Andre D'Avignon

Human neutrophils employ the myeloperoxidase-hydrogen peroxide-chloride system to oxidize α-amino acids to a family of reactive aldehydes: Mechanistic studies identifying labile intermediates along the reaction pathway

We have recently demonstrated that neutrophils oxidize nearly all of the amino acids commonly found in plasma to a corresponding family of aldehydes in high yield. The reaction is mediated by hypochlorous acid (HOCl), the major oxidant generated by the myeloperoxidase-H2O2-Cl−system of phagocytes. We now present evidence for the underlying mechanism of this reaction, including the structural requirements and reaction intermediates formed. Utilizing mass spectrometry and isotopically labeled amino acids, we rule out hydrogen atom abstraction from the α-carbon as the initial event in aldehyde formation during amino acid oxidation, a pathway known to occur with ionizing radiation. Aldehyde generation from amino acids required the presence of an α-amino moiety; β- and ε-amino acids did not form aldehydes upon oxidation by either the myeloperoxidase system or HOCl, generating stable monochloramines instead. UV difference spectroscopy, high pressure liquid chromatography, and multinuclear (1H,15N) NMR spectroscopy established that the conversion of α-amino acids into aldehydes begins with generation of an unstable α-monochloramine, which subsequently decomposes to yield an aldehyde. Precursor product relationships between α-amino acid and α-monochloramine, and α-monochloramine and aldehyde were confirmed by high pressure liquid chromatography purification of the reaction intermediate and subsequent 1H and 15N NMR spectroscopy. Collectively, these results detail the chemical mechanism and reaction intermediates generated during conversion of amino acids into aldehydes by myeloperoxidase-generated HOCl.

p-Hydroxyphenylacetaldehyde, the Major Product of l-Tyrosine Oxidation by the Myeloperoxidase-H2O2-Chloride System of Phagocytes, Covalently Modifies ε-Amino Groups of Protein Lysine Residues

Activated human phagocytes employ the myeloperoxidase-H2O2-Cl−system to convert l-tyrosine top-hydroxyphenylacetaldehyde (pHA). We have explored the possibility that pHA covalently reacts with proteins to form Schiff base adducts, which may play a role in modifying targets at sites of inflammation. Because Schiff bases are labile to acid hydrolysis, prior to analysis the adducts were rendered stable by reduction with NaCNBH3. Purified pHA reacted withN α-acetyllysine, an analog of protein lysine residues. The reduced reaction product was identified asN α-acetyl-N ε-(2-(p-hydroxyphenyl)ethyl)lysine by 1H NMR spectroscopy and mass spectrometry. The compoundN ε-(2-(p-hydroxyphenyl)ethyl)lysine (pHA-lysine) was likewise identified in acid hydrolysates of bovine serum albumin (BSA) that were first exposed to myeloperoxidase, H2O2, l-tyrosine, and Cl− and then reduced with NaCNBH3. Other halides (F−, Br−, I−) and the pseudohalide SCN− could not replace Cl− as a substrate in the myeloperoxidase-H2O2-l-tyrosine system. In the absence of the enzymatic system, pHA-lysine was detected in reduced reaction mixtures of BSA, l-tyrosine, and reagent HOCl. In contrast, pHA-lysine was undetectable when BSA was incubated with l-tyrosine and HOBr, peroxynitrite, hydroxyl radical, or a variety of other peroxidases, indicating that the aldehyde-protein adduct was selectively produced by HOCl. Human neutrophils activated in the presence of tyrosine also modified BSA lysine residues. pHA-lysine formation required l-tyrosine and cell activation; it was inhibited by peroxidase inhibitors and catalase, implicating myeloperoxidase and H2O2in the reaction pathway. pHA-lysine was detected in inflamed human tissues that were reduced, hydrolyzed, and then analyzed by mass spectrometry, indicating that the reaction of pHA with proteins may be of physiological importance. These observations raise the possibility that the identification of pHA-lysine in tissues will pinpoint targets where phagocytes inflict oxidative damage in vivo.

Sickle hemoglobin disturbs normal coupling among erythrocyte O2 content, glycolysis, and antioxidant capacity

Energy metabolism in RBCs is characterized by O2-responsive variations in flux through the Embden Meyerhof pathway (EMP) or the hexose monophosphate pathway (HMP). Therefore, the generation of ATP, NADH, and 2,3-DPG (EMP) or NADPH (HMP) shift with RBC O2 content because of competition between deoxyhemoglobin and key EMP enzymes for binding to the cytoplasmic domain of the Band 3 membrane protein (cdB3). Enzyme inactivation by cdB3 sequestration in oxygenated RBCs favors HMP flux and NADPH generation (maximizing glutathione-based antioxidant systems). We tested the hypothesis that sickle hemoglobin disrupts cdB3-based regulatory protein complex assembly, creating vulnerability to oxidative stress. In RBCs from patients with sickle cell anemia, we demonstrate in the present study constrained HMP flux, NADPH, and glutathione recycling and reduced resilience to oxidative stress manifested by membrane protein oxidation and membrane fragility. Using a novel, inverted membrane-on-bead model, we illustrate abnormal (O2-dependent) association of sickle hemoglobin to RBC membrane that interferes with sequestration/inactivation of the EMP enzyme GAPDH. This finding was confirmed by immunofluorescent imaging during RBC O2 loading/unloading. Moreover, selective inhibition of inappropriately dispersed GAPDH rescues antioxidant capacity. Such disturbance of cdB3-based linkage between O2 gradients and RBC metabolism suggests a novel mechanism by which hypoxia may influence the sickle cell anemia phenotype.

Near-Infrared Dichromic Fluorescent Carbocyanine Molecules†

Central to major advances in biochemical assays, molecularsensor technologies, and molecular optical imaging arefluorescent materials that provide high detection sensitivityformolecularprocesses.Inbiologicalopticalimaging,thelowtissueautofluorescenceandthedeeppenetrationoflightintothetissuesobservedatwavelengthsbetween650 and900nmallow the use of near-infrared (NIR) fluorescent dyes ascontrast agents in heterogeneous systems.

Specific sequence motifs direct the oxygenation and chlorination of tryptophan by myeloperoxidase.

Most studies of protein oxidation have typically focused on the reactivity of single amino acid side chains while ignoring the potential importance of adjacent sequences in directing the reaction pathway. We previously showed that hypochlorous acid (HOCl), a specific product of myeloperoxidase, inactivates matrilysin by modifying adjacent tryptophan and glycine (WG) residues in the catalytic domain. Here, we use model peptides that mimic the region of matrilysin involved in this reaction, VVWGTA, VVWATA, and the library VVWXTA, to determine whether specific sequence motifs are targeted for chlorination or oxygenation by myeloperoxidase. Our results demonstrate that HOCl generated by myeloperoxidase or activated neutrophils converts the peptide VVWGTA to a chlorinated product, WG+32(Cl). Tandem mass spectrometry in concert with high resolution 1H and two-dimensional NMR analysis revealed that the modification required cross-linking of the tryptophan to the amide of glycine followed by chlorination of the indole ring of tryptophan. In contrast, when glycine in the peptide was replaced with alanine, the major products were mono- and dioxygenated tryptophan residues. When the peptide library VVWXTA (where X represents all 20 common amino acids) was exposed to HOCl, only WG produced a high yield of the chloroindolenine derivative. However, when glycine was replaced by other amino acids, oxygenated tryptophan derivatives were the major products. Our observations indicate that WG may represent a specific sequence motif in proteins that is targeted for chlorination by myeloperoxidase.

The use of HMQC-TOCSY experiments for elucidating the structures of bicyclic lactams : uncovering a surprise rearrangement in the synthesis of a key prophe building block

Abstract HMQC-TOCSY experiments were used to unequivocally assign the ring skeletons of several bicyclic lactams. This work demonstrated the power of these techniques for establishing the complete carbon connectivity of peptide building blocks with closely overlapping protons. In addition, it has led to the discovery of a surprise rearrangement reaction and allowed for the correction of a previously misassigned Pro-Phe building block ring skeleton.

Understanding dichromic fluorescence manifested in certain indocyanine green (ICG) analogs

Fluorescence has advanced our understanding in various aspects of biological processes. Fluorescence in the near infrared (NIR) region avoids background autofluorescence from biological samples, leading to improved image quality. In searching for indo-cyanine green (ICG) analogs that can be attached to biomolecules, we observed that dichromic fluorescence manifested in some mono reactive-group-functionalized ICG analogs. The two emission bands are distinctively separate from each other, making it a unique feature of fluorescent probes found in biological studies. We further demonstrated that the dichromism comes from the structure and is transferable from dye to its bioconjugates. In this paper, we used resonance theory and molecular orbital theory to explain the fluorophore photochemistry in an effort to understand the general fluorescence feature of ICG analogs and provide understanding of the secondary emission band.