FRACTIONATION OF PLANT MATERIAL .3. 2 SCHEMES FOR CHEMICAL FRACTIONATION OF FRESH LEAVES HAVING SPECIAL APPLICABILITY FOR ISOLATION OF BULK PROTEIN

Nearly all the protein of broad‐bean leaves other than cell‐wall protein was readily extracted into phenol‐acetic acid‐water mixtures, and further purified by free‐solution eletrophoresis therein, using the continuous apparatus of Hannig. Under these conditions RNA migrated cationically in association with the protein. Adding salt did not greatly improve the separation, but it reversed the relative cationic migration rates of phaeophytin a and protein. Liquid‐liquid partition between phenol and aqueous buffer phases gave good separation of the protein from RNA. Scheme I involved successive extraction of the leaves with 5% (w/v) aqueous trichloroacetic acid, ethanol and phenol‐acetic acid‐water; the bulk of the protein went into this last solvent mixture and did not require further electrophoretic treatment. In Scheme II, components of low molecular weight were washed out from the plasmolysed leaves with water. Extraction was then carried out with phenol‐acetic acid‐water, and this extract was further fractionated by electrophoresis. The non‐migrating fraction should be valuable starting material for studies of a variety of leaf constituents. The extraction residues are promising starting material for studies of the leaf polysaccharides and other cell‐wall components. The protein preparations contained little phosphorus or carbohydrate but were brown and had high C/N ratios and higher ultra‐violet absorption than corresponded to the aromatic amino acids present. This was decreased, but not abolished, by keeping the work under nitrogen. Model experiments showed good electrophoretic separation of protein from polyphenols and tannic acid under the same conditions. The extraneous aromatic material is therefore presumed to be bound covalently to the protein. Coupling of oxidatively polymerising quinones to side‐chains of amino acid residues, especially lysine, seems probable. The occurrence of such reactions in leaves has implications for the nutritive value of their proteins. Such products may also contribute directly to the ‘humic acid’ fraction of soil. Copyright © 1968 John Wiley & Sons, Ltd