Isolation and Purification of Leaf Starch Components

Abstract

A procedure was developed for the separation and purification of amylose and amylopectin isolated from cotton leaves. Cotton leaves were homogenized in 0.02 molar phosphate buffer at pH 7.0 containing HgC2 plus toluene. Crude starch granules were collected by centrifugation and partiaDly purified by treating with acetone and toluene. The starch granules were then dispersed in dimethylsulfoxide and precipitated with ethyl alcohoL The precipitate was suspended in bouling water. Amylose was separated from amylopectin and ceDl wail particles on a Sepharose 2B column and further purWified with thymol and butanol. Amylopectin was then separated from the coloidal ceDl wall contaminants by its specific interaction with concanavalin A. Purities of starch components were verified by specific biochemical and enzymic tests in addition to their iodine-binding capacity. This procedure should also be suitable for purification of starch components from other plant sources.In the purification of starch from either seeds or leaves for physicochemical studies, no specific processes have been employed to remove cell wall contaminants (1,3,11,12,15,22,24). In these procedures dispersed starch has been treated with various precipitants such as butanol (24), butanol plus pentasol 27 (Pennsalt Chemical Corp.)' (9,15), thymol (13), and others (14,28). The complex of amylose plus precipitant is then directly centrifuged to achieve a separation from amylopectin. The butanol and thymol procedures (13,24) were tested with cotton leaves. Unfortunately, the complex of amylose and precipitant was heavily contaminated with cell wall fragments.Banks et al. (3) MATERIALS AND METHODSIsolation of Crude Starch Granules. About 20 g of fresh cotton (Gossypium hirsutum L.) leaves were mixed with 78 ml of cold (4 C) 0.02 M Na-phosphate buffer (pH 7.0), containing 0.01 M HgC12 and 13 ml of toluene. The mixture was homogenized for 3 min at ice bath temperature with a Sorvall Omni-Mixer (35% full speed). The homogenate was then filtered through two layers of cheesecloth and centrifuged at 1,085g for 3 min. The supernatant, including the surface layer of protein, green pigments, and lipids, was discarded. The surface of the starch residue was rinsed with cold water, the starch pellet was then washed twice by resuspension in about 25 ml of cold water and centrifugation at 1,085g for 3 min. The pellet was extracted with acetone three times, resuspended in 10 ml of cold water, and then shaken with an equal volume of toluene for 3 min. The mixture was centrifuged at 1,085g for 3 min and the top toluene-protein layer was subjected to a stream of N2 gas to release the trapped starch granules in toluene. This layer was then removed with a pipette and discarded (decantation caused contamination of the starch residue with denatured protein). The process was repeated four times or until the toluene layer no longer became turbid. The starch granules were again washed twice with cold water and stored under watertoluene in the cold (4 C) until required.Pretreat...

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