Resonance Raman microspectroscopy of myeloperoxidase and cytochrome b558 in human neutrophilic granulocytes.

With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm.     

EPR spin-trapping of a myeloperoxidase protein radical

Incubation of myeloperoxidase (MPO) with H(2)O(2) in the presence of the spin trap DBNBS (3,5-dibromo-4-nitrosobenzenesulfonic acid) results in the EPR-detectable formation of a partially immobilized protein radical. The radical was only formed in the presence of both MPO and H(2)O(2), indicating that catalytic turnover of the protein is required. The changes in the EPR spectrum of the adduct upon treatment with pronase confirm that the spin trap is bound to a protein residue. These results establish that MPO, like lactoperoxidase [Lardinois, O. M., Medzihradszky, K. F., and Ortiz de Montellano, P. R. (1999) J. Biol. Chem. 274, 35441-35448], reacts with H(2)O(2) to give a protein radical intermediate. The protein radical may have a catalytic role, may be related to covalent binding of the prosthetic heme group to the protein, or may reflect a process that leads to inactivation of the enzyme.     

Hemosiderin An EPR study of water-insoluble iron in human and rat liver

EPR spectra of the water-insoluble iron fraction, hemosiderin of human and rat liver are described. The homogenate of freshly prepared perfused rat liver shows a non-heme iron signal at g = 4.3 and a high-spin heme-iron signal around g = 6, whereas the washed and sonicated sample of the insoluble iron fraction shows solely a non-heme iron signal at g = 4.3. This indicates that hemosiderin from rat liver does not contain heme iron.Human-liver preparations from post mortem obtained material show in the homogenates as well as in the washed and sonicated samples an intense high-spin heme iron signal at g = 6.0 and a non-heme iron signal at g = 4.3. A comparative experiment, carried out with “aged” rat liver preparations, reveals the same spectra as with the human preparations. It is concluded that the heme present in the insoluble iron fraction is caused by degradation of hemoglobin in the obduction material, and that heme is not a constituent of the insoluble depot iron.     

Cryopreservation of human granulocytes.

Granulocyte preservation was undertaken using hydroxyethylstarch for both sedimentation of red cells and cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4% (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes. The leukocyte enriched (100–500×) supernatant was frozen at 2.0 °C/min to ?80 °C (and stored frozen up to 3 months). Alternatively, sedimented leukocytes were frozen after a slow addition of 10% DMSO to 5%. Tubes were thawed at 37 °C, and DMSO was removed by dilution with Hank's solution containing CPD and centrifugation. The pellets of granulocytes were resuspended in Normosol.Buffy coat from 10 units yielded 60 ± 9.7% of the available whole blood leukocytes, of which 43 ± 14% were recovered after sedimentation in HES. Freezing in DMSO yielded all, 101% of the prefrozen leukocytes. Postthawed viability of granulocytes was estimated morphologically and by their ability to inhibit the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Approximately 25% of the total available granulocytes in CPD whole blood were recovered.     

Raman microspectroscopic approach to the study of human granulocytes.

A sensitive confocal Raman microspectrometer was employed to record spectra of nuclei and cytoplasmic regions of single living human granulocytes. Conditions were used that ensured cell viability and reproducibility of the spectra. Identical spectra were obtained from the nuclei of neutrophilic, eosinophilic, and basophilic granulocytes, which yield information about DNA and protein secondary structure and DNA-protein ratio. The cytoplasmic Raman spectra of the three cell types are very different. This was found to be mainly due to the abundant presence of peroxidases in the cytoplasmic granules of neutrophilic granulocytes (myeloperoxidase) and eosinophilic granulocytes (eosinophil peroxidase). Strong signal contributions of the active site heme group(s) of these enzymes were found. This paper illustrates the potentials and limitations for Raman spectroscopic analysis of cellular constituents and processes.     

Molecular forms of myeloperoxidase in human plasma.

A radioimmunoassay for myeloperoxidase was established with the use of affinity-purified anti-(human myeloperoxidase) immunoglobulins. By the use of ion-exchange followed by immunoaffinity chromatography a preparation of immunoreactive, catalytically active myeloperoxidase was obtained from fresh human plasma. In non-denaturing gel electrophoresis, the plasma preparation showed about four catalytically active components of mobility very similar to that of the granulocyte enzyme. SDS/polyacrylamide-gel electrophoresis combined with protein blotting showed that the two polypeptides of strongest antigenicity in the plasma preparation corresponded in Mr to the large and the small subunits of the granulocyte enzyme. In addition, the plasma preparation contained a higher-Mr immunoreactive polypeptide, possibly a precursor form of the enzyme, together with another of Mr similar to that of the large subunit of eosinophil peroxidase.     

Interaction of human myeloperoxidase with nitrite.

EPR (electron paramagnetic resonance) and optical spectroscopy show that human neutrophil myeloperoxidase is converted from ferric high-spin to low-spin by the addition of nitrite. The Soret peak shifts from 429 to 447 nm and new peaks appear in the visible region at 573 and 627 nm; the EPR g-values change from 6.84, 5.02, 1.95 to 2.55, 2.31, 1.82. Small differences are seen in the EPR (but, not optical) spectra of myeloperoxidase isoenzyme I compared to isoenzymes II and III. The reaction with nitrite is detectable by EPR in intact neutrophils and is thus a possible in vivo monitor of NO/nitrite production by these cells.     

Cryoinjury in human granulocytes and cytoplasts.

Although most isolated cells can be successfully cryopreserved, human granulocytes have little functional recovery after cryopreservation, even under optimized conditions. Cytoplasts, which are vesicles created from human granulocytes by depletion of organelles including granules and the nucleus, can carry out some of the complex functions of the parent granulocyte such as phagocytosis of bacteria, even after cryopreservation. Human granulocytes and cytoplasts were used in this comparative study of low-temperature responses to assess the relative importance of the plasma membrane and the granules in cryoinjury to human granulocytes. Boyle-van't Hoff plots of cell volume as a function of the reciprocal of osmolality showed that granulocytes and cytoplasts have similar osmometric behavior and equivalent osmotically inactive fractions. The hydraulic conductivities were also similar, indicating that the osmotic properties of the plasma membrane and cytoplasm were retained during preparation of the cytoplasts. Assessment of membrane integrity using fluorescein diacetate after graded freezing stresses showed that the low-temperature responses of cytoplasts were similar to those of human lymphocytes and hamster fibroblasts, with recoveries much higher than those of human granulocytes, particularly after post-thaw incubation at 37 degrees C. The results indicate that the plasma membrane is not the primary site of injury to granulocytes during freezing and thawing, and suggest that activation of cytoplasmic elements, such as granules, may constitute the early events in cryoinjury to human granulocytes. These studies have significance in approaches to the cryopreservation of granulocytes and other types of cells, such as platelets, with increased sensitivity to the conditions encountered during freezing and thawing.     

The interaction of myeloperoxidase with ligands as studied by EPR

1. The reaction of myeloperoxidase with fluoride, chloride and azide has been studied by EPR. 2. Fluoride decreases the rhombicity of the high-spin heme signal of myeloperoxidase and the nuclear spin of the fluoride atom induces a splitting in g parallel of 35 G. This observation demonstrates that fluoride binds as an axial ligand to the heme iron of the enzyme. 3. Addition of chloride to the fluoride-treated enzyme increases the rhombicity of the high-spin heme signal and brings about a disappearance of the splitting at g parallel. The addition of azide to the fluoride-treated enzyme changes the spin state of the heme iron from a high-to a low-spin state (gx = 2.68, gy = 2.22 and gz = 1.80). 4. Upon addition of chloride or fluoride to low-spin azido-myeloperoxidase this compound is converted into the high-spin chlorido- or fluorido-myeloperoxidase. These observations demonstrate that these ligands compete for a binding site at or close to the heme iron of myeloperoxidase.     

Hemosiderin. an EPR study of water-insoluble iron in human and rat liver.

EPR spectra of the water-insoluble iron fraction, hemosiderin of human and rat liver are described. The homogenate of freshly prepared perfused rat liver shows a non-heme iron signal at g=4.3 and a high-spin heme-iron signal around g=6, whereas the washed and sonicated sample of the insoluble iron fraction shows solely a non-heme iron signal at g=4.3. This indicates that hemosiderin from rat liver does not contain heme iron. Human-liver preparations from post mortem obtained material show in the homogenates as well as in the washed and sonicated samples an intense high-spin heme iron signal at g=6.0 and a non-heme iron signal at g=4.3. A comparative experiment, carried out with "aged" rat liver preparations, reveals the same spectra as with the human preparations. It is concluded that that the heme present in the insoluble iron fraction is caused by degradation of hemoglobin in the obduction material, and that heme is not a constituent of the insoluble depot iron.     

An EPR study of the lineshape of copper in cytochrome c oxidase.

The EPR spectrum of copper in cytochrome c oxidase (EC 1.9.3.1) has been studied between 5 and 220 degreesK, and the spectral parameters have been determined for both forms of EPR-detectable copper by computer simulation methods. Numerical methods have been developed to separate the spectra of intrinsic copper and inactive copper. Evidence is presented to show that inactive copper is probably formed by denaturation. The EPR parameters for intrinsic copper were determined as gx = 1.99, gy = 2.03, gz = 2.185, / Ax(Cu) / = 0.0020 cm-1, / Ay(Cu) / = 0.0025 cm-1, / Az(Cu) / = 0.0030 cm-1. The principal values of the g tensor and the small value of /Az(Cu) / are interpreted in terms of mixing of 3d, 4s, and 4p metal orbitals. A flattened-tetrahedral stereochemistry about Cu2+ with an additional rhombic distrotion is in best agreement with all of the data. The peak-to-peak linewidth is found to be orientation dependent, and is described by a tensor with principal values deltaHx = 45G, deltaHy = 65 G, deltaHz = 85 G. A weak dipolar interaction with a low-spin ferric species stereochemistry for the copper ion is consistent with the electron transport function of the enzyme. Broad EPR signals with a very short spin-lattice relaxation time has been observed near g = 14 and g = 3 at 5 degrees K in oxidized cytochrome oxidase but not in the reduced or denatured enzyme. The possibility that these are due to the "EPR-undetectable" iron and copper is raised.     

Iron(II) and hydrogen peroxide detoxification by human H-chain ferritin. An EPR spin-trapping study

Overexpression of human H-chain ferritin (HuHF) is known to impart a degree of protection to cells against oxidative stress and the associated damage to DNA and other cellular components. However, whether this protective activity resides in the protein's ability to inhibit Fenton chemistry as found for Dps proteins has never been established. Such inhibition does not occur with the related mitochondrial ferritin which displays much of the same iron chemistry as HuHF, including an Fe(II)/H(2)O(2) oxidation stoichiometry of approximately 2:1. In the present study, the ability of HuHF to attenuate hydroxyl radical production by the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + OH(-) + *OH) was examined by electron paramagnetic resonance (EPR) spin-trapping methods. The data demonstrate that the presence of wild-type HuHF during Fe(2+) oxidation by H(2)O(2) greatly decreases the amount of .OH radical produced from Fenton chemistry whereas the ferroxidase site mutant 222 (H62K + H65G) and human L-chain ferritin (HuLF) lack this activity. HuHF catalyzes the pairwise oxidation of Fe(2+) by the detoxification reaction [2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH(core) + 4H(+)] that occurs at the ferroxidase site of the protein, thereby preventing the production of hydroxyl radical. The small amount of *OH radical that is produced in the presence of ferritin (相似文献   

Studies on the subunits of human myeloperoxidase.

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.     

Secretion of myeloperoxidase isoforms by human neutrophils.

The human neutrophil lysosomal enzyme, myeloperoxidase (MPO), exists in three major and chromatographically distinct forms, MPO I, MPO II, and MPO III. We used cation-exchange medium-pressure liquid chromatography and kinetic microenzyme assay (or spectrophotometric monitoring) to analyze the secretion of MPO isoforms by neutrophils exposed to N-formylmethionylleucylphenylalanine (FMLP), digitonin, the ionophore A23187, and serum-opsonized zymosan A (SOZ). All three MPO isomers were released into the fluid phase after neutrophils were exposed to these secretagogues. A significant proportional increase in MPO I was released when neutrophils were stimulated with SOZ. MPO I was released in higher proportions than found in the whole cell constituency when neutrophils were stimulated with FMLP + cytochalasin B, A23187, and digitonin, but this was not statistically significant.     

Identification of myeloperoxidase in human colostrum

The properties of a peroxidase in human colostrum were studied using antiserum against human myeloperoxidase. The peroxidase in human colostrum gave a single precipitin line against the antiserum on double immunodiffusion, and this precipitin line fused completely with the precipitin line formed between myeloperoxidase and the antiserum. The peroxidase activity in human colostrum was precipitated completely with anti-myeloperoxidase IgG, like myeloperoxidase activity. The peroxidase of colostral whey was purified to homogeneity. The purified enzyme consisted of two subunits of Mr 59,000 and 15,000, corresponding in size to the two subunits of myeloperoxidase. Immunostaining of a protein blot from a sodium dodecyl sulfate-polyacrylamide electrophoresis gel also showed that the peroxidase in the whey extract consisted of the same two subunits as myeloperoxidase. These results indicate that the peroxidase of human colostrum is identical with myeloperoxidase.     

Galvanotaxis of human granulocytes

The galvanotactic response of human granulocytes was investigated theoretically and experimentally. The basic results are: (i) The granulocytes move towards the anode. (ii) The directed movement has been quantified by two different polar order parameters-the McCutcheon index and the average of cos . (iii) The polar order parameters are a function of the applied electric field (= dose-response curve). (iv) The inverse of the galvanotactic constant of migrating cells (analogous to the Michaelis-Menten constant) has a value of-0.2±0.03 V/mm. (v) The galvanotactic response of granulocytes is a non-cooperative process with a cooperativity coefficient of 1±0.2. (vi) The galvanotactic constant is a function of pH. (vii) The protein essential for the galvanotactic response is very likely a G-protein.