About Sickle Cells

Raman spectroscopy utilizes light scattering to gain information about a molecule’s vibrations. (link to going deeper: Molecular vibrations) These vibrations can then provide information regarding the structure and symmetry of the molecule. In the case of Hb, these vibrations yield information regarding local environment and H-bonding. The vibrations are inferred from the frequency of the scattered light. When light is incident on a molecule, it can interact with the molecule but the net exchange of energy is zero, so that the frequency of the scattered light is the same as the incident light (n = n_o). This process is known as Rayleigh scattering. Conversely, the light can interact with the molecule and the net exchange of energy is the energy of one molecular vibration. If the interaction causes the light photon to gain vibrational energy from the molecule then the frequency of scattered light will be higher than that of the incident light (n = n_o + Dv), this process is known as Anti-Stokes Raman scattering. On the other hand if the interaction causes the molecule to gain energy from the photon then the frequency of the scattered light will be lower than that of the incident light (n = n_o - Dv); this process is known as Stokes’ Raman scattering. This is the type of Raman scattering that is measured in the Mukerji lab.

FIG 8.2 The UV Raman Spectrometer in action
Prof. Mukerji at work with the UV laser system.

In the Mukerji laboratory, we also take advantage of the 'resonance effect.' The Raman signal can be strongly enhanced (at least 3 orders of magnitude) by exciting directly into an absorption band of the molecule. The advantage lies in the fact that by tuning the excitation wavelength, we can now selectively examine different parts of the molecule. In Fig. 8.3, we can see how much difference this can make when obtaining resonance Raman spectra of hemoglobin. If we use an excitation wavelength of 418 nm, this is right in the center of the absorption band for the heme group and therefore the molecular vibrations that are enhanced all arise predominately from the heme group. If an excitation wavelength of 230 nm is used, this primarily enhances the vibrations from tyrosine (Tyr) and tryptophan (Trp) amino acid residues in the protein.

FIG 8.3
Different excitation wavelengths enhance Raman signal from different parts of the molecule
418 nm: Excitation into heme absorption band. RR spectra from the heme
230 nm: Excitation into absorption band of Tyr and Trp. UVRR spectra arises from those residues in the protein

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The Raman spectrum obtained with 230 nm excitation is completely different from the one obtained with 418 nm excitation because of the resonance enhancement. This gives us a great deal of selectivity in our experiments and by choosing the excitation wavelength we can decide which part of the molecule we want to monitor. For most of our experiments, we examine the protein portion of the molecule. We are particularly interested in tyrosine, tryptophan and phenylalanine amino acid residues.