Fourier transform-IR determination of protein contamination in thioglycolic acid lignin from radish seedlings, and improved methods for extractive-free cell wall preparation

Fourier transform (FT)–IR spectroscopy was used to characterize and check for contamination in lignothioglycolic acid (LTGA) preparations from radish seedlings. LTGA, prepared by the conventional technique of extracting plant material first with 50% (v/v) aqueous methanol, was so highly contaminated with proteins that lignin, based on absorptivity at 280 nm, was overestimated by as much as eight-fold. When samples were extracted first with buffer to remove soluble proteins, then with buffer plus 1 M sodium chloride to remove ionically-bound proteins, and finally digested with cellulase and pectinase, the FT–IR spectra of the LTGA samples so obtained were comparable to spectra of LTGA prepared from purified hardwood lignin. Lignin values estimated from benzaldehydes recovered after cupric oxide degradation were similar for both methods of sample preparation. FT–IR analysis of the cell wall material from which LTGA had been extracted showed that this procedure removed all detectable lignin. LTGA values provide an accurate and highly sensitive assay of lignin in radish seedlings and this procedure should be widely applicable to lignin analysis of other young herbaceous tissues, but only if proteins are removed during extractive-free cell wall preparation.