Modification of surface residues of lysine in immunoglobulin G using the spin marker 2,2,5,5-tetramethyl-3-maleimidopyrrolidine-1-oxyl

Exposed lysine residues of human IgG were modified by a spin-label, 2,2,5,5-tetramethyl-3-male-imidopyrrolidine-1-oxyl at pH 9.2. Under these conditions, the degree of modification was about 10 lysine residues per protein molecule. The ESR spectrum of the spin-labeled immunoglobulin was much more mobile than that of spin-labeled immunoglobulin with the modification degree of about 1 residue that was obtained at pH 7.0. Thus, the sharp increase in the modification degree due to the increase in pH by two units leads to a marked loosening of the tertiary structure of the protein in solution, which is just indicated by the mobile ESR spectrum. Lithium chloride added to the solution of spin-labeled immunoglobulin induces a similar "immobilization" of its ESR spectra as sucrose.     

Rotational correlation times of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy spin label with respect to heme and nonheme proteins

Noncovalent spin labeled proteins (ovalbumin, bovine serum albumin, hemoglobin, and cytochrome c) were investigated in order to follow the different type of interactions between the nitroxide radical of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy spin label and functional groups of heme and nonheme proteins as well as the pH influence on molecular motion of the label with respect to these proteins. EPR spectra were recorded at room temperature and the computer simulation analysis of spectra was made in order to obtain the magnetic parameters. Noncovalent labeling of proteins can give valuable information on the magnetic interaction between the label molecule and the paramagnetic center of the proteins. The relevance of this interaction can be obtained from line shape analysis: computer simulations for nonheme proteins assume a Gaussian line shape, whereas for heme proteins, a weighted sum of Lorentzian and Gaussian components is assumed. In the framework of the "moderate jump diffusion" model for rotational diffusion, the rotational correlation time is strongly influenced by pH, because of the electrostatic interactions and hydrogen bonding.     

Electron spin resonance and pulse radiolysis studies on the spin trapping of sulphur-centered radicals

Thiyl radicals are shown to be readily trapped with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO) giving characteristic spin adducts with hyperfine coupling constants aN 1.52-1.58, aH 1.52-1.80 mT, and g values in the range 2.0065-2.0067 for the DMPO adducts and aN 1.50-1.56, aH 1.70-1.92 mT, g 20049-2.0051 for the TMPO adducts. Kinetic data obtained from pulse radiolysis studies show that, in general, thiyl radicals react rapidly with these spin traps with rate constants of the order of 10(7)-10(8) dm3 mol-1 s-1. The tetramethylated spin trap TMPO though giving slightly less intense electron spin resonance (ESR) spectra, produces longer lived adducts, and is suggested to be of greater utility due to the more characteristic nature of the coupling constants of the observed adducts; reaction of certain thiyl radicals with DMPO produces adducts which are superficially similar to the hydroxyl radical adduct to the same trap.     

Reversed-phase ion-pair high-performance liquid chromatography of mercaptoacetate and N-acetylcysteine after derivatization with N-(1-pyrene)maleimide and N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide

We have developed a high-performance liquid chromatographic system capable of resolving mercaptoacetate and N-acetylcysteine as their N-(1-pyrene)maleimide (PM) and N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM) derivatives. Good resolution was obtained by ion pairing with tetramethylammonium hydroxide and chromatography on reversed phase. The detection limits for the thiols were about 50 fmol as their DACM derivatives and about 400 fmol as their PM derivatives. The method is illustrated by chromatography of urinary thiols which indicates that the derivatization and chromatography procedures should be well applicable in bioanalytical work.     

Electron spin resonance studies on the lipoxygenase reaction by spin trapping and spin labelling methods.

The rate of oxygenation and that of trapping linoleic acid free radicals in the lipoxygenase [EC 1.13.11.12] reaction were measured in the presence of linoleic acid, oxygen, and nitrosobenzene at various concentrations, with a Clark oxygen electrode and ESR spectroscopy. The results were interpreted under the assumption that the free radical of linoleic acid, an intermediate of the lipoxygenase reaction, reacts competitively with oxygen or nitrosobenzene. The oxidation of the iron in the active site of lipoxygenase caused by the spin label reagent, 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl, was also observed by ESR- and fluorescence-spectroscopy.     

Photoaffinity labeling of rat liver ribosomes by N-(2-nitro-4-azidobenzoyl)puromycin

N-(2-Nitro-4-azidobenzoyl)-[3H]puromycin (NAB-puromycin) was synthesized as a photoreactive derivative of puromycin in order to detect ribosomal proteins located near the peptidyltransferase centre of rat liver ribosomes. Irradiation of ribosome-NAB-puromycin complexes leads to covalent attachment of the affinity label to proteins of the large ribosomal subunit, in particular to proteins L28/29, and, to a somewhat lower extent, to proteins L4, L6, L10 and L24. The results are discussed in the light of earlier studies performed with other affinity labels that attacked the peptidyltransferase region of rat liver ribosomes.     

Electron spin resonance studies of the reaction of hypochlorite with 5,5-dimethyl-1-pyrroline-N-oxide

The reaction of hypochlorous acid with the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was found to yield 5,5-dimethyl-2-pyrrolidone-N-oxyl (DMPOX). In addition to DMPOX, 5,5-dimethyl-2-hydroxypyrrolidine-N-oxyl (DMPO-OH) and an unidentified chlorine-containing radical species were also observed under neutral and near-neutral conditions. Through the use of [17O]HOCl and the hydroxyl radical scavengers ethanol and formate, it was established that DMPO-OH was derived from hydration of DMPO rather than the spin-trapping of hydroxyl radical. Furthermore, kinetic studies and the incorporation of 17O showed that DMPO-OH was readily oxidized to DMPOX and that this reaction was acid and base catalyzed. Under strongly alkaline conditions, DMPOX reversibly formed another species, presumably the enolate, that had a four-line ESR signal identical to that of DMPO-OH. Eventually, carbon-centered adducts appeared whose ESR signals were consistent with the formation of DMPO condensation products.     

Electron spin resonance studies of ultraviolet irradiation effects on some proteins

Summary Electron spin resonance signals of dry protein samples are enhanced after ultraviolet irradiation at wavelength 3650 Å, which corresponds closely to the energy gap between valence and conduction bands.Proteins denatured with short wave ultraviolet or 8 M urea fail to show the enhancement after irradiation at wavelength 3650 Å. This result supports the hypothesis that hydrogen bond structures are relevant in determining the semiconductor properties of proteins.     

1H-NMR studies of lipid extracts of rat liver mitochondria

Lipid extracts of rat liver mitochondria were studied by 1H-NMR at 200 MHz. Each spectrum, obtained in 6 min with routine acquisition parameters, allowed the estimation of the phosphatidylcholine/phosphatidylethanolamine ratio, the cholesterol/total lipid molar ratio, the average fatty acid length, the unsaturation ratio and the oxidation state of the ubiquinone/ubiquinol couple. 1H-NMR spectroscopy was also used to analyze structural changes of the lipids following peroxidation. In model liposomes subjected to enzymatic peroxidation the appearance of hydroperoxides and conjugated double bonds were detected, whereas in mitochondria no such changes were seen even after a period of relatively high oxidative stress. This suggests that the systems for protection against oxidative damage in mitochondria are able to keep the amount of peroxidation products below the detection limit of the technique.     

Electron spin resonance and magnetic relaxation studies of gadolinium(III) complexes with human transferrin

A human serum transferrin complex was prepared in which Gd(III) was substituted for Fe(III) at the two metal-binding sites. Characteristic changes upon metal binding in both the UV absorption of ligated tyrosines and the solvent proton longitudinal magnetic relaxation rates demonstrated 2/1 metal stoichiometry and pH-dependent binding constants. Binding studies were complicated both by binding of Gd(III) to nonspecific sites on transferrin at pH less than or equal to 7 and by complexation of the Gd(III) by the requisite bicarbonate anion at pH greater than or equal to 6.0. A unique Gd(III) electron spin resonance spectrum, with a prominent signal at g = 4.96, was observed for the specific Gd(III)-transferrin complex. The major features of this spectrum were fit successfully by a model Hamiltonian which utilized crystal field parameters similar to those determined for Fe(III) in transferrin [Aasa, R. (1970) J. Chem. Phys. 52, 3919-3924]. The magnetic field dependence of the solvent proton relaxation rate was measured as a function of both pH and metal ion concentration. An observed biphasic dependence of the relaxation rate on metal concentration is attributed to either sequential metal binding to the two iron-binding sites with different relaxation properties or random binding to two sites that are similar but show conformationally induced changes in relaxation properties as the second metal is bound. The increase in the solvent proton relaxation rate with pH is consistent with a model in which a proton of a second coordination sphere water molecule is hydrogen bonded to a metal ligand which becomes deprotonated at pH 8.5.     

Effect of proteolysis on the electron spin resonance spectra of maleimide spin labeled erythrocyte membrane

The ratio of low-field amplitudes of weakly and strongly immobilized signals of ESR spectra of a maleimide spin label bound to erythrocyte membranes (hw/hs) increases progressively during incubation at 37 degrees C. This increase is due to the 'self-digestion' of membrane proteins by endogenous proteinases and is attenuated by proteinase inhibitors. Digestion of membranes with chymotrypsin also increases the hw/hs ratio. These results suggest a need for a careful interpretation of data from spin-labeled membrane proteins, especially in experiments involving prolonged incubations of membrane preparations when the proteolytic effects may be significant.     

Biochemical studies on sub-fractions of rat liver mitochondria

Highly purified mitochondria from rat liver were separated into six sub-fractions by differential centrifugation. The sub-fractions represent a spectrum from “heavy” to “very light” mitochondria. Enzymes representative of mitochondrial compartments were assayed to see whether functional differences occurred among the various mitochondrial sub-fractions. Respiratory control and NADH oxidase activity, both of which are indicators of mitochondrial structural integrity, were also measured. An enzyme marker for endoplasmic reticulum (glucose-6-phosphatase, G-6-Pase) was also assayed. Specific activities for monoamine oxidase (outer membrane marker), cytochrome oxidase (inner membrane marker) and malate-cytochrome c reductase did not vary within experimental error in all sub-fractions; similarly, for respiratory control and NADH oxidase activity. Malate dehydrogenase, a component of malate-cytochrome c reductase is located within the matrix surrounded by the inner membrane. Specific activity of adenylate kinase (located between the outer and inner membrane) decreased markedly from the “heavy” mitochondria to the “very light” fractions. Specific activity for G-6-Pase, very low in the “heavy” fractions, increased markedly in the “light” to “very light” fractions. Isopycnic density centrifugation on a linear sucrose density gradient of each of the fractions indicated that the correlation coefficient for the sucrose concentrations at which cytochrome oxidase and G-6-Pase activities peaked was 0.995. Thus the “light” to “very light” mitochondria may represent mitochondria whose outer membrane is still contiguous with the endoplasmic reticulum. Microsomes containing the endoplasmic reticulum peaked on the gradient at a significantly lower sucrose concentration than any of the mitochondrial sub-fractions. A buoyant effect of endoplasmic reticulum still attached to any of the mitochondrial sub-fractions would be expected to lower the density of attached mitochondria and thus give rise to “light” and “very light” mitochondria.     

The reaction of N-(1-pyrene)maleimide with sarcoplasmic reticulum.

The excimer fluorescence of the adduct of N-(1-pyrene)maleimide (PMI) with the Ca2+-ATPase was proposed as a probe of ATPase-ATPase interactions in sarcoplasmic reticulum (Lüdi and Hasselbach, Eur. J. Biochem., 1983, 130:5-8). We tested this proposition by analyzing the spectral properties and stoichiometry of the adducts of pyrenemaleimide with sarcoplasmic reticulum and with dithiothreitol and by comparing the effects of various detergents on the excimer fluorescence of the two adducts, with their influence on the sedimentation characteristics, ATPase activity, and light scattering of the pyrenemaleimide-labeled sarcoplasmic reticulum. These studies indicate that pyrenemaleimide reacts nearly randomly with several SH groups on the Ca2+-ATPase, and suggest that the observed excimer fluorescence of pyrenemaleimide-labeled sarcoplasmic reticulum may reflect intramolecular phenomena rather than ATPase-ATPase interactions. Further work is required to establish the relative contribution of intra- and intermolecular mechanisms to the excimer fluorescence.