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Jayanti Pande, Johanna Gomez-Santos, Ajay Pande; Surface-enhanced Raman spectroscopy (SERS) of gamma crystallins: A promising technique to study the redox reactions of cysteine residues in dilute protein solutions. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4961.
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We previously showed the unique role of Raman spectroscopy in tracking specific lens crystallin interactions, especially those related to Cys-SH groups (Pande et al. Biochemistry (2009); 48:4937-45). However, a serious drawback of the technique is the need for high protein concentrations, in the millimolar range. Here we explore an alternate method to overcome this drawback.
Raman data were collected on a Renishaw inVia Raman microscope with laser excitation at 785 nm and 20 mW power at the sample. A gamma crystallin stock solution (60 mg/ml, in sodium acetate buffer, pH 4.5) was diluted with colloidal gold nanoparticles (Au-NPs) which provided the SERS enhancement. Citrate-capped Au-NPs with a plasmonic absorption band at 525 nm were mainly used. Several 2 to 3 microliter droplets of gamma crystallin-Au-NP mixtures were placed on a coverslip which was inverted over a microscope slide with a hollow depression. Each drop was scanned for 10-30 seconds and the data from multiple drops averaged, thus avoiding sample degradation due to laser irradiation.
We measured SERS spectra of gamma crystallin solutions at protein concentrations of ~100 micromolar. This is a significant advantage over normal Raman spectra which require 4-5 millimolar protein concentrations. The vibrational frequencies and intensities of the SERS spectral bands are nearly identical to those in normal Raman spectra. Thus various Raman modes appear to scale up equally in SERS. A significant finding is the presence of a clear Cys-SH frequency in our SERS spectra, which is often missing in the SERS spectra of proteins reported in the literature.
SERS shows great promise for measuring the Raman spectra of crystallins at 20-50 fold lower protein concentrations than those required for conventional Raman measurements. The ready detection of Cys-SH groups in SERS spectra facilitates the tracking of -SH mediated redox reactions of the crystallins. We are extending these studies to other novel forms of Au-NPs for higher SERS signal enhancements than those observed here with citrate-capped Au-NPs.
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