Biomedical subjects

Kenneth B Tomer

Publications and source records attributed to Kenneth B Tomer.

42 records · Page 3Linked to original sources

PubMed2002

Identification of protein-derived tyrosyl radical in the reaction of cytochrome c and hydrogen peroxide: characterization by ESR spin-trapping, HPLC and MS.

The reaction of cytochrome c and H(2)O(2) is known to form a protein-centred radical that can be detected with the spin trap 2-methyl-2-nitrosopropane (MNP). To characterize the MNP/tyrosyl adduct structure that had previously been determined incorrectly [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498-15503], we eliminated unreasonable structure models by ESR studies with a series of (13)C-labelled tyrosines, and photochemically synthesized an authentic MNP/tyrosyl adduct that has its trapping site on the C-3 position of the tyrosine phenyl ring. The observation of the identical ESR spectra for this radical adduct from the UV irradiation of 3-iodo-tyrosine and the adduct from the cytochrome c reaction demonstrated that the radical trapping site of MNP/tyrosyl is located on the equivalent C-3/C-5 positions instead of the C-1 position, as was proposed by Barr et al. In an on-line HPLC/ESR system, an identical retention time (17.7 min) was observed for the ESR-active HPLC peak of the MNP/tyrosyl adduct from the following three reactions: (i) the tyrosine oxidation via horseradish peroxidase/H(2)O(2); (ii) UV irradiation of 3-iodo-tyrosine and (iii) the reaction of cytochrome c with H(2)O(2). This result demonstrated that the radical adducts of all three reactions are most probably the same. The mass spectrometric analysis of the HPLC fractions from reactions (i) and (ii) showed an ion at m/z 267 attributed to the MNP/tyrosyl adduct. We conclude that the cytochrome c-derived tyrosyl radical was trapped by MNP, leading to a persistent radical adduct at the C-3/C-5 positions of the tyrosine phenyl ring.

Animals↗

PubMed2002

Characterization of the tertiary structure of soluble CD4 bound to glycosylated full-length HIVgp120 by chemical modification of arginine residues and mass spectrometric analysis.

The initial step of infection of blood cells with the human immunodeficiency virus, HIV, is the formation of a complex of the viral envelope protein gp120 and its human receptor CD4. We have examined structural features of recombinant soluble CD4 (sCD4) by chemical modification of arginine residues with hydroxyphenylglyoxal and subsequent analysis by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. As R58, R59, R131, R134, R219, R240, R293, and R329 could be derivatized free in solution, these arginine residues were exposed on the surface of the protein. In the noncovalent complex of sCD4 with HIV(SF2)gp120, only R58, R131, R134, R219, R240, R293, and R329 were accessible for the derivatizing agent. R59 was shielded from hydroxyphenylglyoxal and was, therefore, considered to be part of the interaction site with gp120. This indicates that the carbohydrate moieties and the flexible variable loops of the glycosylated full-length gp120 from HIV strain SF2 do not induce a reorganization of CD4 in its binding to gp120 and, therefore, do not appear to significantly affect the structural orientation of the primary receptor in complex with the HIV envelope protein as compared to the binding observed in the crystal structure of CD4 with truncated deglycosylated gp120.

Amino Acid Sequence↗

PubMed2002

Separation and characterization of human high-density apolipoproteins using a nonaqueous modifier in capillary electrophoresis-mass spectrometry.

The separation and characterization of human apolipoproteins and their isoforms was investigated using capillary electrophoresis (CE) in combination with mass spectrometry (MS). The focus of these analyses was the major protein constituents of plasma high-density lipoproteins, apolipoprotein A-I and A-II. Using aqueous buffers in CE, no separation between apolipoprotein A-I and A-II was observed. With the addition of 10-20% acetonitrile, however, the two species could be separated. Furthermore, multiple peaks for each of the apolipoprotein species were observed under these CE conditions. In order to identify and characterize the components, these separations were then coupled with online mass spectrometric detection (CE-MS). Our CE-MS results suggest that the multiple components observed in the acetonitrile-containing CE separation appear to be oxidized forms of the proteins in addition to native forms of the apolipoprotein A-I and A-II. These data are in agreement with previous reports that the methionine residues of the high-density lipoproteins (HDLs) are sensitive to oxidation, which in turn, alters their lipid binding characteristics and secondary structure. In addition to oxidized forms of the proteins, apolipoprotein A-II contained additional components, which varied in mass by 128 Da. The structural differences between these components were determined by proteolytic digestion and tandem MS. Using these techniques, we determined that these components were due to truncation of the C-terminal glutamine amino acid residue on apolipoprotein A-II. These results demonstrate that CE in combination with MS is a promising technique for screening and characterizing isomers of plasma apolipoproteins.

Acetonitriles↗

PubMed2002

MALDI/MS-based epitope mapping of antigens bound to immobilized antibodies.

Proteolytic digestion of proteins bound to immobilized antibodies, combined with matrix assisted laser desorption (MALDI) mass spectrometric identification of the affinity-bound peptides, can be a powerful technique for epitope determination. Binding of the protein to the antibody is done while the protein is in its native, folded state. A purified protein is not required for this procedure, because only proteins containing the antigenic determinant will bind to the antibody in the initial step. The method makes use of the resistance of the antibody to enzymatic digestion. Enzymatic cleavage products of the antigenic protein not containing the epitope are washed off the beads, leaving the epitope-containing fragments affinity bound to the immobilized antibody. Dissociation of the antigen-antibody complex prior to mass spectrometric analysis is unnecessary because the affinity-bound peptides are released by the MALDI matrix crystallization process, although the antibody remains covalently attached to the sepharose beads. This epitope-mapping protocol has been used in the determination of both continuous and discontinuous epitopes on both glycosylated and unglycosylated proteins.

Amino Acid Sequence↗

PubMed2001

Electron spin resonance investigation of semiquinone radicals formed from the reaction of ubiquinone 0 with human oxyhemoglobin.

The redox properties and thiol reactivity of quinones play critical roles in their therapeutic and toxicological properties. The present study was undertaken to investigate the binding activity of ubiquinone 0 (UQ(0)) to human oxyhemoglobin (HbO(2)) using electron spin resonance (ESR). Addition of UQ(0) to HbO(2) resulted in the immediate detection of a five-line ESR spectrum characteristic of the semiquinone radical of UQ(0) (UQ(0)). With time the HbO(2) adduct with UQ(0), which was characterized by a broad immobilized ESR spectrum, was gradually formed. Matrix-assisted laser desorption/ionization time-of-flight mass spectra analysis showed that UQ(0) bound to the beta-chain of HbO(2). Superoxide dismutase dose-dependently suppressed the intensity of the broad spectrum and accelerated its formation. However, N-ethylmaleimide, a thiol-blocking agent, completely eliminated its formation. The nonspecific protease mixture pronase also prevented its formation and resulted in the gradual appearance of a 4-line spectrum from the 5-line spectrum of UQ(0). The structure of the species responsible for the 4-line spectrum was confirmed and identified by the reaction of UQ(0) with reduced glutathione. In human red blood cells, UQ(0) rapidly bound to glutathione but more slowly to HbO(2). These results suggest that UQ(0) reacted with both ferrous heme and the reactive beta-93 cysteinyl residue of HbO(2) to generate its corresponding semiquinone radical. Subsequently UQ(0) bound to the beta-93 cysteinyl residue of HbO(2) to form a covalent-binding adduct responsible for the broad spectrum.

Benzoquinones↗

PubMedDate not supplied

Effect of nonaqueous buffer modifiers on the capillary electrophoresis-mass spectrometry analysis of peptides.

The effect of the addition of nonaqueous modifiers in capillary electrophoresis at varying concentrations was determined using synthetic peptides as model compounds. The electroosmotic flow is compared in each of the binary solvent systems and, in all cases, an increase in the concentration of organic solvent resulted in a decrease in the electroosmotic flow. The electrophoretic mobilities of each peptide were calculated and, in the case of acetonitrile, little or no change was observed in the peptide mobilities. In general, a decrease in the electrophoretic mobilities of the peptides was observed for increasing concentrations of methanol and N-methylformamide. The compatibility of each modifier at 25% (vol/vol) for use with mass spectrometric detection was evaluated. From our data, the addition of acetonitrile to the CE buffer appears to be the most compatible with mass spectrometry due to shorter analysis times and increased sensitivities in comparison to methanol and N-methylformamide.

Buffers↗