Biochem. J. (2008) 416, 441–452 (Printed in Great Britain) doi:10.1042/BJ20070941 441 Tryptophan residues are targets in hypothiocyanous acid-mediated protein oxidation Clare L. HAWKINS 1 , David I. PATTISON, Naomi R. STANLEY and Michael J. DAVIES The Heart Research Institute, 114 Pyrmont Bridge Road, Camperdown, Sydney, NSW 2050, Australia Myeloperoxidase, released by activated phagocytes, forms reactive oxidants by catalysing the reaction of halide and pseudo- halide ions with H 2 O 2 . These oxidants have been linked to tissue damage in a range of inflammatory diseases. With physiological levels of halide and pseudo-halide ions, similar amounts of HOCl (hypochlorous acid) and HOSCN (hypothiocyanous acid) are produced by myeloperoxidase. Although the importance of HOSCN in initiating cellular damage via thiol oxidation is becoming increasingly recognized, there are limited data on the re- actions of HOSCN with other targets. In the present study, the products of the reaction of HOSCN with proteins has been studied. With albumin, thiols are oxidized preferentially forming unstable sulfenyl thiocyanate derivatives, as evidenced by the reversible incorporation of 14 C from HOS 14 CN. On consumption of the HSA (human serum albumin) free thiol group, the formation of stable 14 C-containing products and oxidation of tryptophan residues are observed. Oxidation of tryptophan residues is observed on reaction of HOSCN with other proteins (including myoglobin, lysozyme and trypsin inhibitor), but not free tryptophan, or tryptophan-containing peptides. Peptide mass mapping studies with HOSCN-treated myoglobin, showed the addition of two oxygen atoms on either Trp 7 or Trp 14 with equimolar or less oxid- ant, and the addition of a further two oxygen atoms to the other tryptophan with higher oxidant concentrations (2-fold). Tryptophan oxidation was observed on treating myoglobin with HOSCN in the presence of glutathione and ascorbate. Thus tryptophan residues are likely to be favourable targets for the reaction in biological systems, and the oxidation products formed may be useful biomarkers of HOSCN-mediated protein oxidation. Key words: hypochlorous acid (HOCl), hypothiocyanous acid (HOSCN), myeloperoxidase, protein oxidation, thiocyanate. INTRODUCTION Peroxidase enzymes play an important role in mammalian defence mechanisms by catalysing the formation of oxidants that act as potent antibacterial agents (reviewed in [1]). MPO (myelo- peroxidase) and EPO (eosinophil peroxidase) are released by activated phagocytes [2,3], whereas LPO (lactoperoxidase) is an antimicrobial agent in milk, saliva and tears [4]. However, excessive or misplaced generation of oxidants by peroxidases, particularly MPO and EPO, is believed to contribute to the progression of a number of diseases, including atherosclerosis, chronic inflammation, asthma and some cancers (reviewed in [1]). It is well-established that MPO and EPO utilize chloride ions (Cl − ) and bromide ions (Br − ) respectively, to produce HOCl (hypochlorous acid) and HOBr (hypobromous acid) [2,3]. How- ever, oxidation of SCN − (thiocyanate ions) by MPO and EPO is also important, as SCN − is the preferred substrate for these peroxidases at physiological halide ion concentrations (100– 140 mM Cl − , 20–100 μM Br − , < 1 μM I − and  120 μM SCN − ) [3,5,6]. It has been estimated that approx. 50 % of the H 2 O 2 consumed by MPO oxidizes SCN − under physiological conditions, with most of the remaining H 2 O 2 (approx. 45 %) being used to oxidize Cl − , based on the specificity constants of 1:60:730 for Cl − , Br − and SCN − respectively [5]. HOCl is known to play an important role in the oxidative damage observed in various pathologies, as evidenced by the detection of a specific biomarker for this oxidant, 3-chlorotyro- sine, in diseased tissue (e.g. [7]). Similarly, detection of 3-bromo- tyrosine implies a critical role of HOBr in the tissue damage observed in asthma, allergic reactions, malignancies and infections (reviewed in [1]). In contrast, the role of SCN − -derived oxidants in disease is not well understood, mainly due to a lack of specific biomarkers for SCN − -mediated damage. The importance of SCN − -derived oxidants produced by MPO in atherosclero- sis has been highlighted by the detection of elevated levels of carbamylated proteins in plaques [8]. Moreover, plasma concen- trations of SCN − , which are elevated in smokers [9], correlate significantly with early markers of atherosclerosis, including deposition of oxidized lipoproteins in the artery wall and the formation of lipid-laden macrophages (foam cells) [10,11]. It has been proposed that the oxidation product responsible for the antibacterial activity of the LPO/H 2 O 2 /SCN − system is HOSCN (hypothiocyanous acid) [12–18]. The proposed mechan- ism of SCN − oxidation by the peroxidase and H 2 O 2 are outlined in eqns (1–3). H 2 O 2 + 2SCN − + 2H + → 2H 2 O + (SCN) 2 (1) (SCN) 2 + H 2 O ↽ ⇀ HOSCN + H + + SCN − (2) HOSCN ↽ ⇀ OSCN − + H + (3) The pK a of HOSCN is 5.3 [14], therefore a mixture of both the protonated form and anion OSCN − will exist at physiological pH. HOSCN may not be the only SCN − -derived oxidant, as HOSCN decomposes readily in aqueous solution to form other reactive species [14,16,17]. HOSCN is used to represent this mixture hereon. The nature of these decomposition products remains controversial, with evidence for the production of (SCN) 2 (thiocyanogen) [19,20], HO 2 SCN (cyanosulfurous acid) [13], Abbreviations used: ANS, 8-anilino-1-napthalenesulfonic acid; DTNB, 5,5 ′ -dithio-2-nitrobenzoic acid; DTT, dithiothreitol; EPO, eosinophil peroxidase; LPO, lactoperoxidase; MPO, myeloperoxidase; MS/MS, tandem MS; NFK, N-formylkynurenine; SRM, selective reaction monitoring; STI, soya bean trypsin inhibitor; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; TNB, 5-thio-2-nitrobenzoic acid; XC, cross correlation score. 1 To whom correspondence should be addressed (email hawkinsc@hri.org.au). c  The Authors Journal compilation c  2008 Biochemical Society