Three o-diphenol-O-methyltransferases (OMTs; EC 2.1.1.6) involved in the biosynthesis of lignin have been purified to homogeneity from tobacco leaves. Seven different fractionation steps which included (NH4)2 SO4 precipitation, conventional low-pressure chromatography on Ultrogel AcA34 and DEAE-cellulose columns, high-performance liquid chromatography (HPLC) on three different supports (Mono Q, Mono P, and TSK G-3000 SW columns), and finally preparative electrophoresis were necessary. At each step of purification, the protein content of the enzymatic fractions was analyzed by electrophoresis on polyacrylamide gels under denaturing conditions. Purified OMT I appeared on sodium dodecyl sulfate-polyacrylamide gel as a doublet with electrophoretic mobilities corresponding to molecular weights of 38,500 +/- 2000 and 39,500 +/- 2000. The other two enzymes migrated as single but rather broad bands with molecular weights of 42,000 (OMT II) and 43,000 (OMT III). Polyclonal antibodies were raised in rabbits. The titers of antibodies were measured by an indirect enzyme-linked immunosorbent assay method, and their specificity was demonstrated by immunoblotting enzyme preparations at different stages of purification. Immunodetection of the three enzymes with a specific antiserum suggested serological relationships between the three OMTs of tobacco.

High-performance liquid chromatography was used to purify four different enzymes extracted from tobacco leaves. Phenylalanine ammonia-lyase (E.C. 4.3.1.5) and three O-methyltransferases (S-adenosyl-l-methionine: catechol O-methyltransferases, E.C. 2.1.1.6) were subjected to high-performance chomatofocusing. Parameters effecting the resolution of chromatography and the recovery of enzyme activity were investigated. The speed and high resolving power of chromatofocusing are major advantages for analytical or preparative purposes. The absorbance at 280 nm of chromatographic fractions was shown to arise mainly from small molecules and was not a measurement of protein concentration as indicated by subsequent high-performance size exclusion chromatography. Electrophoretic analysis of the active fractions on sodium dodecyl sulphate—polyacrylamide slab gels demonstrated the high degree of purification achieved by chromatofocusing.

High-performance liquid chromatography (HPLC) was used to purify catalytically-active enzymes present in minor quantities in plant material. The three O-methyltransferases (S-adenosyl-l-methionine:catechol O-methyltransferases, E.C. 2.1.1.6) of tobacco leaves were subjected to high-performance ion-exchange chromatography. Excellent recovery of enzyme activity (70–100%) was obtained. HPLC was tentatively used at both analytical and preparative scales. As an analytical tool HPLC offered major advantages over conventional low-pressure ion-exchange chromatography in both speed and resolving power. For preparative purposes however, pre-purification of plant extracts by conventional means was necessary before HPLC. Purification achieved by HPLC was evidenced by electrophoretical analysis of the active fractions on sodium dodecyl sulphate—polyacrylamide slab gels.