Larry A. Hick

Structural identification of valine hydroperoxides and hydroxides on radical-damaged amino acid, peptide, and protein molecules

We have previously demonstrated the formation of two reactive moieties on proteins during free radical attack: hydroperoxides, and 3,4-dihydroxyphenylalanine (DOPA). Here we have undertaken the structural elucidation of the hydroperoxides of valine, the amino acid which is most susceptible to peroxidation. Exposure of L-valine to free radicals generated by radiolysis in an oxygen-saturated system produced three valine hydroperoxides. Upon treatment with sodium borohydride these were reduced to their corresponding hydroxides, which can be separated and purified by high performance liquid chromatography (HPLC). Based on spectroscopic data from high resolution chemical ionization (CI) mass spectrometry (MS), electrospray (ES) MS, electron impact (EI) MS, proton (1H) nuclear magnetic resonance (NMR) and carbon-13 (13C) NMR studies, the three valine hydroxides have been identified as beta-hydroxyvaline [(2S)-2-amino-3-hydroxy-3-methyl-butanoic acid], (2S,3S)-gamma-hydroxyvaline [(2S,3S)-2-amino-3-hydroxymethyl-butanoic acid], and (2S,3R)-gamma-hydroxyvaline [(2S,3R)-2-amino-3-hydroxymethyl-butanoic acid]. HPLC analysis of O-phthaldialdehyde (OPA) derivatives of the hydroxyvalines provides a sensitive and accurate method for quantitative measurement. This method enabled hydroxyvalines to be detected in the hydrolysates of a tripeptide (glutamyl-valinyl-phenylalanine) and a protein (bovine serum albumin) that had been gamma-radiolysed and treated with sodium borohydride. Hydroxyvaline may be useful as a marker in studying protein oxidation in some biological systems under oxidative stress.

Hawkinsinuria: identification of quinolacetic acid and pyroglutamic acid during an acidotic phase

A second Australian family with the genetic disease Hawkinsinuria has been identified. Affected members excrete hawkinsin and cis- and trans-4-hydroxycyclohexylacetic acid. An infant in this family presented with metabolic acidosis and excreted quinolacetic acid and pyroglutamic acid in the urine together with the tyrosine derived phenolic acids reported in the original index case. It is thought that quinolacetic acid is accumulated as a by-product of the partially defective enzyme, 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) and that pyroglutamic acid indicated lowered glutathione levels.

An electrospray mass spectrometry study of some metal-ion cage complexes

A series of cationic metal complexes of the bicyclic hexaamine cage compound fac-1,5,9,13,20-pentamethyl-3,7,11,15,18,22-hexaazabicyclo[7.7.7]tricosane have been examined by electrospray ionisation (ESI) mass spectrometry, along with metal complexes of related smaller and larger hexaamine cages. The ESI mass spectra are considerably simpler than the corresponding fast atom bombardment (FAB) mass spectra. The most abundant ion in the ESI mass spectra of divalent metal-ion cage complexes is the doubly charged molecular ion [M(cage)]2+. For trivalent metal-ion complexes spectra obtained using a low-resolution quadrupole mass spectrometer suggested that the most abundant ion is of the type [M(cage)3+– H+]2+. However, when the spectra of several of these cage complexes were obtained using a high-resolution sector instrument it can be shown that the most intense peaks are due to mixtures of these and other ions, [M(cage)]2+, formed by reduction of the metal ion in the ion source. The ESI mass spectra of both di- and tri-valent metal-ion complexes also show the presence of ion pairs [M(cage)x++ anion–](x–1)+. In general ions arising from the free cage are not observed which makes the ESI technique suited for characterising the complex cations. However, varying the cone and skimmer potentials can alter the relative abundances of ions, and the degree to which reduction of the central ion occurs, so these parameters must be carefully controlled. The ESI mass spectra of analogous cobalt(III) complexes containing ammonia or ethane-1,2-diamine displayed more extensive fragmentation compared to those of the cobalt(III) cage complexes. This study demonstrates the potential of ESI mass spectrometry for the characterisation of metal cage complexes as a powerful adjunct to NMR spectroscopy and microanalysis.

Ether formation on the tridentate Schiff base ligands of copper(II) complexes

A series of copper(II) complexes, CuL·imidazole, where L2− are tridentate Schiff base ligands formed by condensation of salicylaldehyde with a series of amino acids, have been synthesized. Visible spectral data indicate that copper(II) in these complexes are five coordinate in the solid state and in solution. Electrospray mass spectrometry has been used to show how these complexes react in alcohol/NaOH solutions with and without the presence of d-galactose. In the absence of d-galactose where the amino acid in the ligand is serine, the alcohol group on the ligand is converted to its alkyl ether after sonication of the solution for up to 4 h. In the presence of d-galactose, an alkoxy group is added to the ligands except for the ligand containing serine after sonication of the solutions for up to 4 h. At the same time, d-galactose is oxidized to its aldehyde. Where the ligand contains methionine, oxygen is also added to the ligand, most likely to the thioether sulfur. Graphical Abstract