Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance

Covalent conjugation of superoxide dismutase and catalase with polyethylene glycol (PEG) increases the circulatory half-lives of these enzymes from less than 10 min to 40 h, reduces immunogenicity, and decreases sensitivity to proteolysis. Because PEG has surface active properties and can induce cell fusion, we hypothesized that PEG conjugation could enhance cell binding and association of normally membrane-impermeable enzymes. Incubation of cultured porcine aortic endothelial cells with 125I-PEG-catalase or 125I-PEG-superoxide dismutase produced a linear, concentration-dependent increase in cellular enzyme activity and radioactivity. Fluorescently labeled PEG-superoxide dismutase incubated with endothelial cells showed a vesicular localization. Mechanical injury to cell monolayers, which is known to stimulate endocytosis, further increased the uptake of fluorescent PEG-superoxide dismutase. Endothelial cell cultures incubated with PEG-superoxide dismutase and PEG-catalase for 24 h and then extensively washed were protected from the damaging effects of reactive oxygen species derived from exogenous xanthine oxidase as judged by two criteria: decreased release of intracellular 51Cr-labeled proteins and free radical-induced changes in membrane fluidity, measured by electron paramagnetic resonance spectroscopy of endothelial membrane proteins covalently labeled with 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Addition of PEG and PEG-conjugated enzymes perturbed the spin-label binding environment, indicative of producing an increase in plasma membrane fluidity. Thus, PEG conjugation to superoxide dismutase and catalase enhances cell association of these enzymes in a manner which increases cellular enzyme activities and provides prolonged protection from partially reduced oxygen species.     

Electron spin resonance study of light-induced conformational changes in nitroxide labelled bovine rhodopsin

Bovine rhodopsin was isolated in the unbleached form as a retinal disc membrane suspension and spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl. Both conventional and saturation transfer electron spin resonance methods were used to investigate the sensitivity of the spin-label to conformational changes of rhodopsin induced by both transient and long-term exposure to light. The results indicate that the ESR methods do display sensitivity to such changes. An exponential decay curve with a time constant of 10 s was obtained by following the height of a single peak in the saturation transfer electron spin resonance spectrum in response to a single light flash.     

A novel analytical method to evaluate directly catalase activity of microorganisms and mammalian cells by ESR oximetry

Abstract

Electron spin resonance (ESR) oximetry technique was applied for analysis of catalase activity in the present study. Catalase activity was evaluated by measuring oxygen from the reaction between hydrogen peroxide (H₂O₂) and catalase-positive cells. It was demonstrated that the ESR spectra of spin-label probes, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO) and 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (4-maleimido-TEMPO) in the presence of H₂O₂ were broadened with the concentrations of catalase. It was possible to make a calibration curve for catalase activity by peak widths of the spectra of each spin-label probe, which are broadened dependently on catalase concentrations. The broadened ESR spectra were also observed when the catalase-positive micro-organisms or the mammalian cells originally from circulating monocytes/macrophages were mixed with TEMPOL and H₂O₂. Meanwhile, catalase-negative micro-organisms caused no broadening change of ESR spectra. The present study indicates that it is possible to evaluate directly the catalase activity of various micro-organisms and mammalian cells by using an ESR oximetry technique.     

Permeation of a spin-label phosphate into the human erythrocyte.

The reduction of spin-labels by human erythrocytes can be used to follow their penetration into these cells. The neutral spin-label alcohol Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl) diffuses through the membrane very quickly. The membrane is virtually impermeable to the positively charged spin-label Tempo-choline (N,N-dimethyl-N-(2',2',6',6'-tetramethyl-4'-piperidinyl-1-oxyl)-2-hydroxyethylammonium chloride). The negatively charged spin-label Tempo phosphate (4-phospho-2,2,6,6-tetramethylpiperidinyl-l-oxyl) is reduced at 37 degrees, with a half-time of about 1 hr. The reduction occurs internally following the rate-limiting transport of the label across the erythrocyte membrane. Reduction of this spin-label is greatly diminished by the specific inhibitor of anion transport, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). The rate of transport depends strongly on the transmembrane electrical potential.     

Radiation-induced structural alterations in fish red blood cells

The structural properties of gamma-irradiated fish red blood cells were studied using a spin labelling method. The gradient increase of lipid fluidity with the increasing gamma radiation doses was indicated by methyl 5-doxylpalmitate and methyl 12-doxylstearate spin labels spectra. In turns, the spectra of maleimide spin label (4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl) indicated a modification of the internal proteins and the increased internal viscosity of red blood cells. The results encourage the conclusion that the increase in membrane fluidity may result from theernations in lipid-protein interactions rather than lipid peroxidation.     

Anti-nitroxide immunoglobulin G: analysis of antibody specificity and their application as probes for spin-labeled proteins

Antibodies have been elicited to the nitroxide spin-label 4-maleimido-2,2,6,6-tetramethyl-piperidinyl-1-oxy conjugated, via protein sulfhydryl groups, to bovine serum albumin. Antibody-hapten cross-reactivity was demonstrated by double immunodiffusion and by a broadening of the nitroxide electron paramagnetic resonance spectrum. The specificity of the antibodies with respect to hapten structure was examined by means of a simple filter binding assay. Under these conditions, antibodies were shown to distinguish between the nitroxide and hydroxylamine derivatives and between spin-labels comprising either five- or six-membered ring structures. In addition, protein-bound nitroxide spin-labels were detected at the nanogram level by immunoblotting. By use of this method, the specificity of the antibody-hapten reaction predicted by the filter binding assay procedure was utilized to differentially detect various types of bound spin-label. Finally, antibodies were used to identify protein-bound nitroxide spin-label of protein fractionated by gel electrophoresis.     

Lipopolysaccharide from Proteus mirabilis O29 induces changes in red blood cell membrane lipids and proteins

Alterations in red blood cell (RBC) plasma membranes, i.e. in lipids and proteins, and osmotic fragility of these cells after treatment with Proteus mirabilis O29 endotoxin (lipolysaccharide (LPS)) were examined using a spin labelling method. At the highest concentration of LPS, insignificantly decreased fluidity of membrane lipids was observed. Changes in conformation of membrane proteins were determined by two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (MSL) and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The analysis of spectra of MSL and ISL showed modifications in membrane proteins in red blood cells treated with the highest concentration of lipopolysaccharide. On the other hand, in the case of isolated membranes, disturbances in membrane were observed for all concentrations of LPS. The alterations in membrane lipids and proteins are paralleled in a significant rise in osmotic fragility of RBCs upon endotoxin treatment. These results provide experimental evidence that P. mirabilis O29 LPS causes deleterious changes in membranes of human red blood cells. They show that action of lipopolysaccharide mainly concerns the membrane cytoskeleton.     

The role of the essential sulfhydryl group in assimilatory NADH: nitrate reductase of Chlorella

Incubation of the complex metalloflavoprotein, assimilatory nitrate reductase with N-ethylmaleimide, or a spin-labeled analog, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl, resulted in a time-dependent inactivation of NADH:nitrate reductase and NADH: cytochrome-c reductase activity with no effect on reduced methyl viologen:nitrate reductase activity. Inactivation of the enzyme, which could be prevented by incubation in the presence of NADH, was achieved following modification of a single sulfhydryl group determined from [3H]N-ethylmaleimide incorporation and quantitation of the EPR spectrum of the spin-labeled enzyme. Sulfhydryl group modification precluded reduction of the enzyme by NADH and NAD+ binding. The EPR spectrum of the spin-labeled enzyme revealed the presence of a single species with the nitroxide retaining substantial motional freedom. Cleavage of the spin-labeled enzyme using corn-inactivating protease and separation into its flavin and molybdenum/heme domains followed by EPR spectroscopy revealed the modified sulfhydryl group to be associated with the latter fragment suggesting a close interaction of these domains in the region of the nucleotide-binding site.     

Disruption of erythrocyte membranal organization by superoxide

Human erythrocyte ghosts were covalently labeled with 4-maleimide-2,2,6,6-tetramethylpiperidinooxyl. Electron paramagnetic resonance (EPR) spectrometry revealed two major binding environments representing strongly (S) and weakly (W) immobilized species. The disorder parameter, W/S, determined from the respective peak amplitudes, was shown to be irreversibly elevated following treatment of the labeled ghosts with superoxide, indicating an increase in membrane fluidity. Labeled ghosts reduced with ascorbate showed no nitroxide EPR signals. However, following exposure of these membranes to superoxide, the nitroxide spectrum returned with a W/S ratio of 25. In contrast, the disorder parameter for spin-labeled ghosts decreased following exposure to hydroxyl radicals suggesting decreased fluidity, as a result of lipid peroxidation. This effect could be prevented by the inclusion of mannitol. These changes in membrane fluidity and/or protein mobility observed by EPR are compared with previous results obtained by other methods and provide additional evidence for physiologic alterations initiated by superoxide.     

The mobility and reactivity of maleimide-binding proteins in the rat erythrocyte membrane. Effects of dietary zinc deficiency and incubation with zinc in vitro

Erythrocyte ghosts, prepared from rats fed zinc-deficient diets, were analyzed for the mobility of membrane proteins by electron spin resonance spectroscopy of the sulfhydryl-binding spin probe, 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl. Compared with erythrocyte membranes from rats fed zinc-adequate diets ad libitum or pair-fed, erythrocyte membranes from zinc-deficient rats had a significantly increased ratio of weakly immobilized to strongly immobilized probe-binding proteins. This suggests that dietary zinc deficiency causes a conformational change in erythrocyte membrane proteins. Dietary zinc deficiency did not significantly affect N-ethylmaleimide (NEM)-induced thermal sensitivity or NEM-induced mechanical fragility in rat erythrocytes; however, the addition of zinc in vitro to red cells significantly inhibits NEM-induced mechanical fragility.     

A spin-label study of the effects of drugs on calcium release from isolated sarcoplasmic reticulum vesicles

The effects of caffeine, thymol, and procaine on calcium release from fragmented sarcoplasmic reticulum (FSR) from rabbit skeletal white muscle were investigated by the spin label method at the organellar level. Two thiol-directed spin labels, 4-maleimide-2,2,6,6-tetramethylpiperidinooxyl and 4-(2-iodoacetamide)-2,2,6,6-tetramethylpiperidinooxyl, were used for the labeling of SR proteins. The ratio (W/S) of the weakly (W) and strongly (S) immobilized ESR signals was measured for the maleimide and iodoacetamide labeled FSR. The two labels gave different W/S values, which means that the two labels report conformational changes at different loci of SR proteins. The dependences of the W/S ratios on the concentration of the drugs showed that conformational changes of SR proteins induced by these drugs are not the same. From measurements of the distribution of 5-doxyldecanoic acid methylester between the lipid and water phases, it was found that the conformational changes of the SR proteins caused by thymol or procaine induced a disorder in local regions of the phospholipid bilayers of FSR, while such disordering was not induced by caffeine. On the other hand, caffeine and thymol showed definite effects on calcium release from FSR, while procaine did not. These results indicate that the effects of the drugs on the protein conformations can be well characterized at the organellar level by means of the spin label technique and that some specific changes in the conformations of SR proteins are necessary for calcium release from FSR.     

Carbonylcyanide p-trifluoro-methoxyphenylhydrazone-induced change of mitochondrial membrane structure revealed by lipid and protein spin labeling.

Structural information on the phenomena accompanying uncoupling of oxidative phosphorylation in mitochondria was obtained using lipid and protein spin labels. The event of partitioning, observed with a small lipid spin label, the 4,4-dimethyl-2,2-dipentyl-oxazolidine-3-oxide (6-N-11) has been studied. The ratio of polar/hydrophobic part of the third line of the spectra was decreased in the presence of the uncoupler carbonylcyanide-p-trifluoro-methoxyphenylhydrazone (FCCP), probably indicating a higher proportion of hydrophobic environment of the label. Protein spin labels have been employed to study mobilities and rate of reduction of the labels. A long-chain maleimide spin label, the 3-2-(2-maleimidoethoxy)ethylcarbamoyl-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl, in the presence of carbonylcyanide-p-trifluoro-methoxyphenylhydrazone revealed decreases of mobility and of the rate of reduction. Large amplification of these effects was obtained with a short-chain maleimide spin label, the 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. With this spin label, the effect of the uncoupler could be traced down to a concentration of 0.05 μm. It is concluded that both membrane lipid and protein are changed simultaneously in the uncoupling event.     

Alterations in human red blood cell membrane properties induced by the lipopolysaccharide from Proteus mirabilis S1959

The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant (P<0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly (P<0.05) decreased the relative rotational correlation time of ISL and significantly (P<0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h(w)/h(s) ratio. These results indicated increased membrane protein mobility, mainly in the spectrin-actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.     

Distance estimate of the active center of D-beta-hydroxybutyrate dehydrogenase from the membrane surface

D-beta-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) is a membrane-bound, lipid-requiring enzyme which has a reactive sulfhydryl in the vicinity of the active center. The spin-probe-spin-label technique has been used to estimate the distance of separation of the reactive sulfhydryl of D-beta-hydroxybutyrate dehydrogenase from the bilayer surface. The reactive sulfhydryl of the enzyme was derivatized with the maleimide spin-label reagent 4-maleimido-2,2,6,6-tetramethylpiperidinyl-1-oxy in the presence of the cofactor NAD+. The derivatized enzyme, inserted (inlaid orientation) into phospholipid vesicles, was titrated with spin probes, either Mn2+ or Gd3+, until the spin-label EPR spectrum was reduced in amplitude to its residual (limiting) value. From this limiting amplitude, the dipolar interaction coefficient was obtained, which is related to the reciprocal of the distance to the sixth power. The radial distances of closest approach of the paramagnetic Mn2+ and Gd3+ ions to the spin-label nitroxide on the enzyme were found to be 18 and 16 A, respectively. These calculated distances were in accord with those determined by comparison with a phosphatidylcholine calibration system having 2,2-dimethyloxazolidinyl-1-oxy spin-labels located at selected positions along the sn-2 fatty acyl chain. Since the distal nitroxide moiety of the maleimide spin-label (17 A from the bilayer surface) is 8 A from the sulfhydryl addition site, the two limiting distances of immersion of the reactive sulfhydryl within the bilayer are 9 and 25 A. The shorter distance is considered more compatible with facile access of the coenzyme to the active site of the enzyme.     

Electron spin resonance spin label studies of plasma fibronectin: effect of temperature

Plasma fibronectin was chemically modified by 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (maleimide spin label). Only the free sulfhydryl groups of plasma fibronectin were modified by the label under the experimental conditions. The ESR spectrum of spin-labeled fibronectin showed that the sites of labeling were highly immobilized, suggesting that the sulfhydryl groups of the protein are in small, confined environments. The conversion of the strongly immobilized ESR spectrum into a weakly immobilized one was observed when the spin-labeled protein was heated from 30 to 60 degrees C, indicating the thermal unfolding of the protein molecules. The midpoint temperature for the thermal unfolding of plasma fibronectin is about 50 degrees C. The results suggest that plasma fibronectin is stable to about 40 degrees C and starts unfolding above this temperature. The rotational correlation time estimated from the ESR spectrum of spin-labeled fibronectin at 21 degrees C was about 2.0 X 10(-8) s. The rotational correlation time calculated from the Stokes-Einstein equation, assuming a rigid globular configuration for fibronectin with a Stokes radius of 10 nm, was about 7.8 X 10(-7) s. The differences in rotational correlation time by a factor of 39 between experimental and calculated values do not support a globular configuration for plasma fibronectin.     

Binding of spin-labeled local anesthetics to lobster nerves

Summary The hyperfine coupling constant of spin-labeled local anesthetics, 2-(N-methyl N-(2,2,6,6-tetramethylpiperidinooxyl)) ethyl 4-alkoxybenzoates, showed these compounds to partition betwen the aqueous exterior and the hydrocarbon phase of the membrane. Increased partitioning into the hydrocarbon phase of the membrane was in the order: hexyloxy>butoxy>ethoxy. Since these compounds are known to have different durations of anesthesia in the same order, this suggests that durations of activity and ability to partition into the hydrocarbon region of the membrane are related.     

Ca2+-induced conformational changes of spin-labeled g2 chain bound to myosin and the effect of phosphorylation.

One of the low molecular weight components of myosin, g2, was isolated by alkali treatment of myosin and was chemically modified with a spin label reagent, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. The label on g2 showed a rather weakly immobilized ESR spectrum and it was clearly affected by Ca2+; the half-maximal change was at around pCa 4. The spin-labeled g2 was incorporated into myosin by exchange with the intrinsic g2 of myosin in 0.6 M KSCN or 4 M LiC1. The label on g2 became strongly immobilized on association with myosin. Under the conditions used, ESR spectral change due to Ca2+ occurred at two different concentration ranges, which were as low as pCa 8 and at around pCa 4. Phosphorylated g2 was isolated from myosin after the protein kinase [EC 2.1.1.37]-catalyzed phosphorylation of myosin and it was also modified with the maleimide label. Dephosphorylation of the phosphorylated g2 was performed using E. coli alkaline phosphatase [EC 3.1.3.1]. The effects of Ca2+ on the ESR spectra of phosphorylated and dephosphorylated g2 were investigated on the state associated with myosin. A change in the ESR spectrum from strongly immobilized to weakly immobilized states was observed with both g2 chains on the addition of Ca2+. However, the effective concentration ranges of Ca2+ were quite different; around pCa 4 for the phosphorylated g2 and around pCa 8 for the dephosphorylated g2. The results indicate that g2 undergoes a conformational change at physiological levels of Ca2+ sufficient to saturate troponin, but it does not do so after phosphorylation.     

Probing the topography of lectins with site-specific spin-labeled glycosides

Three new spin-labeled glycosides, spin-label I [1-[4-(beta-D-galactopyranosyloxy)phenyl]-3-(2,2,6,6-tetramethyl-1 -oxypiperidin-4-yl)-2-thiourea], spin-label II (2,2,6,6-tetramethyl-1-oxypiperidin-4-yl alpha-D-galactopyranoside), and spin-label III [1-(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)-3-(2,2,6,6- tetramethyl-1-oxypiperidin-4-yl)-2-thiourea], were investigated as structural probes of Griffonia simplicifolia I isolectins (GS I) A4 and B4, respectively, by electron spin resonance (ESR) and inhibition of guaran isolectin precipitation. The p-aminophenyl beta-galactoside spin-label I was strongly immobilized by the B4 isolectin (Kd = 0.42 mM; 2T parallel = 54.0 +/- 0.3 G), while binding to the A4 isolectin was so weak (KI congruent to 2 mM) that binding was undetectable by ESR. The preference for the B4 isolectin was indicative of a more extended hydrophobic binding locus adjacent to the carbohydrate-specific binding site. The alpha-galactosyl spin-label II bound slightly more strongly to the A4 than to the B4 isolectin, as evidenced in both Kd values and particularly by differences in the degree of immobilization (2T parallel = 53.5 vs. 51.5 G, respectively). The 2-N-substituted methyl galactoside spin-label III was so poor an inhibitor of both isolectins (KI congruent to 1-2 mM) that ESR detection of the bound complex was not feasible. In all cases above, the spin-labels were displaced by specific monosaccharide haptens.     

A spin labeling study of the effects of inorganic ions and pH on the conformation of spectrin

The structure of spectrin from human erythrocytes has been investigated by the EPR technique measuring the mobility of the protein spin label, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Conformational changes in the protein induced by variation of the concentrations of NaCl, Na2SO4, KCl, CaCl2 and MgCl2 and of pH have been studied. It could be demonstrated that both Ca2+ and Mg2+ give rise to structural changes by binding to specific sites, whereas the monovalent cations (K+, Na+) seem to act via ionic strength. A model is used to correlate the spin label mobility with the radius of the protein. In the Ca2+- and Mg2+-binding experiments, the decrease in the spin label mobility has been interpreted on the basis of the theory of multiple chemical equilibria. These experiments have been compared with EPR spectra measured at different pH values. The results support the model in that binding of H+, Ca2+ or Mg2+ reduces the charges located on the protein surface: the 'discharging' reduces the repulsive forces on the surface of the molecule and consequently, the protein contracts in discrete steps.     

A comparative structural analysis of Fab-fragments of monoclonal rheumatoid and non-rheumatoid immunoglobulins M

The spin-labeling method was used to study the Fab- and Fab-RF-fragments of IgM and IgM-RF, respectively. The spin-label 2,2,6,6-tetramethyl-4-dichloro-sym-triazinyl-aminopiperidine-1-oxyl was introduced into the peptide moiety of the proteins. The rotational correlation time t of the spin-label carrier was determined based on the temperature-viscosity dependence of the EPR spectra parameters of the spin-labeled proteins. The tau values for Fab- and Fab-RF-fragments were 21 +/- 2 and 12 +/- 1 ns, respectively. The data strongly suggest that the significantly lower tau value for the Fab-RF-fragment may be due to the local structural flexibility of the fragment, which in turn may explain the peculiarities of IgM-RF as an autoantibody.