"Visualizing" Enzyme Activity in a Polyacrylamide Gel A Biochemistry Laboratory Experiment Richard Kilker Jr. and Gina Llbrettl Drew University, Madison, NJ 07940
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E n z v m e nurification is often a time-consuminz orocedure a n d t h u s is n o t usually a s t u d e n t experiment. Roberts e t al.' detailed a raoid. . . simole orocedure for t h e oartial ourification of yeast invertase (8-fructofuranosidase, E.C. 3.2.1.26). T h e v outlined oolacrvlamide eel electroohoresis a s a technique t o allow ;or t h e visualizkion of t h e purification process. T h i s paper details a polycarylamide gel electrophoresis experiment t h a t employs an assay using a p-nitrophenyl derivative for t h e visualization of a n enzyme activity within a gel. T h i s use of a colorless synthetic substrate that produces a colored product u p o n action b y t h e enzyme is a p o t e n t tool when coupled with t h e electrophoresis of a n enzyme sample
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T h e Experlmsnt The enzyme used is a commercially available yeast u-glucosidase. The activity of the enzyme is determined using the following assay.
added to DH 8.9. Dilute to 5 L with distilied water. Solution 2 (Aerylamide). Dissolve 22.2 g acrylamide and 0.6 g N,N'-methylenehisacrylamide in 90 mL distilled water. Dilute to 100 mL. Store in a dark bottle at 4 'C. Caution: Solid acrylamide and acrylamide solutions are very toxic. Avoid contact with skin and breathing of dust. Skinor eye irritation may result. Solution 3 (Fixing Solution). Dissolve 57 g trichloroacetic acid and 17 g sulfosalicylie acid in a solution of 150 mL methanol and 350 mL distilled water. Solution4 (Staining Solution). ~ i 1.25 ~cwmassie ~ bril.~ lianthlue ~ - 2 5 0 i "a solution con. taining 227 mL methanol and 227 mL distilled water. Add 46 mL glacial acetic acid.
a-D-glucose p-nitrophenol (colorless) ( @ p H> 10, yellow)
This assay demonstrates the commonly used practice of determining a hydrolytic enzyme's activity by using a colorless, synthetic substrate with a built-in chromophor (PNPG). When the ehromophor is cleaved from the substrate, under appropriate conditions, a color appears. In the above assay, the ehramophor is the p-nitrophenol moiety, which, once released from PNPG by the action of the enzvme. has a vellow color at DH >lo. This assav can also allow for the appearanreof a yellow band in apol~arrylnrnidrgeiwhere there : i ~ ~ - g l u c ~ d activity, asr henwallowing for the rnpid determinatmn uf the v n 7 ~ m cIhcillion in the gel. Reasen& Solution 1 (Trislglyeine Buffer Stock). Dissolve 75.1 g glycine and 2.5 g sodium azide in about 3 L distilled water. Solid Tris was
'Roberts, C. A,; et al. J. Chem. Educ. 1976, 53,62. Fehrnstrom, H.; Moberg, U. "SDS and Conventional Polyacrylamide Gel Electrophoresis with LKB 21 17 Multiphor"; LKRProdukter AB: Brornma, Sweden: Application Note 306.
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Procedure Prepomtion of Gels. Mix 7.5 mL distilled water, 33 rnL Solution 1,22.2 mL Solution2, andO.l mL N,N,N',N'-tetramethylethylenediamine (TEMED) in a 125 mL suction flask. Deaerqte, and quickly add 3.2 mL freshly prepared 1.5%ammonium persulfate solution. Immediately transfer some of this mixture to glass gel tubes (13 em X 0.5 cm). Overlay the unpolymerized gel surface (+I with deaerated water, and allow the gels to polymerize (-45 min) wlucosidase gel assayed for enforming polymerized gels il em zyme activitylfixedlstainedldestained. high (7.5% acrylamide). I'rcpnrnlion r./ E n z ~ r n eSolurum Dissolve 2 5 mg n-glucowdnse ryrasr; lyophllized pwder; Sigma:Type I ) in 0.25 ml.Sarnple I3uflrr t5 mL Solutron I d h e d to It10 ml. with distilled water,. T o this add 0.025 mL glycerol and 0.01 mL 0.25% hromophen~lblue in Sample Buffer. Electrophoresis. Place the gel tubes containing 7.5% polyacrylamide in the electrophoresis unit, e.g., Bio Rad Model #150A. Prepare 1800 mL of Running Buffer (1 part Solution 1 1 part distilled water) and fill the upper and lower chambers oftheelectrophoresis unit. Rinse the surface of the gels and carefully layer 0.020 mL of the enzyme solution on the surface of each gel. Electrophoresis is carried out at 4 rnAIgel tube until the tracking dye is approximately 1 em from the bottom of the gel. Remove the gels from their tubes and cut them at the dye front. Assay Gel for n-Glueasidose Aetiuity. Place a gel into a test tube with sufficient 5 mM p-nitrophenyl-u-D-glucopyranosidesolution in 10 mMimidazole buffer, pH 6.8, to cover the gel. Stopper the test tube andgently rock it untiladistinet yellow hand appearsin thegel (-2min). Quicklyremove thegel from the tuhe and mark the center . . of the yellow band by pie& the gel using a thin, short piece of wire. Then fixlstainldestain the gel as below. Fixing the Gel. Place agelinto atest tube withsufficient Solution 3 to cover thegel. Allow thegel toremain in this solution for 60min. Staining Gel for ProteinlDestaining. Place a gel that has been fixed into a test tuhe filled with Solution 4. After 30 min transfer the
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gel to a test tube containing ethano1:acetic acid:water (15:5:30). Continue to change the solution in the test tube (at P5-h intervals) until it remains free of blue color. Resuns
A typical gel that contained a sample of a-&ucosidase electrophoresed as above, assayed for enzyme activity and then fixedlstainedldestained appears in the figure 1. It can he seen that the enzyme sample contains numerous protein hands, i.e., this is not a purified sample of u-glucosidase. The area of the gel that stained yellow when assayed with the PNPG is indicated in the figure. The Rr = 0.19 protein hand corresponds most closely to the a-glucosidase position although one cannot mle out that the Rr = 0.17 hand or a hand too faint to be seen may he the enzyme band. This points up the necessity for marking the yellow baud as soon as i t appears in order to identify it closely with a single protein
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Journal of Chemical Education
hand. Waitine too lona results in a broad band whose center is difficult to estimate. Presumably, if a purification was carried out and an enzyme sample was electrophoresed a t each step all the protein bands except for the u-glucosidase baud would disappear from the gel. Possible purification techniques are ammonium sulfate precipitation. DEAE-cellulose ion-exchange chromatography, and molecular-sieve chromatography.'.Vhis would allow for the final assignment of a protein hand to the enzyme. The experiment demonstrates (1) the technique of polyacrylamide gel electrophoresis used routinely in enzyme purification procedures, ( 2 ) a auick-and easy assay for an a-glucosidase, and (3) how these.two procedu;es toiether allow-for the "visualization" of enzyme activity in a polyacrylamide gel. SkO. M.; Lovgren. T. Acta Chern. Scand. 1978, 832,447.
