RESONANCE RAMAN MICROSPECTROSCOPY OF MYELOPEROXIDASE AND CYTOCHROME B(558) IN HUMAN NEUTROPHILIC GRANULOCYTES

With (resonance) Raman microscospectroscopy, it is possible to investi gate the chemical constitution of a very small volume (0.5 fl) in a li ving cell. We have measured resonance Raman spectra in the cytoplasm o f living normal, myeloperoxidase (MPO)-deficient, and cytochrome b(558 )-deficient neutrophils and in isolated specific and azurophilic granu le fractions, using an excitation wavelength of 413.1 nm. Similar expe riments were performed after reduction of the redox centers by the add ition of sodium dithionite. The specific and azurophilic granules in b oth redox states appeared to have clearly distinguishable Raman spectr a when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b(558)-deficient neutrophils showed Raman spectra s imilar to that of the isolated MPO. The spectra of the specific granul es and the MPO-deficient neutrophils corresponded very well to publish ed cytochrome b(558) spectra. The resonance Raman spectrum of the cyto plasmic region of normal neutrophilic granulocytes could be fitted wit h a combination of the spectra of the specific and azurophilic granule s, which shows that the Raman signal of neutrophilic granulocytes main ly originates from MPO and cytochrome b(558), at an excitation wavelen gth of 413.1 nm.