PURIFICATION OF LYSOZYME

In 1922 Alexander Fleming while working in his laboratory even though he had a cold, accidentally
added a few drops of nasal mucus to a bacterial culture !o much for systematic research" #o his
surprise something in the mucus killed the bacteria" #he active substance was the en$yme lyso$yme"
%yso$yme is present in eye tears, egg white, and a number of other sources" #he en$yme causes the
hydrolysis of bacterial cell walls and constitutes a defense mechanism against bacterial infections" #he
en$yme cleaves the glycosidic bond between carbon number 1 of &acetylmuramic acid '&A() and
carbon number * of &acetyl+glucosamine '&A,)" In vivo, these two carbohydrates are polymeri$ed
to form the cell wall polysaccharide
#he procedure takes advantage of lyso$yme-s remarkably high pI 'over 11). even well above neutral p/,
lyso$yme has a net positive charge, resulting in its retention by a cation exchange matrix when most
other proteins would run through due to a net negative charge" 0ation exchange matrices that might be
used include phosphocellulose and carboxymethyl or sulfopropylsubstituted cellulose, !ephadex, or
!epharose 'I use !!epharose)" For cation exchange chromatography, the buffer used should consist of
neutral and anionic species"
1e will isolate lyso$yme from chicken egg white and perform a spectrophotometric assay" It is a single
polypeptide chain, crosslinked by four disulfide bonds, and has a molecular weight of 1*,233 +altons"
#he shape of its active site, the mode of substrate binding, and the mechanism of its reaction with
bacterial cell walls have all been thoroughly studied" 1e will purify it further by ionexchange
chromatography on 0('carboxymethyl)!ephadex, a cationexchange resin" 4ecause lyso$yme-s
isoelectric point is 11, the en$yme is applied in buffer at p/ 5"2 and then eluted with buffer at p/ 13"6"
!amples will be collected and stored on ice and then analy$ed for protein concentration, en$yme activity,
and electrophoretic mobility"
!ample 17 Filtered egg white
!ample 27 0(!epharose Flowthrough
!ample 27 0(!epharose 8lution
+ividing the number of en$yme activity 'units9m%) by the protein concentration 'mg9m%) for each
sample, the ratio of en$yme activity 9 :protein; can be determined" #his ratio should increase at each step
of purification to demonstrate the purification of lyso$yme from other protein components in egg white"
Procedure:
1" 8ach group of students should separate one egg white 'from nonirradiated egg) and filter
it through a double layer of cheese cloth by gently 'don-t add pressure) passing the cheese cloth over
the rim of a 133 or 163 m% beaker, which is placed in an ice bath"
2" #urn on the !cience workshop interface and turn on the computer" Attach a p/ meter to
the interface, !tart the !cience1orkshop program, and calibrate a using p/ * and p/ 13 buffers"
2" (ake 633 m% of 3"36( &a0l, 3"36( #ris buffer in a <33 m% beaker on a magnetic
stirrer and ad=ust the p/ to 5"2 with dropwise addition of /0l" #ris, short for
#ris'hydroxymethyl)aminomethane, has a formula of 0*/11&>2 and a molecular weight of
121"12g9mole"
*" (ake 263 m% of elution buffer that is 3"36( &a0l, 3"2( &a20>2 and ad=ust the p/ to
13"6"
6" #ransfer 16 m% of the egg white filtrate to a second beaker and dilute it 176 with 3"36(
&a0l, 3"36( #ris/0l 'p/ 5"2) buffer" (ix thoroughly"
<" ?ass the mixture through a plug of glass wool placed in a funnel" !ave 12 m% and label
as !ample 1"
@" >btain 6 g of dry 0(!epharose and add deioni$ed water to allow swelling to approx" 2
times the volume 'check bottle for swelling specifications)" Alternatively, if pre hydrated 0(
!epharose is available proceed to next step"
5" ?lace a glass wool plug 'or filter plug) in a chromatographic column and then pour the
preswollen resin into the column and allow the resin to settle" #he liAuid chromatography pump can
be used to help settle the 0( !epharose" Fill the column so approximately 6 m% of empty space
remains at the top of the column"
9" 1ith the intake placed in the buffer, pump 63 m% of 3"36( &a0l, 3"36( #ris/0l 'p/
5"2) buffer through the column to prepare it for the egg white" Bemove the water at the top of the
column and add 6 m% of the filtered egg white, reassemble and start the pump"
13" After the sample has
entered the column, pump buffer
through the column and collect 6 m%
flow through fractions in labeled test
tubes"
?ump started with p/
5"2 buffer C egg white
11" (ark the graph when each flow through fraction collection starts and stops" %abel the
flow through fraction with the highest absorbance '253 nm) as !ample 2"
12" 1hen the absorbance '253 nm) of the flow through fractions decreases to that of the
buffer level, stop the pump and remove any buffer from above the resin" Fill the top with elution
buffer '3"36( &a0l, 3"2( &a
carbonate 'p/ 13"6), start the pump
and collect 6 m% elution fractions"
12" (ark the graph when flow
through fractions collections start and
stop" %abel the elution fraction with
the highest absorbance '253 nm) as
!ample 2"
?ump started after buffer
change to p/ 13"6
Spectrophotometric Protein Ass!:
#here are several assay techniAues for proteins, 4iuret, %owry, 4radford, to name a few. however, this
spectrophotometric protein assay relies on the fact that proteins strongly absorb ultraviolet light of
wavelength 253 nm" #he absorbance of known concentrations of bovine serum albumin, 4!A, is
measured and a calibration curve of absorbance vs" concentration is constructed" 4y measuring the
absorbance of the collected lyso$yme solution, a good estimate of the amount of lyso$yme in the
solution can be determined"
1" (ake a stock protein solution 'solution 1) consisting of "3633 g of 4!A in 63 m% of
buffer" Dsing a volumetric 13 m% pipette and 63 m% volumetric flasks, make 6 solutions each 196 as
concentrated as the previous" I"e" solution 2 E 13 m% stock F *3 m% buffer, solution 2 E 13 m%
solution 2 F *3 m% bufferG""
2" In=ect the 6m% of the buffer in to the input port on the spectrophotometer and $ero the
chart recorder" #he buffer serves as the blank"
2" !tart the recorded and in=ect 6m% of the lyso$yme solution into the spectrophotometer
and measure the pen deflection" 4e sure to save all of this solution that passes through the
spectrophotometer" 0ontact the instructor if any solution causes an off scale deflection, as
ad=ustments may be reAuired to the eAuipment or procedure"
*" In=ect the most dilute 4!A solution 'solution 6) into the spectrophotometer and measure
the pen deflection"
6" Bepeat for solutions * through 1"
<" ,raph the scale reading vs" protein concentration and determine the concentration of the
lyso$yme through application of 4eerHs law"
S"S#PA$E E%ectrophoresis An%!sis o& L!so'!me
In this part of experiment, students prepare purified lyso$yme for analysis on a tenlane !+!?A,8
electrophoresis gel" ?recipitate 1 m% of the purified lyso$yme solution with * m% of acetone, centrifuge,
separate, and then dissolved the precipitated lyso$yme in 3"6 m% of electrophoresis buffer in a boiling
water bath" (ix 16 microliters of the lyso$ymeelectrophoresis buffer solution with 16 microliters of
blue sample buffer and deposit 16 microliters of this mixture into your assigned gel lane" +onHt puncture
the bottom of the gel well" 8ach group will be assigned one lane" A standard protein mixture,
commercial lyso$yme, and albumen proteins will also be loaded" #he gel will be fixed, stained, de
stained, and a photocopy made of the resulting gel" +etermine lyso$yme-s molecular weight using a
semilog plot" http799a22"lehman"cuny"edu9molbioIcourse9std"htm
#he %yso$yme !tructural Analysis with all links and Auestions can be found at7
http799chemserv"ventura"cc"ca"us9doliver29chem12b9exercises9lyso$ymeIanalysis"htm" 8ach group
should go through the analysis and answer the Auestions at the end of the exercise" Jou should read the
Auestions before your start so answer them as you go through the analysis"
0ool pictures of lyso$yme crystals growing can be seen at7 http799shelx"uni
ac"gwdg"de9Ktpape9lowres"htm"