Egg White Lysozyme - Partial Purification and Characterization
(1998 Spring Semester)
Robert Cluss, Kathy Jewett, Stephen Sontum
The enzyme lysozyme is very well-described. The x-ray crystal structure of hen egg lysozyme was the first to be determined, so it should of no surprise that it's catalytic mechanism is defined. Hopefully, you recall that we examined the catalytic mechanism of this enzyme last semester in CH 322. Lysozyme hydrolyses the [beta] (1->4) glycosidic linkage of N-acetylmuramic acid -> N-acetyl-glucosamine and therefore is capable of destroying the cell wall of some bacterial species. The enzyme is commonly associated with tears and mucus membranes, and is believed to play a protective role at these anatomical sites.
Lysozyme is a stable enzyme, 14.6 kDa in size, and has an unusually high pI of 11. The conformational integrity of this monomeric enzyme is maintained, in part, by four disulfide bonds (see Voet and Voet Figs. 14-9 and 14-10 or the tutorial "http://alta1.middlebury.edu/chemistry/class/bio").
Lysozyme activity will be determined from several sources. The assay employs a bacterial suspension as the substrate, and activity is monitored by the lysis of the organisms. An essentially pure lysozyme preparation from Sigma (L 6876) will be used initially to establish the parameters for the assay. These parameters will then be used for assaying the lysozyme activity of crude egg white as well as partially purified preparation from egg white. In addition, each group will test a known inhibitor of lysozyme, N,N,N-triacetylchitotriose (chitotriose) (Sigma T 2144) at three concentrations. You will also navigate a tutorial of lysozyme and chitotriose that was prepared for CH 313 by Professor Steve Sontum and Ryan David ('01). The tutorial involves the use of RasMol, a molecular modeling program designed for visualizing protein structure. Lastly, each member of the class will conclude the two week lysozyme laboratory with a self-designed experiment that will investigate some aspect of lysozyme (using either the pure or semi-purified preparation).
Week 1 Day 1 Establish the necessary parameters for the lysozyme assay - use pure lysozyme
Week 1 Day 2 Winter Carnival - combine with Day 1 lysozyme assay of egg white, assay in the presence of chitotriose
Week 2 Day 1 Partial purification of egg white lysozyme using affinity
chromatography
Determination of specific activity for the partially
purified egg white lysozyme
BCA protein assay
Analysis of
purification steps by SDS-PAGE
Week 2 Day 2 Analysis of purification steps by SDS-PAGE
(continued)
Independent work - alteration of an assay parameter or simple
characterization of the hen egg lysozyme
- protein assay
- assay of lysozyme activity from crude material and partially purified preparations- ion-exchange chromatography
- UV detection of protein from column eluants- characterization of partially purified material
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
- assessment of enzyme activity in various samples and under different conditions
- plots of enzyme (either pure or partially purified in our lab) + or - chitotriose - determination of specific activity
- analysis of SDS-PAGE data
Discussion: Overview of the Experiment
General Topics
-
methods for cultivating bacteria
- the assay of lysozyme activity
The gram positive coccus, Micrococcus luteus (lysodeikticus) (ATCC strain # 4698) is used as the substrate for assaying lysozyme. This bacterium is exquisitely sensitive to lysozyme and lyses rapidly when exposed to the enzyme. The assay is straightforward and turbidometric; that is lysozyme activity is monitored by a decrease in absorbance at 450 nm, as organisms lyse in the cuvette.
- the assay is performed in a 1 ml cuvette; combine phosphate buffer, enzyme material, and the substrate to a final volume of 1 ml in all assays.
- prepare a fresh suspension of M. luteus from the overnight culture provided. Use this during Week 1. You will need to prepare your own suspension for Week 2 (details to be provided in lab).
- prepare the M. luteus suspension in 0.1 M phosphate buffer (pH 6.24)
- commercial lysozyme (Sigma) at 2 mg/ml in phosphate buffer - will be provided
- the assay is performed by recording the change in absorbance vs time. Start with a concentration of M. luteus that gives an absorbance of 0.700 to 0.800 at 450 nm. A unit of lysozyme activity is defined as the amount of enzyme that produces a [Delta]A450 of 0.001 per minute at pH 6.24 at 25deg.C. Attempt to generate linearity for 3 to 5 minutes with an [Delta]A450 decrease of between 0.50 and 0.100 per minute - you must experimentally determine an appropriate dilution of the preparation (be it "pure" or "crude") in order to satisfy this condition.
A - obtain linear results with the pure enzyme
B - test the egg white. Start by preparing a 1:5 dilution in phosphate buffer (1 part egg white to 4 parts of buffer). Proceed as you deem necessary.
Preparation of the egg white (all the steps below may be conducted at the
bench)
- separate the yolk from the white portion of the egg, remove
particularly viscous material from the egg white
- place the sample in a
graduated cylinder mix by pipetting up and down remove 4 ml and dilute 1:5 with
phosphate buffer (total vol of 20 ml) in a 50 ml conical tube
C - test the inhibitor chitotriose. A 2 mM working stock solution in 0.1 M phosphate buffer (pH 6.24) is provided. Run the initial assay with chitotriose at 1 uM.
D - Access Steve and Ryan's web site tutorial as follows:
http://alta1.middlebury.edu/chemistry/
Select Class Material
Biochemistry
The Coulson Tutorial Rasmol (IMPORTANT DO THIS - will teach you how to use RasMol and teach you about protein secondary structure)
Lysozyme Chime if you did not learn RasMol or Lysozyme RasMol if you did
E - Down load a lysozyme pdb file from Molecules-R-us (use the Web-sites button)
edit the pdb file using Word to cut and paste the amino acid sequence into your lab report
underline the catalytic amino acid responsible for breaking the cell wall.
This assay uses bicinchoninic acid to detect complexes of Cu+ and protein that form under alkaline conditions. It is based on the Lowry method (employed by thousands of researchers since the 1950's) but is simpler to use, less time-sensitive, and is affected by fewer inhibitory substances. We will use the BCA assay in a microtiter plate format which greatly simplifies data collection and subsequent calculations.
Reagents, Standards, and Samples for the BCA Protein Assay (Microtiter Plate Format)
1. BCA Protein Assay Reagent
Reagent A: alkaline reagent, sodium carbonate, sodium bicarbonate, BCA detection reagent, and sodium tartrate in 0.2 N NaOH
Reagent B: 4% copper sulfate solution (w/v)
2. Protein Standard
Bovine Serum Albumin (BSA) solution at 2 mg/ml
3. Egg white
Assay at 1:5 and 1:20 in phosphate buffer
BCA Assay Procedure:
Prepare the Working Reagent - mix 50 parts of Reagent A with 1 part of Reagent B. How much should you prepare? Each assay requires 200 ul of working reagent and it is best to prepare some extra. So, for example, if you need to do 20 assays (including blanks), then you will need a minimum of 4 ml of Working Reagent - therefore prepare 5 ml.
The assay calls for 10 ul of each standard, blank, or unknown sample. Set up as follows:
Protein Standard Curve (assay all points in duplicate)
BSA
|
dH2O
|
0.1M
Phosphate Buffer, pH 6.24
|
BCA
Working Reagent
|
BSA
Content
| |
| 0
ul
|
10
ul
|
10
ul
|
200
ul
|
0
ug (assay blank)
| |
| 2
ul
|
8 ul
|
10
ul
|
200
ul
|
4 ug
| |
| 4
ul
|
6 ul
|
10
ul
|
200
ul
|
8 ug
| |
| 6
ul
|
4 ul
|
10
ul
|
200
ul
|
12 ug
| |
| 8
ul
|
2 ul
|
10
ul
|
200
ul
|
16 ug
| |
| 10
ul
|
0 ul
|
10
ul
|
200
ul
|
20 ug
|
Dilute the egg white in phosphate buffer
Egg white
Phosphate
Buffer
dH2O
BCA
Working Reagent
1:5 dilution 10 ul
0
ul
0
ul
200
ul
1:5 dilution 2 ul
8
ul
8
ul
200
ul
1:50 dilution 10 ul
0
ul
0
ul
200
ul
1:50 dilution 2 ul
8
ul
8
ul
200
ul
Microtiter Plate Protocol
1. Prepare working assay reagent
2. Pipet BSA, H2O, phosphate buffer, or egg white material into the appropriate wells. Avoid the wells on the border of the plate.
3. Add 200 ul of BCA working reagent to each well. Mix once by pipetting up and down. Discard the tip before adding reagent to the next well.
4. Cover the microtiter plate and incubate at 37deg.C for 30 minutes.
5. Read the absorbance at 562 nm (A562) with a microtiter plate reader. If a 562 nm setting is unavailable studies show that readings taken at wavelengths between 540 and 590 nm work with this method.
6. Calculate the protein content of your bacterial lysate from the BSA standard curve.
(Dilution factor) (x ug/ul) (1000 ul/ml) = ug/ml Convert to mg protein per ml of sample
in phosphate buffer. Store the sample @ 4deg.C.
Discussion -ion exchange chromatography; principles and
practice
-preparation of lysozyme partially purified from egg
white
-determination of specific activity for the partially purified
preparation
Lysozyme will be partially purified from one egg white by ion exchange chromatography using one of two resins:
DEAE-Sephadex (Pharmacia/LKB) anion exchanger
CM-Sephadex (Pharmacia/LKB) cation exchanger
Use 1.25 g of resin per 1:5 dilution of egg white.
- add 1.0 g of either the DEAE or CM resin to 10 ml of the 1:5 dilution of egg white
- mix for 15 min (by rocking or gentle swirling)
- assemble a small disposable column (Pierce) and carefully transfer the resin and egg white protein solution into the column. Wash the 15 ml conical with an additional 1 ml of phosphate buffer to recover the remaining resin and transfer to the column.
- spin the column in the Beckman GPR tabletop centrifuge for 1 min at a speed setting of 1 (approx. 1,000 rpm)
- pipette off and reserve most of the buffer above the resin. This will be tested for protein content and lysozyme assay
- the bed volume should be around 8-10 ml.
- elute the phosphate buffer to a level just above the bed, should be less that 1 ml, discard
- elute the column batchwise with either NaCl or KCl as follows:
10 ml of 0.125 M salt
10 ml of 0.250 M salt
10 ml of 0.500 M salt
10 ml of 1.0 M salt
10 ml of 2.0 M salt
- collect the entire volume of each fraction into a 15 ml conical tube
- assay each of the fractions for lysozyme activity, as well as the 1:5 dilution of egg white that you originally mixed with the resin (you should have about 10 ml remaining) and the material that did not absorb to the resin (the phosphate buffer supernatant obtained after column centrifugation)
1. Voet, Donald and Judith. 1995. Biochemistry 2nd. ed. New York: John Wiley.
2. Cheetham, J.C., P.J. Artymiuk, and D.C. Phillips. 1992. Refinement of an enzyme complex with inhibitor bound at partial occupancy. Hen egg white lysozyme and tri-N-acetylchitotriose at 1.75 Angstroms resolution. J. Mol. Biol. 224, 613-628.
3. Wolfson, A.J., M. L. Hall, and T.R. Branham. 1996. An integrated biochemistry laboratory, including molecular modeling. J. Chem. Ed. 73, 1026-1029.