Comparison of salicylate and D-phenylalanine for detection of hydroxyl radicals in chemical and biological reactions

Summary

Hydroxylation of salicylate and D-phenylalanine was measured to test the usefulness of these compounds for hydroxyl radical (HO^?) detection in chemical and biological systems. When HO^? were produced by the photolytic decomposition of hydrogen peroxide, nearly equal amounts of 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were produced from salicylate, with catechol as a minor product. In the photolytic reaction, nearly equal concentrations of p-,m-, and o-tyrosine were formed from D-phenylalanine. When salicylate or D-phenylalanine was present with Fenton reagents or in iron(II) autoxidation systems, the relative proportions of hydroxylated products were similar to those observed after photolysis, although less total products were usually detected. In contrast, when similar experiments were conducted with isolated hepatic microsomes and perfused livers, 2,5-DHBA was the primary product from salicylate, and p-tyrosine was the major product from D-phenylalanine. Cytochrome P-450 enzymes can hydroxylate salicylate to produce 2,5-DHBA, and it is likely that phenylalanine hydroxylase produces most of the p-tyrosine detected in hepatic tissues. Thus, although both salicylate and D-phenylalanine are useful probes for hydroxyl radical formation in chemical systems, hydroxylated products formed from enzymatic reactions complicate interpretation of data from both compounds in vivo.     

Quantification of hydroxyl radical-derived oxidation products in peptides containing glycine, alanine, valine, and proline

Proteins are a major target for oxidation due to their abundance and high reactivity. Despite extensive investigation over many years, only limited quantitative data exist on the contributions of different pathways to the oxidation of peptides and proteins. This study was designed to obtain quantitative data on the nature and yields of oxidation products (alcohols, carbonyls, hydroperoxides, fragment species) formed by a prototypic oxidant system (HO^?/O₂) on small peptides of limited, but known, amino acid composition. Peptides composed of Gly, Ala, Val, and Pro were examined with particular emphasis on the peptide Val-Gly-Val-Ala-Pro-Gly, a repeat motif in elastin with chemotactic activity and metalloproteinase regulation properties. The data obtained indicate that hydroperoxide formation occurs nonrandomly (Pro > Val > Ala > Gly) with this inversely related to carbonyl yields (both peptide-bound and released). Multiple alcohols are generated at both side-chain and backbone sites. Backbone fragmentation has been characterized at multiple positions, with sites adjacent to Pro residues being of major importance. Summation of the product concentrations provides clear evidence for the occurrence of chain reactions in peptides exposed to HO^?/O₂, with the overall product yields exceeding that of the initial HO^? generated.     

Evidence of reactive oxygen species generation in synovial fluid from patients with temporomandibular disease by electron spin resonance spectroscopy

Abstract

Reactive oxygen species (ROS) have been implicated in the pathogenesis of temporomandibular disorders. In the present study, we provide the first evidence of ROS generation in the synovial fluid from human temporomandibular disorder patients, as shown by electron spin resonance (ESR) and spin trapping. Three distinct ESR spectra of DMPO spin adducts were observed in the synovial fluid. They corresponded to three free radical species: hydroxyl radical (HO^?), hydrogen radical (H^?), and carbon-center radical (R^?). Among them, the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH spectrum was the most prominent, suggesting that HO^? was dominantly generated in the synovial fluid from temporomandibular disorder patients. Desferrioxamine (DFO), an iron chelator, strongly depressed the DMPO-OH signal intensity in the synovial fluid from patients with temporomandibular disorders. We successfully demonstrated ROS-induced oxidative stress in the synovial fluid from temporomandibular disorder patients. ROS generation in the temporomandibular joint could lead to exacerbation of inflammation and activation of cartilage matrix degrading enzymes that proceed to degenerative change of the temporomandibular joint. Thus, iron-dependent generation of HO ^? might have a crucial role in the pathogenesis of temporomandibular disorders.     

Mechanism of Hydroxyl Radical Scavenging by Rebamipide: Identification of Mono-hydroxylated Rebamipide as a Major Reaction Product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (^?OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against ^?OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H²O²), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a ^?OH-trapping agent after UVB exposure (305?nm) to H²O² for 1?min in the presence of rebamipide. The signal intensity of ^?OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62?×?10¹⁰, 8.16?×?10⁹ and 1.65?×?10¹⁰?M^-1?s^-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the ^?OH generated by UVB/H²O². Specific formation of this product explained the molecular characteristics of rebamipide as a potential ^?OH scavenger.     

Hydroxyl Radical-Mediated Oxidation of Serotonin: Potential Insights into the Neurotoxicity of Methamphetamine

Abstract: When incubated with a hydroxyl radical (HO^?)-generating system (ascorbic acid/Fe²⁺-EDTA/O₂/H₂O₂), 5-hydroxytryptamine (5-HT; serotonin) is rapidly oxidized initially to a mixture of 2,5-, 4,5-, and 5,6-dihydroxytryptamine (DHT). The major reaction product is 2,5-DHT, which at physiological pH exists as its keto tautomer, 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). Rapid autoxidation of 4,5-DHT gives tryptamine-4,5-dione (T-4,5-D), which reacts with the C(3)-centered carbanion of 5-HEO to give 3,3′-bis(2-aminoethyl)-5-hydroxy-[3,7′-bi-1H-indole]-2,4′,5′-3H-trione (7). The latter slowly cyclizes to 3′-(2-aminoethyl)-1′,6′,7′,8′-tetrahydro-5-hydroxyspiro[3H-indole-3,9′-[9H]pyrrolo[2,3-f]quinoline]-2,4′,5′(1H)- trione (9). A minor amount of T-4,5-D dimerizes to give 7,7′-bi-(5-hydroxytryptamine-4-one) (7,7′-D). In the presence of GSH, the reaction of T-4,5-D with 5-HEO is diverted and, in the presence of sufficient concentrations of this tripeptide, completely blocked. This is because GSH preferentially reacts with T-4,5-D to give 7-S-glutathionyltryptamine-4,5-dione (11). The results of this investigation suggest that 5,6-DHT, 5-HEO, 7, and 9 are products unique to the HO^?-mediated oxidation of 5-HT. Thus, the observation of other investigators that 5,6-DHT is formed in the brains of rats following a large dose of methamphetamine (MA) suggests that this drug might evoke HO^? formation. However, the present in vitro study indicates that 5,6-DHT is a rather minor, unstable product of the HO^?-mediated oxidation of 5-HT and suggests that detection of 5-HEO, 7/9, and 11 in rat brain following MA administration could provide additional support for HO^? formation. Furthermore, one or more of the intermediates and major products of oxidation of 5-HT by HO^? might, in addition to 5,6-DHT, contribute to the MA-induced degeneration of serotonergic neurons.     

Effect of methionine-35 oxidation on the aggregation of amyloid-β peptide

Aggregation of Aβ peptides into amyloid plaques is considered to trigger the Alzheimer’s disease (AD), however the mechanism behind the AD onset has remained elusive. It is assumed that the insoluble Aβ aggregates enhance oxidative stress (OS) by generating free radicals with the assistance of bound copper ions. The aim of our study was to establish the role of Met35 residue in the oxidation and peptide aggregation processes. Met35 can be readily oxidized by H₂O₂. The fibrillization of Aβ with Met35 oxidized to sulfoxide was three times slower compared to that of the regular peptide. The fibrils of regular and oxidized peptides looked similar under transmission electron microscopy. The relatively small inhibitory effect of methionine oxidation on the fibrillization suggests that the possible variation in the Met oxidation state should not affect the in vivo plaque formation. The peptide oxidation pattern was more complex when copper ions were present: addition of one oxygen atom was still the fastest process, however, it was accompanied by multiple unspecific modifications of peptide residues. Addition of copper ions to the Aβ with oxidized Met35 in the presence of H₂O₂, resulted a similar pattern of nonspecific modifications, suggesting that the one-electron oxidation processes in the peptide molecule do not depend on the oxidation state of Met35 residue. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aβ–Cu(II) complex.     

The Radical SAM Superfamily

ABSTRACT

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe–4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe–4S]^{1 +} –SAM complex to [4Fe–4S]^{2 +}–Met and the 5′ -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.     

Hydroxyl radical oxidation of guanosine 5′-triphosphate (GTP): requirement for a GTP–Cu(II) complex

Abstract

Levels of oxidized guanosine base in DNA have become a hallmark biomarker in assessing oxidative stress implicated in a variety of disease and toxin-induced states. However, there is evidence that the guanosine in the nucleotide triphosphate pool (GTP) is more susceptible to oxidation than guanosine residues incorporated into nucleic acids and this causes a substantial amount of the oxidized product, 8-oxoguanosine 5′-triphosphate (oxo⁸GTP), to accumulate in cell-free and in cell-culture preparations. Electron paramagnetic resonance (EPR) spectroscopy and direct EPR analysis of free radical production by copper sulfate and L-ascorbic acid demonstrates that the hydroxyl radical (HO^?) is produced via oxidation of Cu⁺ to Cu²⁺ while in a complex with GTP. This HO^? production is dependent on the availability of oxygen and the presence of GTP in the reaction milieu. Verification of free radical-mediated production of oxo⁸GTP is presented using HPLC with electrochemical detection and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry (MALDI-LTOF-MS). The sum of these results is presented in a novel mechanism of GTP oxidation by Cu²⁺ and L-ascorbic acid. A better understanding of the chemistry involved in this oxidative modification of GTP facilitates a more comprehensive understanding of its potential physiological consequences.     

Brucin,an antibacterial peptide derived from fruit protein of Fructus Bruceae,Brucea javanica (L.) Merr

A novel antibacterial peptide specific to Streptococcus pyogenes was produced from dried fruit protein of Brucea javanica (L.) Merr. A mixture of active peptides from the fruit protein was produced in vitro by pepsin hydrolysis. The hydrolysate was purified by reverse‐phase HPLC, and antimicrobial peptides active against Gram‐negative and Gram‐positive bacteria were analysed using SDS‐PAGE and nanoLC‐MS/MS. Here, four possible peptides were obtained and chemically synthesized for comparative study of the growth inhibition of Strep. pyogenes. One chemically synthesized peptide with a molecular mass of 1168·31 Da, His‐Thr‐Leu‐Cys‐Met‐Asp‐Gly‐Gly‐Ala‐Thr‐Tyr, showed the most potent antibacterial activity against Strep. pyogenes. This 11‐amino acid peptide was named Brucin. Its bacterial inhibitory activity was 16‐fold and 12·5‐fold higher than penicillin G and chloramphenicol, respectively, with a MIC value of 20 μmol l^?1. The results suggest that Brucin, a potent antibiotic peptide, may be developed as an alternative drug for the treatment of the disease caused by Strep. pyogenes.

Significance and Impact of the Study

An antibacterial peptide, named Brucin with specificity for Streptococcus pyogenes, was produced in vitro from dried fruit protein of Brucea javanica (L.) Merr. by pepsin‐catalysed hydrolysis. Its inhibitory activity towards the Gram‐positive bacteria was higher than penicillin G and chloramphenicol. The result suggested that Brucin may be applied for the treatment of the disease caused by Strep. pyogenes^*.     

Relative reactivity of lysine and other peptide-bound amino acids to oxidation by hypochlorite

Antibacterial and inflammatory responses of neutrophils and macrophages produce hypochlorite as a major oxidant. Numerous side chains of amino acids found in extracellular proteins can be modified by hypochlorite, including His, Arg, Tyr, Lys, Trp, and Met. We studied the relative reactivity of each of these amino acid residues in short N-blocked peptides, where other residues in the peptide were highly resistant to hypochlorite attack. Hypochlorite treatment led to modified peptides in each case, which were detected by changes in retention on reversed-phase HPLC. A distinct single product, consuming two equivalents of hypochlorite per equivalent of peptide, was obtained from the Lys-containing peptides. UV spectroscopy, nuclear magnetic resonance (NMR), and electrospray/mass spectroscopy identified this product as the dichloramine at the epsilon-amino group of the Lys side chain. The dichloramine at Lys did not decompose to form a detectable amount of carbonyl reactive with dinitrophenylhydrazine. The dichloramine at Lys did however quantitatively revert back to Lys during HCl digestion of the tetrapeptide for amino acid analysis, with simultaneous modification of the adjacent Phe residue. The formation of the dichloramine at Lys was not blocked by peptides or acetylated amino acids that contained Tyr, His, or Arg. In contrast, the presence of equimolar Met-containing peptide, or N-Acetyl-Trp, both inhibited the formation of the dichloramine at Lys. Thus, Met and Trp side chains of proteins might be able to protect Lys from chloramine formation under some circumstances, but this interpretation must consider that Met and Trp are typically found in relatively inaccessible hydrophobic sites, whereas lysine is typically exposed on the protein surface. The hierarchy of amino acid reactivities examined here will aid in the prediction of residues in biological samples most likely to be modified by hypochlorite.     

Synthesis and antioxidant properties of diphenylmethane derivative bromophenols including a natural product

Bromination of bis(3,4-dimethoxyphenyl)methanone (5) gave four products (6–9) with mono, di, tri, and tetra Br under different conditions. Reduction and demethylation reactions of product 9 with tetra Br were performed, consecutively and a natural product, 5,5′-methylene bis(3,4-dibrombenzene-1,2-diol) (1), was obtained with a 53% yield. Five derivatives, (13–17) (bromophenols), of 1 were also synthesised. The antioxidant and radical scavenging activities of bromophenols 1 and 13–17 were determined by employing various in vitro assays such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH^?), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS^?+), N,N-dimethyl-p-phenylenediamine dihydrochloride radical cation (DMPD^?+), and superoxide anion radical (O₂^?-) scavenging, reducing ability determination by the Fe³⁺-Fe²⁺ and Cu²⁺-Cu⁺ cupric reducing antioxidant capacity (CUPRAC) transformation methods, hydrogen peroxide scavenging, and ferrous ion (Fe²⁺) chelating activities. Moreover, these activities were compared to those of synthetic standard antioxidant compounds such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol, and trolox. The results showed that the synthesised bromophenols had effective antioxidant power.     

Radical scavenging and chemical repair of rutin observed by pulse radiolysis: as a basis for radiation protection

Abstract

Pulse radiolysis was conducted to investigate: several fundamental reactions of a natural flavonoid, rutin, and its glycosylated form (αG-rutin) as a basis for their radiation protection properties; the reactions with ^?OH (radical scavenging) and dGMP radical, dGMP^? (chemical repair), which was used as a model of initial and not yet stabilised damage on DNA. Three absorption peaks were commonly seen in the reactions of the flavonoids with ^?OH, showing that their reactive site is the common structure, i.e. aglycone. One among the three peaks was attributed to the flavonoid radical produced as a result of the removal of a hydrogen atom. The same peak was found in their reactions with dGMP^?, showing that dGMP^? is chemically repaired by obtaining a hydrogen atom supplied from the flavonoids. Such a spectral change due to the chemical repair was as clear as never reported. The rate constants of the chemical repair reaction were estimated as (9?±?2)×10⁸ M^?1 s^?1 and (6?±?1)×10⁸ M^?1 s^?1 for rutin and αG-rutin, respectively. The rate constants of the radical scavenging reactions towards ^?OH were estimated as (1.3?±?0.3)×10¹⁰ M^?1 s^?1 and (1.0?±?0.1)×10¹⁰ M^?1 s^?1 for rutin and αG-rutin, respectively. In addition, there was no obvious difference between rutin and αG-rutin, indicating that the glycosylation does not change early chemical reactions of rutin.     

PAS‐cal: A repetitive peptide sequence calibration standard for MALDI mass spectrometry

Mass spectrometers equipped with matrix‐assisted laser desorption/ionization (MALDI‐MS) require frequent multipoint calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS‐cal tailored for MALDI‐MS based bottom‐up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C‐terminal arginine residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multipoint calibration of MS spectra using PAS‐cal peptides compares well to current commercial reagents for protein identification by PMF. Calibration of tandem mass spectra from LC‐MALDI experiments using the longest peptide, PAS‐cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS‐cal standard generically useful for bottom‐up proteomics.     

Disclosure of new and recurrent microbial metabolites by mass spectrometric methods

The application of modern mass spectrometry methods (SI-CID-MS/MS; MS ⁿ ) in the disclosure of new and recurrent microbial metabolites is discussed. Spray ion (SI) sources coupled to different kinds of mass analyzers enable the determination of molecular weights and chemical formulas of given samples even in mixtures. Diagnostic fragment formation by collision-induced dissociation (CID-MS/MS) and MS ⁿ experiments using ion trap mass analyzers are shown as another indispensable source of structural information. Due to the development of benchtop-type mass spectrometers coupled to high-performance liquid chromatography (HPLC), MS can be practised in almost every laboratory as a powerful tool in natural product analysis. Examples are given for special MS applications in identification of bioactive metabolites from screening strains. Journal of Industrial Microbiology & Biotechnology (2001) 27, 136–143. Received 21 September 1999/ Accepted in revised form 19 January 2000     

Gamma Ray Irradiation of the Vasoactive Peptide Bradykinin Reveals a Residue- and Position-Dependent Structural Modification

In this work the effect of radical species generated by gamma ray irradiation of aqueous solution upon structure of vasoactive peptide bradykinin (BK, RPPGFSPFR) was investigated. Increasing doses of 1–15 kGy Co⁶⁰ gamma radiation were applied to BK solutions and a progressive degradation of its structure in a non-linear mode was observed. Two main peptide derivatives generated by these treatments were isolated and characterized through a combined amino acid analysis and daughter ion scanning mass spectrometry approach. Notably, it was observed that only the Phe residue located at position 8 and not 5 of BK was oxidized by reactive hydroxyl radical species given rise to Tyr⁸-BK and m-Tyr⁸-BK analogues. Comparative circular dichroism (CD) experiments of these peptides revealed that BK presents greater conformational similarity to Tyr⁸-BK than to m-Tyr⁸-BK. These results are in agreement with the biological potencies of these compounds measured in rat uterus and guinea pig ileum muscle contractile experiments. In summary, gamma irradiation of BK solutions revealed a residue- and surprisingly, position-structural modification effect of reactive radicals even in small peptides. Also of value for peptide chemistry field, the approach of applying controlled strong electromagnetic radiation in solution seems to be an alternative and unique strategy for generating, in some cases, peptides derivatives with uncommon structures and valuable for their further therapeutic potential evaluations.     

Position 45 influences the peptide binding motif of HLA-B*44:08

Position 45 represents a highly polymorphic residue within HLA class I alleles, which contacts the p2 position of bound peptides in 85% of the peptide–HLA structures analyzed, while the neighboring residues 41 and 46 are not involved in peptide binding. To investigate the influence of residue 45 at the functional level, we sequenced peptides eluted from recombinant HLA-B*44:08^{41Ala/45Met/46Ala} molecules and compared their features with known peptides from B*44:02^{41Thr/45Lys/46Glu}. While HLA-B*44:02 has an anchor motif of E at the p2 anchor position, HLA-B*44:08 exhibits Q and L as anchor motif. The 45^Met/Lys polymorphism contributes to the alteration in the peptide-binding motif and provides further evidence that mismatches at position 45 should be considered as nonpermissive in a transplantation setting.     

A method for simple identification of signature peptides derived from polyUb-K48 and K63 by MALDI-TOF MS and chemically assisted MS/MS fragmentation

Summary. A simple method is described to identify signature peptides derived from polyubiquitin (polyUb) chains. The method is based on MALDI-TOF MS/MS analysis after chemically assisted fragmentation, and works on peptides isolated from polyacrylamide gels. PolyUb chains branched at K48 and K63 were chosen as models for Ub-protein conjugates. They were resolved by SDS-PAGE, and their tryptic peptides (in-gel-trypsinolysis) derivatized with 3-sulfopropinic acid NHSester to obtain chemically assisted fragmentation during the MS/MS analysis. PolyUb-K63 produced a single peptide identified as ⁵⁵TLSDYNIQK⁶³ (GG)ESTLHLVLR⁷². PolyUb-K48 produced two branched signature peptides identified as ⁴³LIFAGK⁴⁸(GG)QLEDGR⁵⁴ and ⁴³LIFAGK⁴⁸(LRGG)QLEDGR⁵⁴. The recovery of signature peptide with LRGG as branched chain underscores the need to take limited proteolysis into account in the search for detection of ubiquitinated peptides in proteomics studies. In conclusion, a simple method has been described allowing the identification of signature peptides, which are diagnostic markers of the majority of polyUb-conjugated proteins. In principle, the method should be applicable also for other more rare signature peptides.     

Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones

In biological systems, both proteolysis and aminolysis of amide bonds produce activated intermediates through acyl transfer reactions either inter- or intramolecularly. Protein splicing is an illustrative example that proceeds through a series of catalyzed acyl transfer reactions and culminates at an O- or S-acyl intermediate. This intermediate leads to an uncatalyzed acyl migration to form an amide bond in the spliced product. A ligation method mimicking the uncatalyzed final steps in protein splicing has been developed utilizing the acyl transfer amide-bond feature for the blockwise coupling of unprotected, free peptide segments at methionine (Met). The latent thiol moiety of Met can be exploited using homocysteine at the α-amino terminal position of a free peptide for transthioesterification with another free peptide containing an α-thioester to give an S-acyl intermediate. A subsequent, proximity-driven S- to N-acyl migration of this acyl intermediate spontaneously rearranges to form a homocysteinyl amide bond. S-methylation with excess p-nitrobenezensulfonate yields Met at the ligation site. The methionine ligation is selective and orthogonal, and is usually completed within 4 h when performed at slightly basic pH and under strongly reductive conditions. No side reactions due to acylation were observed with any other α-amines of both peptide segments as seen in the synthesis of parathyroid hormone peptides. Furthermore, cyclic peptide can also be obtained through the same strategy by placing both homocysteine at the amino terminus and the thioester at the carboxyl terminus in an unprotected peptide precursor. These biomimetic ligation strategies hold promise for engineering novel peptides and proteins. © 1998 John Wiley & Sons, Inc. Biopoly 46: 319–327, 1998     

Free radical reactions involving the angiotensin converting enzyme inhibitor captopril

Summary

Using the pulse radiolysis technique, absolute rate constants have been obtained for the reaction of captopril with several free radicals. The results demonstrate that although captopril reacts rapidly with a number of free radicals, such as the hydroxyl radical (k = 5.1 × 10⁹ dm^?3mol^?1s^?1) and the thiocyanate radical anion (k = 1.3 × 10⁷ dm^?3mol^?1s^?1), it is not exceptional in this ability. Similarly, the reactions with carbon centred radicals although rapid are an order of magnitude slower than those observed with glutathione. Additional lipid peroxidation studies further demonstrate that captopril is a much less effective antioxidant than glutathione. The data go some way to supporting the view that any attenuation of reperfusion injury by captopril is not through a direct free radical scavenging mechanism but may be afforded by other, non-radical-mediated mechanisms.