Christine A. Roberts' Clenton Jones Evlyn J. Spencer2 Giselle C. Bowman3 and David Blackman Federal City College Washington D.C. 20005
II
Polyacrylamide Gel Electrophoresis of Yeast lnvertase A biochemistry laboratory experiment
A ranid., simnle . nrocedure for the nartial nurification of yeast kvertase (i-fructofuranosidase, E.c.' 3.2.1.26) has been described hv Melius (.I .) . He allowed the veast cells to autolyze overnight in 0.1 M bicarbonate; after removal of the cell debris by centrifugation, the crude extract was subjected to a single purification step, namely gel filtration on Senhadex G-200. while the Melius procedure gives a mathematically observable ourification (our students have routinelv observed an increase in specific activity of about three-fold over the crude extract) it is limited in that student experimenters do not get a feel for the physical significance of this pnrification; i.e., they cannot see the elimination of contaminating proteins in the gel filtration step. T o remedy this situation we have extended Melius' experiment to include polyacrylamide gel electrophoresis of hoth the crude extract and the partially purified gel filtrate. In this way it is possible for students to visualize the purification procedure and acutally estimate the number of extraneous proteins removed in the Process. We have also develoned an assav for invertase in the electrophoresed gel, whichallows a correlation hetween the location of enzvme activitv and one or more protein hands.'
dent is directed to pool those samples containing maximum amounts of invertase activity. From the column elution data the student estimates the concentration of protein (in mglml) and invertase (in unitslml) in the pooled fractions; by comparison with the crude extract it is possible to calculate both purification and yield. Four eels are nrenared . . for electronhoresis: two are loaded with crude extract and two with the pooled fractions. After electronhoresis at o H 6.8 one eel of each nair is fixed " with trichloroacetic acid and stained for protein using Coomassie blue, while the remaining gels are (qualitatively) assayed for invertase, again using the DNS reagent. The student is then asked to compare the two protein-stained gels for the number and intensity of hands, and likewise to compare the enzyme-stained gels. Finally, by comparing the protein gels with the corresponding enzyme gels, he can attempt to correlate the positions of enzyme activity with specific protein bands.
The Experiment
LCurrentaddress: Department of Biology, University of Califarnia at San Diego, La Jolla, California. ZCurrent address: College of Medicine. University of West Indies, Jamaica, B.W.I. :'Current address: Department of Pathology, University of Marvland. Baltimore. Md. '\kc nre'indrhred r;, m r of the referees of the Small Cmnti Program for suggcsliny n sperita ASM). oi thus type.
Invertase is prepared as described by Melius, except that Bio Gel P-100 is substituted for Sephadex G-200 in order to eliminate the nossihilitv of bacterial degradation of the " gel filtration medium. After assaying the gel filtration fractions for nrotein bv a modified Lowrv method ( 2 ) and for enzyme activity using 3,5-dinitrosali&late (DNS); the stu-
62 / Journal of Chemical Education
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Reagents Electrophoresis buffers: 10X buffer-0.2 M phosphate, p H 6.8; Running huffer-200 ml of 10X buffer, diluted to
solution. This solution is changed every few hours until 2.0 1 with distilled water.5 Approximately one-fourth to destaining is complete and discrete protein bands are visione-half of this solution should be frozen into cubes before ble. The gels may he stored under destaining solution for the start of the experiment. several months. Gel solution^:^ Solution l-Dissolve 5.98 g tris(hydroxyGels 2 and 4 are soaked in acetate huffer for approximethy1)aminomethane (TRIS) and 0.46 ml N,N,N',N'-temately 3 miu in order to lower the p H from 6.8, a t which tramethylethylenediamine (TEMED) in distilled water, electrophoresis took place, to 4.8, the optimum for enzyme adjust to p H 6.8 with HCI and dilute t o 100 ml; Solution activity. The buffer is removed, and the gels are incubated 2-Dissolve 28 g acrylamide7 and 0.735 g N,N'-methylenea t room temperature with sucrose acetate solution for exbisacrylamide (BIS) in distilled water and dilute to 100 ml. actly 5 min, after which the substrate solution is replaced Solution 3-Dissolve 0.14 g ammonium persulfate in disby the DNS reagent. The samples are then heated for 5 min tilled water and dilute to 100 ml. These solutions should be in a hoiling water bath to develop the color. Examination of kept in amber bottles and stored in the refrigerator. Soluthese gels should proceed immediately after cooling and retions 1 and 2 are stable for two months or more; solution 3 moval of the DNS solution, without any further soaking or should not be kept for more than two weeks. rinsing, since the DNS reduction product is readily diffusiTracking dye-0.01% bromphenol blue in 50% aqueous ble. Iuvertase activity appears as a ruhy-red hand over a glycerol. Sucrose, which is traditionally used to create a background of yellow-orange. high density solution must not be used here since it is the substrate for the enzyme and will obscure the subsequent nne,,1*e . activity assay in the gel. The chemistry of the reaction of DNS with reducing sugGel assay solutions: Soaking buffer-0.1 M acetate, p H ~ ~ ars ~ has ~ recently ~ ~ been ~ ~reviewed (4). Electrophoresis of ei4.8; Sucrose-0.3 M, in soaking buffer; D N S - as ther the crude or partially purified preparation a t p H 6.8 described by Melius (I); Fixative-Trichloroacetic acid yields two DNS-positive bands, corresponding to the two (TCA), 10% in distilled water; Protein stain-~oomassie invertase isozymes elaborated by yeast cells (5). The "exblue, 0.1% in 7% acetic acid; and &staining solution ti^ ternal" form appears a few mm from the top of the gel, acid, 7% in distilled water. while the "internal" form migrates about 30 mm further Procedure (6). Both gels stained for protein show several bands; it is Casting of Geb clear, however, that fewer protein components are present Solutions 1,2, and 3 are mixed in the volume ratio of 1:1:2; imin the gel carrying the pooled fractions from the Bio Gel mediately after mixing, 1.0 ml is dispensed into each of 12 clean, P-100 column than in the gel bearing the crude extract. dry, 3 in. long gel tubes. As rapidly as possible, the gel surfaces are The bands arising from the gel filtrate are also sharper and then layered with distilled water. Even though only four gels are more discrete than those from the crude extract. In this used in the experiment, it is useful to prepare several spares so that students may develop proficiency in removal of the gels after way the effects of gel filtration are immediately visualized. the electrophoretic run. I t also becomes apparent to students that a single purification step is far from sufficient, and that the gel filtrate is Electrophoresis not nearly homogeneous. As a result it is impossible to After gel polymerization (45 min at room temperature) and subidentify the protein bands which contain invertase in either sequent removal of the water layer, samples are applied as follows. the crude or gel-filtered sample, although, based on a comA volume of crude yeast extract containing 0.5-0.6 mg of protein is parison of gels 2 and 4, enzyme activity can be localized applied to each of gels 1 and 2; a volume of the pooled invertase within a relatively small number of components. fractions containing 0.5-0.6 mg of protein is applied to each of gels Comolete instructions for this exoeriment are available 3 and 4. Tracking dye (0.01 ml) is added to each sample, and the gel tubes are inserted in the electrophoresis apparstus. Each tube from the last-named author. This work was supported by is gently filled with running buffer, taking care to avoid dilution of the Division of Chemical Education-duPont Small Grants the protein samples; both chambers of the electrophoresis apparaProgram of ACS and by MBS Support Grant RR08005-01 tus are filled with running buffer and buffer cubes, the latter to from the General Research Support Grant Division of Reminimize the effects of ohmic heating. Electrophoresis is carried search Resources, National Institutes of Health. out at 150 V (37.5 Vlgel) for 2 hr, during which time the current
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may be turned off temporarily to allow further addition of buffer cubes as needed.
Assays
Following electrophoresis the gels are removed from their tubes and placed in labeled 13 X 100-mm test tuhes. Gels 1 and 3 arestained for protein, while gels 2 and 4 are assayed for invertase activity. Gels 1 and 3 are completkly immersed in TCA solution, and allowed to stand 30 min at room t e m ~ e r a t u r eto ensure complete precipitation of protein hands. Removal of the fixative (readily accomplished h s use of a Pasteur pipet attached to a sink aspirator by a length of vacuum &bing) is fo~~owed by soaking overnight in Coomassie blue solution. After removing the staining solution, the gels are rinsed twice with distilled water and covered with 7% acetic acid
Literature Cited I I I Meliui. P . , J CHEM. RnUUC., 48,785 119711. 121 Miller. G. I...Anal. L'hrm.. 31,964 119591. 13, "Chemical Fsrmulacica~rIcr Disc Electn~phoresis."Canalc~#Curp..S~ptrmher. 1968. I d ) l3iumnn.R 1 CHRM.RDLIC..il,J6IiliidI. 15, Neumann. N.P.. and Lampen. .I. 0.. Hioi~lwm;rrr?.6.468 119671. (61 Garcan. S.. Neumann. N. P..snd Lampen. J . O . J . R i d Chem.. 243.1573 (1968).
...
% most formulations glycine is added as a trailing ion; our results have, however, been satisfactory in its absence. "These correspond to "Stock Solutions B, C, and G," respectively, for standard 7% gels. Formulations are taken directly from Ref. ,"\
7These solutions are generally prepared either by support personnel or by the students themselves. In either case, extreme caution should be advised for handling acrylamide, since it can cause skin and eye irritation.
Volume 53. Number 1. January 1976 I 63
