Rapid Histone-Catalyzed DNA Lesion Excision and Accompanying Protein Modification in Nucleosomes and Nucleosome Core Particles.

C5'-Hydrogen atoms are frequently abstracted during DNA oxidation. The oxidized abasic lesion 5'-(2-phosphoryl-1,4-dioxobutane) (DOB) is an electrophilic product of the C5'-radical. DOB is a potent irreversible inhibitor of DNA polymerase ß, and forms interstrand cross-links in free DNA. We examined the reactivity of DOB within nucleosomes and nucleosome core particles (NCPs), the monomeric component of chromatin. Depending upon the position at which DOB is generated within a NCP, it is excised from nucleosomal DNA at a rate 275-1500-fold faster than that in free DNA. The half-life of DOB (7.0-16.8 min) in NCPs is shorter than any other abasic lesion. DOB's lifetime in NCPs is also significantly shorter than the estimated lifetime of an abasic site within a cell, suggesting that the observed chemistry would occur intracellularly. Histones also catalyze DOB excision when the lesion is present in the DNA linker region of a nucleosome. Schiff-base formation between DOB and histone proteins is detected in nucleosomes and NCPs, resulting in pyrrolone formation at the lysine residues. The lysines modified by DOB are often post-translationally modified. Consequently, the histone modifications described herein could affect the regulation of gene expression and may provide a chemical basis for the cytotoxicity of the DNA damaging agents that produce this lesion.

Probing interactions between lysine residues in histone tails and nucleosomal DNA via product and kinetic analysis.

The histone proteins in nucleosome core particles are known to catalyze DNA cleavage at abasic and oxidized abasic sites, which are produced by antitumor antibiotics and as a consequence of other modalities of DNA damage. The lysine rich histone tails whose post-translational modifications regulate genetic expression in cells are mainly responsible for this chemistry. Cleavage at a C4'-oxidized abasic site (C4-AP) concomitantly results in modification of lysine residues in histone tails. Using LC-MS/MS, we demonstrate here that that Lys8, -12, -16, and -20 of histone H4 were modified when C4-AP was incorporated at a hot spot (superhelical location 1.5) for DNA damage within a nucleosome core particle. A new DNA-protein cross-linking method that provides a more quantitative analysis of individual amino acid reactivity is also described. DNA-protein cross-links were produced by an irreversible reaction between a nucleic acid electrophile that was produced following oxidatively induced rearrangement of a phenyl selenide derivative of thymidine (3) and nucleophilic residues within proteins. In addition to providing high yields of DNA-protein cross-links, kinetic analysis of the cross-linking reaction yielded rate constants that enabled ranking the contributions by individual or groups of amino acids. Cross-linking from 3 at superhelical location 1.5 revealed the following order of reactivity for the nucleophilic amino acids in the histone H4 tail: His18 > Lys16 > Lys20 ≈ Lys8, Lys12 > Lys5. Cross-linking via 3 will be generally useful for investigating DNA-protein interactions.

Deconvoluting the reactivity of two intermediates formed from modified pyrimidines.

Generation of the 5-(2'-deoxyuridinyl)methyl radical (6) was reexamined. Trapping by 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl confirms that 6 is generated. However, trapping by methoxyamine reveals that the respective carbocation (10) is also produced. Examining the effects of these traps on products in DNA reveals that the carbocation and not 6 yields interstrand cross-links. Cross-link formation from the carbocation is consistent with DFT calculations that predict that addition at the N1 position of dA is essentially barrierless.