Abstract
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.
Full Text
The Full Text of this article is available as a PDF (96.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Andersson L. A., Loehr T. M., Lim A. R., Mauk A. G. Sulfmyoglobin. Resonance Raman spectroscopic evidence for an iron-chlorin prosthetic group. J Biol Chem. 1984 Dec 25;259(24):15340–15349. [PubMed] [Google Scholar]
- Babcock G. T., Ingle R. T., Oertling W. A., Davis J. C., Averill B. A., Hulse C. L., Stufkens D. J., Bolscher B. G., Wever R. Raman characterization of human leukocyte myeloperoxidase and bovine spleen green haemoprotein. Insight into chromophore structure and evidence that the chromophores of myeloperoxidase are equivalent. Biochim Biophys Acta. 1985 Mar 22;828(1):58–66. doi: 10.1016/0167-4838(85)90009-3. [DOI] [PubMed] [Google Scholar]
- Baehner R. L., Nathan D. G. Leukocyte oxidase: defective activity in chronic granulomatous disease. Science. 1967 Feb 17;155(3764):835–836. doi: 10.1126/science.155.3764.835. [DOI] [PubMed] [Google Scholar]
- Bakkenist A. R., Wever R., Vulsma T., Plat H., van Gelder B. F. Isolation procedure and some properties of myeloperoxidase from human leucocytes. Biochim Biophys Acta. 1978 May 11;524(1):45–54. doi: 10.1016/0005-2744(78)90101-8. [DOI] [PubMed] [Google Scholar]
- Bolscher B. G., Denis S. W., Verhoeven A. J., Roos D. The activity of one soluble component of the cell-free NADPH:O2 oxidoreductase of human neutrophils depends on guanosine 5'-O-(3-thio)triphosphate. J Biol Chem. 1990 Sep 15;265(26):15782–15787. [PubMed] [Google Scholar]
- Borregaard N. The human neutrophil. Function and dysfunction. Eur J Haematol. 1988 Nov;41(5):401–413. doi: 10.1111/j.1600-0609.1988.tb00219.x. [DOI] [PubMed] [Google Scholar]
- Cross A. R., Curnutte J. T. The cytosolic activating factors p47phox and p67phox have distinct roles in the regulation of electron flow in NADPH oxidase. J Biol Chem. 1995 Mar 24;270(12):6543–6548. doi: 10.1074/jbc.270.12.6543. [DOI] [PubMed] [Google Scholar]
- Curnutte J. T., Whitten D. M., Babior B. M. Defective superoxide production by granulocytes from patients with chronic granulomatous disease. N Engl J Med. 1974 Mar 14;290(11):593–597. doi: 10.1056/NEJM197403142901104. [DOI] [PubMed] [Google Scholar]
- Fenna R., Zeng J., Davey C. Structure of the green heme in myeloperoxidase. Arch Biochem Biophys. 1995 Jan 10;316(1):653–656. doi: 10.1006/abbi.1995.1086. [DOI] [PubMed] [Google Scholar]
- Hurst J. K., Loehr T. M., Curnutte J. T., Rosen H. Resonance Raman and electron paramagnetic resonance structural investigations of neutrophil cytochrome b558. J Biol Chem. 1991 Jan 25;266(3):1627–1634. [PubMed] [Google Scholar]
- Isogai Y., Shiro Y., Nasuda-Kouyama A., Iizuka T. Superoxide production by cytochrome b558 purified from neutrophils in a reconstituted system with an exogenous reductase. J Biol Chem. 1991 Jul 25;266(21):13481–13484. [PubMed] [Google Scholar]
- Lehrer R. I., Cline M. J. Leukocyte myeloperoxidase deficiency and disseminated candidiasis: the role of myeloperoxidase in resistance to Candida infection. J Clin Invest. 1969 Aug;48(8):1478–1488. doi: 10.1172/JCI106114. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nauseef W. M. Myeloperoxidase deficiency. Hematol Pathol. 1990;4(4):165–178. [PubMed] [Google Scholar]
- Puppels G. J., Garritsen H. S., Kummer J. A., Greve J. Carotenoids located in human lymphocyte subpopulations and natural killer cells by Raman microspectroscopy. Cytometry. 1993;14(3):251–256. doi: 10.1002/cyto.990140303. [DOI] [PubMed] [Google Scholar]
- Puppels G. J., Garritsen H. S., Segers-Nolten G. M., de Mul F. F., Greve J. Raman microspectroscopic approach to the study of human granulocytes. Biophys J. 1991 Nov;60(5):1046–1056. doi: 10.1016/S0006-3495(91)82142-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Puppels G. J., de Mul F. F., Otto C., Greve J., Robert-Nicoud M., Arndt-Jovin D. J., Jovin T. M. Studying single living cells and chromosomes by confocal Raman microspectroscopy. Nature. 1990 Sep 20;347(6290):301–303. doi: 10.1038/347301a0. [DOI] [PubMed] [Google Scholar]
- Rotrosen D., Yeung C. L., Leto T. L., Malech H. L., Kwong C. H. Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. Science. 1992 Jun 5;256(5062):1459–1462. doi: 10.1126/science.1318579. [DOI] [PubMed] [Google Scholar]
- Salmaso B. L., Puppels G. J., Caspers P. J., Floris R., Wever R., Greve J. Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human eosinophilic granulocytes. Biophys J. 1994 Jul;67(1):436–446. doi: 10.1016/S0006-3495(94)80499-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Segal A. W., West I., Wientjes F., Nugent J. H., Chavan A. J., Haley B., Garcia R. C., Rosen H., Scrace G. Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes. Biochem J. 1992 Jun 15;284(Pt 3):781–788. doi: 10.1042/bj2840781. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sibbett S. S., Hurst J. K. Structural analysis of myeloperoxidase by resonance Raman spectroscopy. Biochemistry. 1984 Jun 19;23(13):3007–3013. doi: 10.1021/bi00308a025. [DOI] [PubMed] [Google Scholar]
- Smith R. M., Curnutte J. T. Molecular basis of chronic granulomatous disease. Blood. 1991 Feb 15;77(4):673–686. [PubMed] [Google Scholar]
- Stump R. F., Deanin G. G., Oliver J. M., Shelnutt J. A. Heme-linked ionizations of myeloperoxidase detected by Raman difference spectroscopy. A comparison with plant and yeast peroxidases. Biophys J. 1987 Apr;51(4):605–610. doi: 10.1016/S0006-3495(87)83385-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sumimoto H., Sakamoto N., Nozaki M., Sakaki Y., Takeshige K., Minakami S. Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein. Biochem Biophys Res Commun. 1992 Aug 14;186(3):1368–1375. doi: 10.1016/s0006-291x(05)81557-8. [DOI] [PubMed] [Google Scholar]
- Wever R., Plat H. Spectral properties of myeloperoxidase and its ligand complexes. Biochim Biophys Acta. 1981 Oct 13;661(2):235–239. doi: 10.1016/0005-2744(81)90009-7. [DOI] [PubMed] [Google Scholar]
- Winterbourn C. C., Garcia R. C., Segal A. W. Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride. Biochem J. 1985 Jun 15;228(3):583–592. doi: 10.1042/bj2280583. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yazdanbakhsh M., Eckmann C. M., Koenderman L., Verhoeven A. J., Roos D. Eosinophils do respond to fMLP. Blood. 1987 Aug;70(2):379–383. [PubMed] [Google Scholar]