Gel Permeation Chromatography of Wheat Germ Acid Phosphatase An Undergraduate Biochemistry Experiment Richard A. Smith State University College, Geneseo, NY 14454
A procedure for the isolation of an acid phosphatase from wheat germ is described in a t least three popular hiochemistry laboratory manuals.' The procedure includes several separation steps based upon differential solubilities of the enzvme and its contaminants, yielding afinalproduct that is significantly enriched in phosphataseactivitr.. Howe\,er, the schemedoes not include the biochemist's most effective tool for enzyme puriticution-one or more of the varieties of column chromatuyraphy. Cooperlc hassuggested further purification of the ghoiphatase by gel permeation chromatography on a 2.5- X 35-cm agarose column after reducing the volume of the vrevaration. a ~ r o c e d u r ethat is too l e n ~ h y . . for the undergraduate laboratory. This paper descrihks vrocedure that can be completed in 3-31? hours, starting with prepared columns. Gel permeation chromatography (also known as molecular exclusion chromatography, or gel filtration) separates molecules primarily according to size, on porous hydrophilic resof molecules dissolved in an aqueous mobile i n ~A. mixture ~ phase is applied to the gel in a column and eluted. Large &olecules&e excluded from the pores in the gel and emerge from the column rapidly; smaller molecules diffuse freely into - - ~ the ~ ~-~~ - nores and a r e retarded as thev travel down the column, emerging later. In this experiment the technique provides a simple, fast, and effective purification method. ~
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Experlrnental Solutions and Resin Phosphotose assay cocktail. 0.10 M sodium acetate huffer, pH 5.7, containing 0.01 M MgClz and 5 mM p-nitrophenyl phosphate. This solution is provided in a bottle with a dispensing pump set to deliver 4.9 mL.
' (a)Crandall. G. D. Selected Exercises for the Biochemistry Lab
oratory; Oxford: New York. 1983; p 24. (b) Stenesh. J. Experimental Biochemistv; Allyn 8 Bacon: Boston. 1984; p 181. (c) Cooper, T. G. The Tools of Biochemistry: Wiley: New York, 1977; p 391. (a) Reiland, J. In Methods in Enzymology; Jacoby, W. B., Ed; Academic New York, 1971; Vol. 22, pp 287-321. (b)Hirs, C. H. W. In Methods in Enzymology; Hirs, C. H. W.; Timasheff, S. N., Eds.; Academic: New York. 1977; Vol. 47. pp 97-107.
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Elution profile of wheat germ acid phosphatase on Bio-Gel P-100.
Journal of Chemical Education
Protein assay. Coomassie Blue G-250 reagent concentrate (BioRad Lahoratories), diluted fivefold. Eluant. 5 mM NuzH2EDTA. Column Preparation Bio-Gel P-100 Fine, polyacrylamide gel (Bio-Rad) is allowed to swell 24 h at ambient temperature in the eluant solution. Sufficient slurried resin is poured into a 1.5 X 30-cm glass column (Bio-Rad)to give a gravity-packed bed height of 20 cm. The column should he equilibrated with 75-100 mL of eluant. Chromatography Kiuant is allowed t o drain jusr to the top of rhe resin, and 1 mL of the tinol diaiy7cd enzyme fraction (1) is carefully applied with a Pasteur ~ipet.Thisorowin solution is caref~llv overlaid with 1 mL of eluant, and elutibn into a 10-mL graduated cylinder is begun. Once the enzyme and overlay solutions have flowed into the resin the column may be filled with eluant. It should be kept filled throughout the chromatographic run in order to maintain an even flow rate. A total of 7 mL is collected in the graduated cylinder; the
flow rate (aooroximatelv 12 mLh) is determined as this occurs. Fractions oii mL are tLen collected. Several students can arrange therrcolumnsarounda single iraction collector;minor differencesin flowrate will not affectthe resulcs.The bulk of the protein and611of the phosphatase elute in a total volume of 23 mL, Proteln Analysls Aliquats of 0.2 mL of each fraction are mixed with 3 mL of Coomassie Blue reagent. After a 5-min wait the absorbance at 595 nm is determined. Phosphatase Analysis Aliquoul of01 mL of each fraction are mixed with 4.9 mL of the assay rucktail at 37 'C. The reaction isquenched after 3 min lw the addition of 2.5 ml. of0.5 .M KOH. Theamallamount ofprecipitate is allowed to settle, and the absorbance of the clear supematant solution is determined at 405 nm. Results Absorbance values for both protein and phosphatase activity are plotted versus elution volume. The elution profile shown is typical. Phosphatase activity appears as a nearly symmetrical peak corresponding to the void volume of the
column (oreviouslv determined with Blue Dextran). Thus wheat acid
