Deep ultraviolet resonance Raman spectroscopy as a tool for biomolecule analysis
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Cholesterol is a major component of membranes, reducing their fluidity at higher concentrations. The UV Raman vibrational mode of the carbon-carbon double bond in cholesterol occurs at 1675 cm-1 is strongly resonance enhanced in deep ultraviolet resonance Raman (dUVRR) spectra of natural and model membranes at an excitation wavelength of 197 nm. DUVRR is a vibrational spectroscopic technique that is used to gather information on protein secondary structure and in this work is extended to study systems in the presence of the membrane component cholesterol and the cholesterol analog 5α-cholestan-3ß-o, which lacks the carbon-carbon double bond. DUVRR studies of melittin indicated that little loss of helical structure occurred with increasing temperature as was suggested by circular dichroism (CD), another structurally sensitive spectroscopic technique. A cholesterol analog was introduced to decrease the fluidity of membrane in a similar manner to cholesterol, while minimizing spectroscopic interference from the resonant enhanced carbon-carbon double bond vibration. With the introduction of the cholesterol analog a loss in helical structure of the melittin was seen in both the dUVRR and CD. Lipoproteins transport cholesterol through the blood stream. Studies have shown that an individual’s lipoprotein levels are correlated with their risk of cardiovascular disease. The strongly resonantly enhanced dUVRR feature of the carbon-carbon double bond was used to quantify cholesterol in lipoprotein particles isolated from whole blood. Like cholesterol, unsaturated lipids give rise to a strongly enhanced vibrational mode in dUVRR spectra, which is slightly downshifted from that of cholesterol. The vibrational mode of unsaturated lipids occurs in the region of 1655-1661 cm-1, depending on the type of lipid. The feature arising from the unsaturated lipids was found to be sufficiently resolved from cholesterol’s feature to allow quantitation of cholesterol in model membranes and lipoproteins. Isolation and characterization of lipoproteins is an emerging field as studies show that cardiovascular disease risk is a complex function of not just cholesterol and lipoprotein levels but also their size distribution. Spectroscopic methods are readily coupled with chromatographic techniques. Size exclusion chromatography (SEC) was employed to separate lipoproteins based on their size from a series of blood serum samples. Each volume fraction was spectroscopically characterized via dUVRR spectroscopy producing a two-way data matrix for each sample, which could then be analyzed using higher order multivariate methods. Parallel factor (PARAFAC) analysis and multivariate curve resolution-alternating least squares (MCR-ALS) were employed to resolve the underlying spectroscopic and chromatographic profiles. The resolved profiles were attributed to lipid/cholesterol, protein and buffer components.
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