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Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: deceptive implications for free radical detection.

ABSTRACT: Ceruloplasmin (ferroxidase) is a copper-binding protein known to promote Fe(2+) oxidation in plasma of mammals. In addition to its classical ferroxidase activity, ceruloplasmin is known to catalyze the oxidation of various substrates, such as amines and catechols. Assays based on cyclic hydroxylamine oxidation are used to quantify and detect free radicals in biological samples ex vivo and in vitro. We show here that human ceruloplasmin promotes the oxidation of the cyclic hydroxylamine 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) and related probes in Chelex-treated phosphate buffer and rat serum. The reaction is suppressed by the metal chelators DTPA, EDTA, and desferal, whereas heparin and bathocuproine have no effect. Catalase or superoxide dismutase additions do not interfere with the CPH-oxidation yield, demonstrating that oxygen-derived free radicals are not involved in the CPH oxidation mediated by ceruloplasmin. Plasma samples immunodepleted of ceruloplasmin have lower levels of CPH oxidation, which confirms the role of ceruloplasmin (ferroxidase) as a biological oxidizing agent of cyclic hydroxylamines. In conclusion, we show that the ferroxidase activity of ceruloplasmin is a possible biological source of artifacts in the cyclic hydroxylamine-oxidation assay used for reactive oxygen species detection and quantification.

SUBMITTER: Ganini D

PROVIDER: S-EPMC3448871 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: deceptive implications for free radical detection.

Ganini Douglas D Canistro Donatella D Jiang JinJie J Stadler Krisztian K Mason Ronald P RP Kadiiska Maria B MB

Free radical biology & medicine 20120721 7

Ceruloplasmin (ferroxidase) is a copper-binding protein known to promote Fe(2+) oxidation in plasma of mammals. In addition to its classical ferroxidase activity, ceruloplasmin is known to catalyze the oxidation of various substrates, such as amines and catechols. Assays based on cyclic hydroxylamine oxidation are used to quantify and detect free radicals in biological samples ex vivo and in vitro. We show here that human ceruloplasmin promotes the oxidation of the cyclic hydroxylamine 1-hydroxy ...[more]

PMID: 22824865

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Project description:Bimolecular rate constants have been measured for reactions that involve hydrogen atom transfer (HAT) from hydroxylamines to nitroxyl radicals, using the stable radicals TEMPO(*) (2,2,6,6-tetramethylpiperidine-1-oxyl radical), 4-oxo-TEMPO(*) (2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl radical), di-tert-butylnitroxyl ((t)Bu(2)NO(*)), and the hydroxylamines TEMPO-H, 4-oxo-TEMPO-H, 4-MeO-TEMPO-H (2,2,6,6-tetramethyl-N-hydroxy-4-methoxy-piperidine), and (t)Bu(2)NOH. The reactions have been monitored by UV-vis stopped-flow methods, using the different optical spectra of the nitroxyl radicals. The HAT reactions all have |DeltaG (o)| < or = 1.4 kcal mol(-1) and therefore are close to self-exchange reactions. The reaction of 4-oxo-TEMPO(*) + TEMPO-H --> 4-oxo-TEMPO-H + TEMPO(*) occurs with k(2H,MeCN) = 10 +/- 1 M(-1) s(-1) in MeCN at 298 K (K(2H,MeCN) = 4.5 +/- 1.8). Surprisingly, the rate constant for the analogous deuterium atom transfer reaction is much slower: k(2D,MeCN) = 0.44 +/- 0.05 M(-1) s(-1) with k(2H,MeCN)/k(2D,MeCN) = 23 +/- 3 at 298 K. The same large kinetic isotope effect (KIE) is found in CH(2)Cl(2), 23 +/- 4, suggesting that the large KIE is not caused by solvent dynamics or hydrogen bonding to solvent. The related reaction of 4-oxo-TEMPO(*) with 4-MeO-TEMPO-H(D) also has a large KIE, k(3H)/k(3D) = 21 +/- 3 in MeCN. For these three reactions, the E(aD) - E(aH) values, between 0.3 +/- 0.6 and 1.3 +/- 0.6 kcal mol(-1), and the log(A(H)/A(D)) values, between 0.5 +/- 0.7 and 1.1 +/- 0.6, indicate that hydrogen tunneling plays an important role. The related reaction of (t)Bu(2)NO(*) + TEMPO-H(D) in MeCN has a large KIE, 16 +/- 3 in MeCN, and very unusual isotopic activation parameters, E(aD) - E(aH) = -2.6 +/- 0.4 and log(A(H)/A(D)) = 3.1 +/- 0.6. Computational studies, using POLYRATE, also indicate substantial tunneling in the (CH(3))(2)NO(*) + (CH(3))(2)NOH model reaction for the experimental self-exchange processes. Additional calculations on TEMPO((*)/H), (t)Bu(2)NO((*)/H), and Ph(2)NO((*)/H) self-exchange reactions reveal why the phenyl groups make the last of these reactions several orders of magnitude faster than the first two. By inference, the calculations also suggest why tunneling appears to be more important in the self-exchange reactions of dialkylhydroxylamines than of arylhydroxylamines.

Project description:Frequently, in rugby, players incorporate deceptive motions (e.g., a side-step) in order to pass their opponent. Previous works showed that expert defenders are more efficient in detecting deceptive motions. Performance was shown to be correlated with the evolution of the center of gravity of the attacker, suggesting that experts may rely on global motion cues. This study aims at investigating whether a representation of center of gravity can be useful for training purposes, by using this representation alone or by combining it with the local motion cues given by body parts. We designed an experiment in virtual reality to control the motion cues available to the defenders. Sixteen healthy participants (seven experts and nine novices) acted as defenders while a virtual attacker approached. Participants completed two separate tasks. The first was a time occlusion perception task, occlusion after 100ms, 200ms or 300ms after the initial change in direction, thereafter participants indicated the passing direction of the attacker. The second was a perception-action task, participants were instructed to intercept the oncoming attacker by displacing medio-laterally. The attacker performed either a non-deceptive motion, directly toward the final passing direction or a deceptive motion, initially toward a false direction before quickly reorienting to the true direction. There was a main effect of expertise, appearance, cut off times and motion on correct responses during both tasks. There was an interaction between visual appearance and expertise, and between motion type and expertise during the perception task, however, this interaction was not present during the perception-action task. We observed that experts maintained superiority in the perception of deceptive motion; however when the visual appearance is reduced to global motion alone the difference between novices and experts is reduced. We further explore the interactions and discuss the effects observed for the visual appearance and expertise.

Dataset Information

Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: deceptive implications for free radical detection.

Publications

Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: deceptive implications for free radical detection.

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