Raman Spectra and Sulfhydryl Ionization Constants of Thiogiycolic Acid and Cysteine

Raman spectra of thioglycolic acid and cysteine in different states of ionization are reported, with quantitative determinations of the intensities of the Raman bands in a photoelectric Raman spectrophotometer. Depolarization measurements are also reported. A method of calibrating polarization measurements with known reference substances is described. Ionization of the sulfhydryl group in thioglycolic acid not only abolishes the S-H stretching frequency at 2580 cm⁻¹ but also is accompanied by marked increases in the intensities of a number of frequencies below 1000 cm⁻¹. In several of these frequencies, although not all, there is a parallel increase in the depolarization of the Raman bands. Apparent ionization constants (pK′ values) for the S-H group at high ionic strengths are calculated from the intensity of the 2580 cm⁻¹ frequency as a function of pH, the reference standard of intensity being either (1) an attenuated beam from the primary light entering the Raman tube or (2) a reference line, assumed independent of pH, in the Raman spectrum of the substance itself. The two standards give results in reasonably close agreement; obviously the first is preferable in principle, but there are practical difficulties in applying it. The four microscopic pk values describing the ionization of the sulfhydryl and amino groups in cysteine were evaluated from the data, in the ionic strength range 0.5–1.0. The values found are in surprisingly close agreement with those obtained at much lower ionic strength, by Benesch and Benesch (1955), from ultraviolet absorption spectra.