Electrochemical nitration of myoglobin at tyrosine 103: structure and stability.

Nitration in proteins is a physiologically relevant process and the formation of 3-nitrotyrosine was first proposed as an in vivo marker of the production of reactive nitrogen species in oxidative stress. No studies have been published on structural changes associated with nitration of myoglobin. To address this deficiency the electrochemical nitration of equine skeletal muscle (Mb) at amino acid tyrosine 103 has been investigated for the evaluation and characterization of structural and thermal stability changes. Y103 in Mb is one of the most exposed tyrosine residues and it is also close to the heme group. Effects of Y103 nitration on the secondary and tertiary structure of Y103 have been studied by UV-Vis, circular dichroism, fluorescence and NMR spectroscopy and by electrochemical studies. At physiological pH, subtle changes were observed involving slight loosening of the tertiary structure and conformational exchange processes. Thermal stability of the nitrated protein was found to be reduced by 5 °C for the nitrated Mb compared with the native Mb at physiological pH. Altogether, NMR data indicates that nitrated Mb has a very similar tertiary structure to that of native Mb, although with a slightly open conformation.

The endogenous production of hydrogen sulphide in intrauterine tissues.

BACKGROUND: Hydrogen sulphide is a gas signalling molecule which is produced endogenously from L-cysteine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The possible role of hydrogen sulphide in reproduction has not yet been fully investigated. It has been previously demonstrated that hydrogen sulphide relaxes uterine smooth muscle in vitro. The aim of the present study was to investigate the endogenous production of hydrogen sulphide in rat and human intrauterine tissues in vitro. METHODS: The production of hydrogen sulphide in rat and human intrauterine tissues was measured in vitro using a standard technique. The expression of CBS and CSE was also investigated in rat and human intrauterine tissues via Western blotting. Furthermore, the effects of nitric oxide (NO) and low oxygen conditions on the production rates of hydrogen sulphide were investigated. RESULTS: The order of hydrogen sulphide production rates (mean +/- SD, n = 4) for rat tissues were: liver (777 +/- 163 nM/min/g) > uterus (168 +/- 100 nM/min/g) > fetal membranes (22.3 +/- 15.0 nM/min/g) > placenta (11.1 +/- 4.7 nM/min/g), compared to human placenta (200 +/- 102 nM/min/g). NO significantly increased hydrogen sulphide production in rat fetal membranes (P < 0.05). Under low oxygen conditions the production of hydrogen sulphide was significantly elevated in human placenta, rat liver, uterus and fetal membranes (P < 0.05). Western blotting (n = 4) detected the expression of CBS and CSE in all rat intrauterine tissues, and in human placenta, myometrium, amnion and chorion. CONCLUSION: Rat and human intrauterine tissues produce hydrogen sulphide in vitro possibly via CBS and CSE enzymes. NO increased the production of hydrogen sulphide in rat fetal membranes. The augmentation of hydrogen sulphide production in human intrauterine tissues in a low oxygen environment could have a role in pathophysiology of pregnancy.

A magneto-optic route toward the in vivo diagnosis of malaria: preliminary results and preclinical trial data.

We report the development of magneto-optic technology for the rapid quantitative diagnosis of malaria that may also be realizable in a noninvasive format. Hemozoin, the waste product of malarial parasitic action on hemoglobin, is produced in a form that under the action of an applied magnetic field gives rise to an induced optical dichroism characteristic of the hemozoin concentration. Here we show that precise measurement of this induced dichroism may be used to determine the level of malarial infection because this correlates, albeit in a complex manner throughout the infection cycle, with the concentration of hemozoin in the blood and tissues of infected patients. Under conservative assumptions for the production of hemozoin as a function of parasitemia, initial results indicate that the technique can match or exceed other current diagnostic techniques. The validity of the approach is confirmed by a small preliminary clinical trial on 13 patients, and measurements on live parasitized cells obtained from in vitro culture verify the possibility of producing in vivo diagnostic instrumentation.

Specific electrochemical iodination of horse heart myoglobin at tyrosine 103 as determined by Fourier transform ion cyclotron resonance mass spectrometry.

The iodination of proteins remains a useful tool in biochemistry for radiolabelling. However, chemical or enzymatic iodination is difficult to control and can give deleterious polyiodination. Previously, we have shown that electrooxidation with nitrite is a rapid method for the selective nitration of tyrosine residues in proteins. In principle, it should be possible to substitute a number of electrooxidisable anions into the tyrosine phenol ring. Electrochemical iodination is more difficult to control than nitration because the rapid anodic oxidation of I(-) leads to persistent formation of the iodinating triiodide anion. However, application of pulsed electrooxidation and reduction cycles is shown to be an effective procedure for the selective mono and double-iodination of myoglobin, which may have general application to other proteins in controlling of the level of iodination. Mono- and double-iodination of myoglobin by this method was confirmed by electrospray FT-ICR mass spectrometry. Infrared multiphoton dissociation (IRMPD) enabled localization of the site of mono-iodination to be restricted to either His97 or Tyr103. More extensive sequence coverage was obtained with electron capture dissociation (ECD), allowing unambiguous assignment of the site of iodination to Tyr103.

Nitration of lysozyme by ultrasonic waves; demonstration by immunochemistry and mass spectrometry.

Solutions containing hen egg white lysozyme (HEWL) and nitrite were exposed to ultrasonic irradiation in order to study the possible sonochemical modifications. This is the first demonstration of the nitration of tyrosine residues in a protein (lysozyme) by the use of an ultrasonic field alone. Sonochemically nitrated lysozyme was detected using the immunochemical techniques dot blot immunodetection and enzyme-linked immunosorbent assay (ELISA). The sonically oxidised and nitrated protein solutions were analysed by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Hydroxylated species were found in the absence of nitrite, whereas nitration was the major modification in the presence of nitrating agent, implying a competing mechanism between hydroxyl radicals and nitrite. Circular dichroism (CD) indicated that the ultrasonic experimental conditions chosen in this study had little effect on the tertiary and secondary structures of HEWL. Whilst enzymatic assay showed that the presence of nitrite provided a protective effect on the inactivation of the protein under ultrasonic irradiation, nevertheless partially purified, sonically nitrated lysozyme showed a dramatic decrease in lytic activity.

Retention of enzyme activity with a boron-doped diamond electrode in the electro-oxidative nitration of lysozyme.

In this paper we report the successful use of a non-metallic electrode material, boron-doped diamond (BDD), for the anodic electro-oxidative modification of hen egg white lysozyme (HEWL). Platinum electrodes can give rise to loss of activity of HEWL in electrosynthetic studies, whereas activity is retained on boron-doped diamond which is proposed as an effective substitute material for this purpose. We also compare literature methods of electrode pre-treatment to determine the most effective in electrosynthesis. Our findings show a decrease in total nitroprotein yield with decreasing nitrite concentration and an increase with increasing solution pH, confirming that, at a BDD electrode, the controlling factor remains the concentration of tyrosine phenolate anion. Purification of mono- and bis-nitrated HEWL and assay of enzymic activity showed better retention of activity at BDD electrode surfaces when compared to platinum. The products from electro-oxidation of HEWL at BDD were confirmed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FT-ICR) mass spectrometry, which revealed unique mass increases of +45 and +90 Da for the mono- and bis-nitrated lysozyme, respectively, corresponding to nitration at tyrosine residues. The nitration sites were confirmed as Tyr23 and Tyr20.

Mass spectrometry in demonstrating the site-specific nitration of hen egg white lysozyme by an improved electrochemical method.

In producing a method for selective protein nitration, we previously demonstrated the electrochemical nitration of hen egg white lysozyme to be at Tyr23 initially, followed by bisnitration at Tyr20, but with no trisnitration at Tyr53. The nitration site was determined by sequencing a tryptic peptide that included Tyr23 and Tyr20, but possible effects on other regions of the protein were not determined. Moreover, the electrooxidation conditions were harsh, involving an oxidation potential of +1.2V (vs. saturated calomel electrode [SCE]), no added nitrogen source except the lysozyme itself, and long reaction periods with copper flag electrodes. Here we report a gentler procedure using much shorter reaction times with nitrite as the nitration source, a lower potential (+0.85V vs. SCE), and a platinum basket electrode. Intact protein analysis by electrospray Fourier transform ion cyclotron resonance mass spectrometry identified mono- and bisnitration products with mass increases of +45 and +90 Da, respectively, consistent with the substitution of NO(2) for H. In addition, the results revealed that no other covalent change in the protein occurred following electrooxidation. Nozzle skimmer dissociation of the intact mononitrated species localized the modification site to Tyr20 or Tyr23. Matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization time-of-flight analysis of the tryptic peptides of mononitrated lysozyme identified the site of nitration as Tyr23.