A Novel Idea for Practical UV Resonance Raman Measurement with a Double Monochromator and its Application to Protein Structural Studies

Abstract

A practical system for UV resonance Raman (RR) measurements based on the combined use of the first-/second-order dispersions of two gratings in an ordinary double monochromator is proposed. This system rejects visible stray light completely and gives four times larger throughput than the combination of the normal second-order dispersion of two gratings and a visible-cut filter. Newly designed f/1.1 Cassegrainian mirror optics and a sensitive spinning cell using an ESR sample tube are combined with this spectrometer and applied to measurements of UV RR spectra of hen egg white lysozyme. The continuous-wave 244-nm excited spectra of lysozyme yielded Raman bands of tryptophan (Trp) and deprotonated tyrosine (Tyr−) residues in 10 min with high signal-to-noise ratios, and there was no sample degradation. It is demonstrated that the intensity of the Tyr−v8 aband in the 244-nm excited spectrum can be used to quantify the number of deprotonated Tyr residues in the stepwise ionization of a protein.