Spinach column chromatography

Chi is purified from chloroplast extracts, usually obtained from spinach leaves, by dioxane precipitation method (51) and conventional sugar column chromatography (52). For rapid and easy preparation, the method recently developed by Omata and Murata (53) is satisfactory synthetic (DEAE-) Sepharose is substituted for sugar on the column. Besides using spectroscopic criteria (52), the purity of Chi samples can be checked readily by means of silica-gel thin layer chromatography (54). Colorless contaminations in Chi... 

For an introduction to thin layer and gravity column chromatography, we separate the pigments from frozen spinach. This is one green experiment that is literally green. A number of variations of this classic procedure have been reported (P). We have made only minor changes in the procedure of Pavia and co-authors 9d). However, since we obtain the UVA is spectrum of the carotene isolated from the spinach, implementation of this experiment facilitates a discussion of ultraviolet/visible spectroscopy. 

Preparation of a Photoaffinitv Label Containing Chlorophvllide a. ChiorophyHide a was prepared from chlorophyll a extracted from spinach and purified by powdered sucrose column chromatography by the method of Strain and Svec (16). An acetone powder of chlorophyllase was prepared from spinach by the method of Tanaka et al. (17) and used to hydrolyze the purified chlorophyll a. The product was extracted with acetone 0.01 M ammonia (9 1) and washed with n-hexane to remove unreacted chlorophyll. It was then covalently attached to Sepharose CL-4B (Pharmacia) after activation of the gel with CNBr by the method of Richards et al. 

Wi ckowski S, Droba M (1978) Heterogeneity of the thylakoid membrane fractions derived from spinach chloroplasts by the action of Triton X-100 in low salt medium. II. Isolation of / -carotene by the DEAE cellulose column chromatography. Plant Sc.Lett. 13, 397-404. 

Fig. 5. Gel-permeation chromatography (on Bio-Gel P-2) of soluble extracellular material from spinach cell suspension cultures that had been fed L-[l- H]arabinose [32]. (a). Total H-labelled material in the culture filtrate - mainly [ Hjpolysaccharides (K. =0.00) and unincorporated [ HJarabinose (K,=0.96). (b) The intermediate fractions (shown in black in Fig. a) were pooled, concentrated and le-chromatographed on the same column and the fractions were assayed specifically for [ HjXGOs.

The 30 kDa protein of Euglena is quite similar to the 33 kda protein of spinach in several aspects, including the isoelectric point and behavior in high-performance liquid chromatographies with ion-exchange and reversed phase columns. Purification of the protein is now in progress. 

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