Polarized resonance Raman measurements for accurate heme protein differentiation

• Polarized Raman spectra of heme proteins were recorded with 405 nm excitation. • Parallel and perpendicular measurements provide accurate heme protein differentiation. • The intensity ratios ν 7 /ν 15 and ν 15 /ν 3 distinguish between Hb, Ngb, Cgb, and Cytc. In the present work, we introduce polarized resonance Raman measurements of the following heme proteins: hemoglobin (Hb), cytoglobin (Cgb), neuroglobin (Ngb), and cytochrome c (Cytc). These proteins are essential for various physiological processes, being involved in the transport and storage of oxygen and other gaseous molecules, energy production, and cellular protection. Despite their diverse functions, heme proteins share similar structural features centred around the heme group, a planar iron-porphyrin complex crucial for their activity. Those structural similarities make them extremely difficult to distinguish using the classical spectroscopic approach. We are the first to provide polarized resonance Raman spectra of these four standards of biologically essential heme proteins using a 405 nm wavelength. Excitation within the Soret absorption band for the heme group, followed by measurements of scattered Raman light polarized parallelly and perpendicularly to the incident light, allowed us to track subtle structural differences between these proteins. We propose two intensity ratios – ν 7 /ν 15 and ν 15 /ν 3 – calculated from the polarized Raman spectra, which allowed differentiation between Hb, Cgb, Ngb, and Cytc and may facilitate their detection in more complex biological in vitro and ex vivo samples.