Resonance Raman Microspectroscopy of Myeloperoxidase and Cytochrome b558 in Human Neutrophilic Granulocytes

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.

Raman spectroscopy: techniques and applications in the life sciences

“…For example, Salmaso et al used resonance Raman spectroscopy to study the enzyme eosinophil peroxidase in living immune cells [197]. Sijtsema et al were able to distinguish redox states in single immune cells by measuring resonance Raman signals from cytochrome b 558 and myeloperoxidase [198].…”

New Ways of Imaging Uptake and Intracellular Fate of Liposomal Drug Carrier Systems inside Individual Cells, Based on Raman Microscopy

“…It has been demonstrated that Raman microspectroscopy is capable of imaging cellular organelles such as the nucleus or chromatin, 1,2 mitochondria, 3 and lipid bodies, 4,5 as well as following metabolic processes or enzymatic activity inside cells. [6][7][8] Apart from imaging subcellular features, Raman imaging may provide an opportunity to follow the uptake of molecules in cell cultures. One way to distinguish incorporated molecules inside cells is to use deuterated compounds.…”

Nonresonant Raman imaging of protein distribution in single human cells

“…7,8 Resonant Raman microspectroscopy has also been used to study eosinophyl peroxidase in eosinophilic granulocytes 9 and NADPH oxidase and myeloperoxidase in neutrophilic granulocytes. 10 The temporal changes in the redox state of the enzymes in these leukocytes could also be followed in vivo using resonant Raman scattering. 11 Nonresonant Raman scattering is 3-4 orders of magnitude weaker than resonant Raman scattering.…”