Purification of Lysozyme by Linear Salt Gradient and SDS Gel Electrophoresis Michael 0. Hurst Georgia Southern University, Statesboro, GA 30460 Michael V. Keenan University of Wisconsin-River Falls, River Falls, WI 54022 Chae C. S o n St. George's University School of Medicine, St. George's, Grenada I n order to provide the students in the introductory biochemistry lab with a sense of the approaches used to characterize and study biomolecules, we are adopting a project format focused on each class of biomolecules. The protein purification project is based upon the purification of hen egg white lysozyme. The purification of hen egg white lysozyme is routinely used to introduce the student to iou-exchange chromatography and enzyme purification. We have develoved a ~urificationof lvsozvme . . that utilizes salt gradient elution ion-exchange chrorn:it(~@xphy l to s e w r a t e und characterize nnd SDS n ~ eiectro~horesis white by &oionic point, molecular of hen the weight and by enzyme activity. Egg white lysozyme is purified on a carboxymethyl(CM)-Sephadexcolumn eluted with a linear NaCl gradient. The resulting fractions are assayed for protein and lysozyme activity i n order to identify t h e lysozyme active fractions. The lysozyme-active fractions are analyzed with SDS polyacrylamide gel electrophoresis for purity, and the molecular weights of the isolated proteins are determined.
egg
Reagents and Equipment SDS-polyacrylamide gel electrophoresis was run on an Ephortec electrophoresis apparatus from Buchler. Spectral readings for the Bradford protein assays and the lysozyme activity assays were done on a Bausch and Lomb Purification of Lysoyrne Fraction
[protein] (mg/mL)
applied to column column wash elution fractionb2 elution fraction 3 elution fraction 13 elution fraction 14 elution fraction 15 elution fraction 16 elution fraction 17
3.14 1.29 0.129 0.131
elution fraction 18 elution fraction 19 elution fraction20
0.133
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total activity
unit^)^ 7410 246 0 0 23.0 42.4 53.7 206 185 116 94.5 39.1
Hen egg white extract was preparid according to the procedure of Stenesh (I).A 20-mL portion of the extract was applied to a 1.5 x 10.3 CM-Sephadex C-25 column that had been equilibrated with 0.050 M Tris10.050 M NaC1, pH 8.2. The column was washed with one column volume of the same buffer, then eluted with a 50-mL total salt gradient, 0.050-1.0 MNaCl, in 0.050 Tris buffer, pH 8.2. Twenty 2.6-mL fractions were collected manually. The extract, column wash, and fractions were stored a t 4 "C. The fractions were assayed during the next laboratory period for protein by the Bradford assay (21, and for lysozyme activity by monitoring the decrease i n light scattering form the hydrolysis of M~crococcusleisodeikticus cell walls according to Stenesh (I),0.5 mL of fraction being used i n the assay. The cell wall suspension used as a substrate should be stirred before use, but the settling rate i s slow enough so that i t will not affect the results during the five minutes of t h e assay. Initial velocities were determined by least squares analysis with SLIDEWRITE PLUS. The results of
118 7.29 -
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671
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unitsiminute. b ~ l l20 fractions were assayed, but only those with measurable protein or enzyme activity were recorded.
Journal of Chemical Education
Ion-exchange linear salt gradient purification of lysozyrne
specific activity (unitsimg)
'One unit of enzyme activity is defined as a change of 0.001 absorbance
850
Spectronic 20 spectrophotometer. Eggs were obtained from a local IGA supermarket the day of the purification. All reagents were obtained from the Sigma Chemical Company and were of reagent or electrophoresis grade. Gradient makers were made by the department glassblower a t Georgia Southern University.
Figure 1. SDS-polyacrylamide gel electrophoresis of lysozyme elution fractions:A(molecularweightstandards);0, D. F, H, J (fractions 1612, 25 pL applied);C, E, G, I (fractions 16-13, 5 pL app~ed); K (columnwash, 5 pL); M, N (columnwash. 10 pL); 0,P (column wash, 25 and 10 pL);Q (egg white extract, 25 WL).
distance moved immi
Figure 2. Standard curve for molecular weight determination of lysozyme fractions bn SDS gel. Alpha-lactalburnin, MW = 14,200; soybean trypsin inhibitor, MW = 20.100: trypsinogen, MW = 24,000; carbonic anhydrase. MW = 29,000;glyceraldehyde-3-phosphatedehydrogenase, MW = 36,000;ovalburnin, MW = 45.000; bovine albumin, MW = 66,000. the assays are shown in the table. Although fraction 16 had the highest total activity, fraction 18 was chosen to calculate soecific activitv. because the rote in concentration in fractcon 16 was toi'low to be detkted by the Bradford assav. Molecular weight and protein purity determination by SDS-eel clectroohoresis: Twodiscontinuous 1 2 O i SIIS mlva c r y l k d e 5136 gels were prepared by the proceduke if Lacmmli (31.Samoles were orepared bv the addition of 20 pL of a solution oi7.5% be&-m&captdethanol, 7.5% SDS, 0.5% bmmphenol blue, and 25% glycerol to 50 pL of sample, and then boiled for 2 min. Samples (5 or 25 pL) were aoplied with a n Eppendorf adiustable micropipet. Eledmp'hbresis was run'fbr 5 h at $00 V, and thesample.; were made vi~iblewith thc Coomassie Brilllanr Blue stain (41. One of the gcls is shown in F~gure1. Molecular weights were determined with a standard curve made with SLIDEWRITE PLUS utilizlne standard molecular weieht markers from Sigma !Fig. 2; The most prominent bands in the eee white extract had molecular weiehts of 65.3OO.4O.500. ;a; 14,800. As one ean see from ~ i 6 i - e1,the'14,800 band (lvsozvme) has been retained on the column and does not " " appear in the column wash, and only the lysozyme band appears in the lysozyme active fractions. Discussion The experiment is designed to be completed in three three-hour sessions, and to combine several different tech-
niques. The linear salt gradients functioned quite well and can be completed in less than three hours. The and activity assays served both to screen the fractions for lysozyme activity prior to electrophoresis and to introduce these techniques to the students. The total recovery of lysozyme was low, due i n part to the length of time (one week) between sessions, but the enzyme was active enough to measure and characterize with electrophoresis. The Ephortec electrophoresis system is used readily by students and allows us to run 40 samoles a t a time. This allows us to run all of our students' active samples. The students were able to do most of the work of preparing and running the gels themselves with tlie instructor stopping the electrophoresis after the lab. If possible students come back after the lab to stop the electrophoresis and stain the gel. The project is, thus, much more of a working experiment than the demo-type experiments that most undergraduate electrophoresis uses are. In this experiment, the students determine the elution from a n ion-exchange column, identify profile of those fractions that contain enzyme activity and analyze the fractions using electrophoresis. In addition to determining the purification and specific activity of the enzyme a t various stages in the purification, the students are also able to characterize the molecular weight distributions of the orotein-containing samoles. This. in turn. enables the students to gain a better understanding of ion-exchange chromatography in comparison to gel filtration chromatography. The project nature of this experiment integrates a varietv of techniaues and cornoutations and makes the purification of l$ozyme a n excellent subject for a n indepth laboratory report. Caution: S t u d e n t s should be told t h a t betamercaptoethanol is toxic. The boiling of the protein samples for electroohoresis should be done in the hood. kcrylamide is a neurotoxin, and the students should use care in preventing skin contact with the monomer.
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Acknowledgment We would like to acknowledge Clair Colvin, the Georgia Southern deoartment chairman and elassblower. for the making of t
