L4cAPPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 7. 157 116 (19821
Affinity Separationof Enzymesfrom
Mixtures Containing SuspendedSolids
Comparisonsof Magneticand Nonmagnetic
Techniques
BnnNnnn L. HmscHBEINt nNn Gponcs M. WHTTESTnBS*
Department of Chemistry, Massachusetts Institute of Technology,
Cambridge,Massachusetts,
02I 39
ReceivedMay 5, 198I ; AcceptedJune23, lgSl
Abstract
Agarosebeadscontainingimmobilized enzymesor affinity ligandshave been made
magneticallyresponsiveby adsorbingfreshlyprecipitatedmagnetiteon their surface.
Thesebeadsare usedfor affinity adsorptionof proteinsfrom complex mixturescontainingsuspended
solids.The magneticallyresponsive
beadsand the unwanted(diamagnetic)solids are then separatedby magneticfiltration. This magneticadsorption
schemefor direct affinity separationof enzymesfrom mixturescontainingsuspended
solidsis comparedwith a similar, but nonmagnetic,schemein which the affinity matrix is supportedon fiberglasscloth. The enzymeis allowed to adsorbin this matrix,
and the matrix is simply removedphysicallyf rom the suspension
to achieveseparation
from the unwantedsolids.The two methodsseemcomparablein their abitity to separate a desiredenzymaticactivity. The magneticmethodsare technicallythe more
complexof the two, but are significantlythe more rapid. The efficiencyof separation
of diamagneticand ferrimagneticsolids in thesebiological systemsby high gradient
magneticfiltration is good.
Index Entries: Affinity separation,of enzymesvia magnetictechniques;separation, of enzymesby magneticaffinity techniques;enzymes,separationby magnetic
affinity techniques;solids,affinity separationof enzymesfrom mixturesof suspended;
magneticseparation,of enzymesfrom suspendedsolids.
tSeeref. /.
roCopyright 1982 by The Humana press Inc.
AII rights of any nature whatsoever reserved.
0273 -2289 t 82/0500-0 I 5 7$04. 00
151
158
HIRSCHBEIN AND WHITESIDES
Introduction
is that of isolatingsolubleproA ubiquitousproblemin biochemicalseparations
solids.This problemis encounteinsfrom complexmixturescontainingsuspended
tered, for example,in the early stagesof enzymeseparations(wherethe preparations contain cellular debris), in the recoveryof enzymesused as catalystsin
andin certaintypesof analyticalanddiagandfood processing,
organicsyntheses
nostic procedures.Conventionalaffinity adsorptionbasedon affinity ligandsatsince
tached,e .g., to gel particles,is only partiallyeffectivein thesecircumstances
reisolationof the affinity ligand-containingmatrix from the mixtureby filtration or
by othersolid compocentrifugationcommonlyleadsto extensivecontamination
basedon magmethods
here
two
We
describe
mixtures.
heterogeneous
nentsof the
suscontaining
mixtures
proteins
from
of
netic filtration for the affinity separation
on
depend
not
do
others
that
pendedsolids,and comparethesemethodswith two
magneticfiltration techniques.The potentialadvantageof magneticseparation
techniquesin biochemistryis basedon the fact that most biologicalsystemsare
diamagnetic.Para-,ferro-, or ferrimagneticcomponentsintentionallyintroduced
into biologicalsystemscan thereforebe manipulatedmagneticallywith relatively
little interferencefrom the naturally occurringcomponents.
This work relieson high gradientmagneticfiltration( HGMF) f or magneticseparations(2-9). This techniqueprovidesa methodfor the rapid isolationof srnall
in water(or
particlesf rom suspensions
( I - I 00 pm) ferro-,ferri-, andparamagnetic
other diamagneticmedia).It effectivelytrapseven weakly magneticparticlesin
filaments
regionsof very high magneticfield gradientthat surroundf-erromagnetic
placedin a saturatingmagneticfield, and provideshigh filtrationratesfor soft or
solidsof the type that oftenclog the face of conventionalfilters. [n
compressible
practicesuchmagneticfilters often simply consistof steelwool packedin a tube
placedbetweenthe polesof a magnet.
we requireda simple method of
To utilize HGMF for enzyme separations,
preparingmagneticallyresponsivematricescontainingeither affinity ligandsor
immobilized enzymes.Ferrimagneticmatricescontainingaffinity ligandshave
been discussedpreviously (10-12), and magnetic supportsfor enymes (10,
l 3 - 1 6 ) , f o r o t h e r c a t a l y s(t 1s 7 , l 8 ) , a n d f o r c e l ls e p a r a t i o n( 1s9 - 2 4 1 h a v e b e e n d e scribed.The magneticmethodsreportedhereusefunctionalizedagarosewith adsorbed magnetite particles. The nonmagneticmethods are based on PAN
gel developedpreviously tor
Ipoly(acrylamide-co-lr{-acryloxysuccinimide)l-a
enzymeimmobilizatton(25).We haveexaminedand comparedfour systems.
agarosebeads(preparedby precipitatingmagl. Magneticallyresponsive
netiteon the beadsurface)containingaffinity ligands.We referto separations
"
basedon this affinity matrix as "magnetic affinity adsorption.
2. Similar magneticallyresponsivebeadscontainingimmobilized carThis immobilizedproteinis usedto adsorbselectivelysolubonic anhydrase.
ble proteinsmodified by covalentattachmentof aryl sulfonamidegroups.We
havepreviouslydescribeda similarprocedure(omittingthe magneticcomponent) as "generahzedaffinity chromatography"(26). Separationsbasedon
A F F I N I T Y S E P A R A T I O NO F E N Z Y M E S
159
this magneticmatrix will be referredto as "magnetic generahzedaffinity
adsorption."
3. Nonmagneticpolyacrylamidegels, containingaffinity ligands,coated
as thin films on fiberglasscloth. We will refer to thesematerialsas "affinity
sponges" for easeof reference.
4. Nonmagneticgels,containingimmobilizedcarbonicanhydrase,
coated
"
on fiberglass.Thesematerialswill be called"generalizedaffinity sponges.
In all of the methods,the solubleproteinof interestadsorbsbiospecficiallyto the
gel. The gelscontainingaffinity ligandsfollow theusualprinciplesof affinity chromatography.Thosecontainingimmobilizedcarbonicanhydraseare usefulprimarily for the recoveryof enzymesusedas catalystsin organicsynthesis:they usea
single',well-definedassociation
(thatof carbonicanhydrase
for aryl sulfbnamides)
to adsorbmodified proteins.The first two methodsrely on HGMF to removethe
magnetic affinity or generalizedaffinity matrices from mixtures containing
unwantedsuspended
solids;the secondtwo usedirectphysicalremovalof the fiberglasscloths to separatethe attachedaffinity or generalizedaffinity matrices
from the media.
To evaluatethe relativeperformanceof theseseparationtechniques,we have
utilizedtwo typesof modelsystems.in one. we measurethe efficiencyof separation of a soluble enzyme from a well-definedmixture containingsuspended
nonmagneticagarosebeads(representing
contaminating
solids)and anothersoluble protein(representing
solublecontaminants).
For convenience
in the following
separations,we label the nonmagneticbeadswith an easily assayedenzyme
(B-galactosidase
or hexokinase)
to facilitatedetection.In a secondmodel system,
we substitutea crude yeasthomogenatefor the nonmagneticagarosebeads.
Experimental
Materials
Sepharose
68-100 (averagemolecularweightexclusionlimit 4 x 106,particle
size in swollen state 40-120 pm), Baker's yeast (YSC-2), ATP, NADP,
o-nitrophenyl-B-o-galactopyranoside,
carbonic anhydrase(bovine), glucose-6(Baker'syeast),hexokinase
phosphate
dehydrogenase
(Baker'syeast),peroxidase
(horseradish),
(E. c'oli)werepurchased
and B-galactosidase
from SigmaChemical
Company. Hexokinase-Arso2NH2Q6) and PAN-1000 (25) were preparedas
describedpreviously.Waterwasdeionizedanddistilledusinga CorningModel 3B
still. All otherreagentswere purchasedfrom commercialsourcesand were used
without further purification.
H i gh-GradientM agneticF ilter
The magneticfilter usedin this work consistedof ca. 5 g of stainlesssteelwool
(International
SteelWool Co.p., fine grade)packedlooselyin a glassburette( I -in.
160
HIRSCHBEIN AND WHITESIDES
diameter)placedvertically in the pole gap of an electromagnet(Varian V-4004)
that possesseda field strengthof l0 kG when operatedat 3.0 A.
Assayfor Carbonic Anhydrase (27)
Water ( 1.7 mL), 4-nitrophenylacetate
solution( I .00 mL, 3 mM) and300 pL of 0. I
phosphate
pH
M
buffer,
7.0, were addedto a quartzcuvetteand the rateof change
of absorbanceat 348 nm measuredspectrophotometrically
at 26"C. The carbonic
anhydrase-containing
sample(5-50 pL) was added,the cuvetteagitated,and the
rateof changeof adsorbance
againmeasured.The molar absorbtivityat 348 nm (an
isobesticpoint for 4-nitrophenoland 4-nitrophenylate)
is 5.4 x 103M- I cm-'.
4-Nitrophenyl acetatealso absorbsslightly at this wavelength(e : 0.4 x 103
M-t cm-l). If measurements
are made in a l-cm cuvette, the increasein
absorbanceat 348 nm divided by 5 givesthe concentration(millimolar) of 4-nitrophenol and 4-nitrophenylateanion, independentof pH.
Assayfor Hexokinase(28)
To a 3-mL quartzcuvettecontaining3.00 mL of a solution(pH 7.6) thatwas0. I M
in Hepesbuffer,0.22M rn glucose,and0.01 M in MgCl2,wasadded200 pL of a
solutionthat was 40 mM in ATP and I I rnM in NADP. Glucose-6-phosphate
(5 prl-of a solutionof 1000u/ml-) wasadded,the cuvetteagitated,
dehydrogenase
and the rate of change of absorbancemeasuredat 340 nm and 26"C. The
hexokinase-containing
sample(5-50 pL) was added,the cuvetteagitated.andthe
rateof changeof absorbance
againmeasuredat 340 nm. The molar absorptivityof
NADH is 6220M-t cm- I at this wavelensth.
Assayfor Peroxidase(29)
To a 4-mLquartzcuvettewasadded3.00 mL of 0.1 M phosphate
buffer (pH 7.0),
50 pL of l8 mM guaracolsolution,and 5-50 pL of the peroxidase-containing
sample.The cuvettewasequilibratedat26"C in the spectrophotometer.
Hydrogen
peroxide(a0 p.L of an 8rnll4solution)was added,the cuvetteagitated,and the rate
of changeof absorbanceat 436 nm measured.The molar absorptivityusedin calculationswas 6.39 x 103M- t cm-t.
Assayfor $-Galactosidase(30)
To 1.00 mL of the substratecontainingsolution (0.75 g o-nitrophenyl-B-ogalactopyranoside/L
in 0.05 M Tris-HCl buffer, pH 7.5,0.1 M NaCl,0.0l M
MgCl2) was added5-30 pL of the sample.The assaymixture was incubatedat
room temperaturewith stirringfor 5 min. The reactionwasquenchedwith I .00 mL
of 1.0 M Na2CO3solution, and the absorbanceat 420 nm measured.The molar
absorptivityof a-nitrophenylateion is 30OOM ' cm-r.
Immobilization of Enzymeson Agarose (34)
Carbonic anhydrase (100 ffig, 103 U), hexokinase (20 ffig, 5000 U), or
(20 mg,680 U) was dissolvedin 2OmL of 0.2 M NaHCO3buffer,
B-galactosidase
pH 9.5, andstirredwith a magneticstirrerin a 250-mL beakerequilibratedin an ice
AFFINITY SEPARATION OF ENZYMES
161
bath. A slurry of l0 mL of Sepharose68 was washedwith 3000 mL of water and
50 mL of 2 M Na2CO3,then filtered with suctionusing a fritted glassfilter. The
moist gel was transferredto a 100-mL beakercooledin an ice bath and equipped
with a magneticstirrer. Water (10 mL) and 2 M Na2COt Q0 mL) were added.
While the slurry was stirredrapidly, a solutionof 1.6 g of cyanogenbromide (a
very toxic material)in 0.8 mL of acetonitrilewas addedall at once.After 3 min of
rapid stirring, the slurry was filtered with suctionand washedimmediatelywith
100 mL each of 0.1 M NaHCOT(pH 9.5), HzO, and again with the NaHCO3
buffer. After the last wash the moist cake was addedto the beakercontainingthe
enzymesolution, stirredbriefly, and storedat 4'C for 20 h. The immobilized enzymeswere packedin columnsand washedsuccessively
with 100mL eachof 0. I
M phosphatebuffer, pH 7.0, 0.1 M phosphate
buffer, pH 7.0 and 0.5 M in KCl,
and then again with the 0.1 M phosphatebuffer, pH 7.0. Activities were:
agarose:carbonicanhydrase,8.4 U/mL gel (82o/o);agarose:hexokinase,
52
U/mL gel (ll7o); agarose:B-galactosidase,
59 U/mL gel (86Vo).
(33)
Agarose:ArSOzNHz
68 ( l6 g of sluny) waswashedwith waterandfilteredwith suction.The
Sepharose
resulting moist sepharose(ca. l0 g) and 14.3 mL of a J07o aq solution of
1,4-butanediol
diglycidyletherwerestirredrapidlyfor 14.5h with l0 mL of 0.6 M
NaOH solutioncontaining20mg of NaBHa.The gel was collectedby filtration
underreducedpressure,washedwith I .6 L of water, and transferredto a 200-mL
round-bottomed
flask. Sulfanilimide(l .14 g,6.62 mmol) in 35 mL of carbonate
buffer,pH I 1.0, wasaddedandthe suspension
stirredfor 36 h at 70'C. The gel was
filtered and washedwith 100 mL eachof 0.5 M NaHCOr, pH 9.5 and 1.0 M in
NaCl, water,0.05 M glycrne'HCl,pH 3.0 and l.fJ M in NaCl, and againwith
water. The sepharose
was packedin a column and washedwith 700 mL of 0.05 M
glycine'HCl,pH 3.0 and I M rn NaCl, andfinally washedwith 800 mL of water.
F ejOa :Agarose: CarbonicAnhydraseand F e-1Oa:
Agarose:ArSO2NH2
(17, I8)
(ca. 10 mL gel) was
Either agarose:carbonic
anhydraseor agarose:ArSO2NH2
washedwith water.FeCl2'4H2O(0.20 g, 1.0 mmol) and 0.54 g (2.0 mmol) of
FeCl3'6H2Oweredissolvedin25 mL of waterand heatedto 70'C. NaOH (0.5 g)
in 5 mL of water was added with stirring and a black precipitateof magnetite
formed. The suspensionwas allowed to cool to ambienttemperature,neutralized
with HCl, addedto the modified agarose,and stirredfor I h. The suspensionwas
filtered with suctionusinga coarseglassfrit, which passesthe free magnetite,and
the gel washedwith I .0 L of water.The resultingbrown-coloredagarosebeadsare
easily manipulated with a weak bar magnet. The magnetite particles are
unobservableby scanningelectronmicroscopy(seeFig. 2).
4 -SulfonamidobenzoyI Chloride
Thionyl chloride(50 mL, 0.685 mol) was addedto a suspension
of 50.0 g (0.2a8
mol) of 4-carboxybenzenesulfonamide
in 200 mL of dry dioxaneunderargon.The
mixture was heatedat reflux for 1.5 h, cooled,andconcentrated
by rotaryevapora-
t62
HIRSCHBEIN AND WHITESIDES
tion. The resultingpale pink solid was trituratedwith cold toluene,filteredwith
suction, and washed with cold toluene. The solid was recrystallizedfrom
toluene:dioxaneyielding 50 g (93Vo) of product as off-white flakes, mp
1 3 9 - 1 4 2 " Cl,i t . m p l 4 l - 1 4 3 ( 3 1 ) . I R ( n u j o l ,c m - r ; : 3 3 3 0 , 3 2 5 A , 3 1 0 0 ,1 1 2 2 ,
1 3 5 0 ,a n d 1 1 6 0 .' H N M R ( a c e t o n e - d 0 , 6 ) : 8 . O - 8 . 5 (d4oHf, d ) , 6 . 8 - 7 . 1 ( 2 H , b ,
s).
FG:PAN:ArSO2NH2
(TET, l0 mmol) in acetone
To l0 mL of a 1.0 M solutionof triethylenetetramine
stirredvigorouslyin a 50-mL round-bottomedflask in an ice bathwas addeddropwise a solutionof I . I g (5 mmol) of 4-sulfonamidobe
nzoylchloridein l0 mL of
acetone.The ice bathwasremovedandthe mixturestirredan additionalI .5 h. The
precipitatethat formed was dissolvedby additionof 3 mL of water, the mixture
concentrated
to a viscousoil by rotaryevaporation.
thendilutedto l0 mL by addition of 0 .3 M Hepesbuffer,pH 7.5. To a stirredsolutionof 4 g of PAN-1000in l5
mL of 0 .3 M Hepes,pH 7.5 wasadded2.5 mL of thesolutionprepared
above.The
mixturewas stirredfor ca. 15 s, appliedto ca. 48 in.r of fiberglasscloth with a
rubberroller. andstoredin a moistatmosphere
for l. 5 h. The resultingFG : PAN :
ATSO2NH2
waSwashedseveraltimeswith 50 mM Hepesbufter IpH 7.5. 50 mM
(NH4)2SOaland storedin this buffer overnightat 4oC. The affinitv spongewas
thenwashedseveraltimeswith0.l Mphosphatebuffer.pH 7.0. andstoredin this
buffer at 4'C.
F G : PAN : Carbonic:Anhydrase
P A N 1 0 0 0( 3 . 0 0g ) w a sd i s s o l v e rdn 1 2 m L o f 0 . 3 M H e p e sb u f f e r .p H l . 5 . i n a
(TET)
25-mL beaker.With stirring,,2.25 mL of 0.5 M aq. trieth,vlenetetramine
was added.After ca. 15 s. a solutionof 500 mg of carbonicanhydrase
in 2 mL of
0.3 M Hepesbuffer.pH 7.5, wasadded,thesolutionstirredan additionall0 s. and
appliedto ca. 40 in.2 of fiberglasscloth with a rubber roller. The resulting
FG : PAN : carbonicanhydrasewas storedin a moist atmosphere
firr I h. washed
severaltimeswith 50 mM Hepesbuffer [pH 7.5, 50 mM in (NH1):SO+]andstored
at 4"C in this buffer overnight.[t wasthenwashedseveraltirneswith 0 . I M phosphatebuffer, pH 7.0. and storedin this buffer at 4"C.
M agneticSeparationof F e30a :Agarose'.Carbonic Anht'druse.fi'ont
Suspension
(15 mL) consistingof agarose:hexokinase
(ca. 5 mL of gel, 80.6
A suspension
(27.3
U), 5 mL of FejOa:agarose:carbonic
anhydrase
U), and peroxidase(ca.
1 tng,132 U) in I mL phosphatebuffer, pH 7.0 was prepared.The peroxidase,
carbonicanhydrase,and hexokinaseactivitieswere assayed.The suspension
was
dilutedto 100mL with I mM phosphate
buffer, pH 1.0, passedthroughthe magnetic filter with a flow rate of 90 mL min-r, and washedwith additionalphosphatebuffer. The suspensionpassedby the magneticfilter was filtered through a
glassfrit and the fraction passedby this filter assayedfor eachof the enzymatic
activities.The materialretainedby the conventionalfilter was resuspended
in l0
AFFINITY SEPARATIONOF ENZYMES
163
mL of 0.1 M phosphate
buffer andassayed
for eachof the enzymes.The magnetic
filter was flushed by turning off the electromagnetand pouring 50 mL of I mM
phosphate
buffer, pH7.0, throughit. This suspension
wasalsoassayed
for eachof
the enzymaticactivities.
MagneticAffinity Adsorptionof CarbonicAnh.vdrase
from a Mixture Containing
SuspendedSolids
(5 mL of 9e1,54.6U) was suspended
Agarose:hexokinase
in 0. 1M phosphate
buffer, pHl.0, containingperoxidase(ca. 3 -g, 343 U) and carbonicanhydrase
(ca. 37 mg, 35.l U). The resulting25.O-mLsuspension
wasassayed
for eachof the
enzymaticactivities.Fe3Oal?g?roSe
:ArSO2NH2(ca. l0 mL of gel) was added
and the suspension
stirredfor I h. The suspension
was then subjectedto consecutive magneticandconventionalfiltrationsandthe fractionsassayedas in the previous experiment.In this case,however,the magneticallyresponsivesuspension
collectedfrom the magneticfilter waspackedin a columnandwashedwith 50 mL
of 0.1Mphosphatebuffer,pH 7.0. The carbonicanhydrase
wasthenelutedwith
50 mL of 0. I M acetate
buffer,pH 5.15 and 1.0M in NaCl. The resultingsolution
was dialyzedtwice against2 L of I rnM phosphatebuffer. pH 7.0. and then
assayed.
Magnetic GeneralizedAffinity Adsorptiortof'He.rokinuse-ArSO2l',1H,
front ct
M ixture Containing SuspendedSolids
(20 mL. ll.1 U) was addedto a solutionof
A slurryof agarose:B-galactosidase
(ca. 3 mg, 292U) andhexokinase-ArSO2NHzQ2.8
peroxidase
U) in 8 mL of I
mM phosphate
buffer,pH 7.0, andtheresultingsuspension
wasassayed
for eachof
the enzymaticactivities.Fe3Oa:agarose:
carbonicanhydrase(ca. I mL of gel)
and 30 mL of 0.1 M phosphatebuffer, pH 7.0. were added.The suspension
was
stirred for I h at room temperature.then subjectedto consecutivemagneticand
conventionalfiltrations.The magneticallyresponsive
suspension
flushedfrom the
magneticfilter was packedin a column. washedwith 50 mL of O.I M phosphate
buffer (pH 7.0), elutedwith 50 mL of 0. I M acetate
buffer (pH 5.15, I M NaCl),
and then assayed.
Adsorptionof CarbonicAnhydraseJrom o Mi.rture ContainingSuspendedSolids
Using the Affinify Sponge
A g a r o s e : h e x o k i n a(scea . 5 m L g e l , l 2 6 U ) w a s s u s p e n d ei d
n 9 5 m L o f 0 . 0 1M
phosphate
buffer, pH7.0, containingcarbonicanhydrase
(ca. 50 mg, 43.9 U) and
(ca. 3 -9, l72U). The resultingsuspension
peroxidase
wasstirredfor 8 h at 4'C in
a 500-mL crystallizing dish containing the affinity sponge, FG : PAN :
ATSO2NHzir,a.48
in2).The spongewasremoved,washedbrieflywith 100mL of
phosphate
0.01 M
buffer, pH 7.0, and stirredfor 8 h at 4'C in another500-mL
crystallizing
dishcontaining100mL of 0.1 M acetate
buffer(pH 5. 15, 1 M NaCl).
The spongewas removedand26 mL of the solutiondialyzedseveraltimes against
4L of I mM phosphate
buffer, pH7.0. The originalsuspension,
the washsolution,
and the dralyzedsolutionwere assayedfor eachof the threeenzymaticactivities.
164
HIRSCHBEIN AND WHITESIDES
d
Adsorption of H exokinase- ArSO2NH 2from a M ixture Containing Suspende
Affinity
Sponge
the
Generalized
Solids Using
(ca. 5 mL gel, 151U) was suspended
in 95 mL of 0.01
Agarose:B-galactosidase
M phosphatebuffer, pH 7.0, containinghexokinase-ArSOzNHz64.0 U) and
peroxidase(ca. 3 mg, 167 U). The resultingsuspensionwas stirredfor 8 h at 4'C
with the generalizedaffinity sponge(FG:PAN:carbonic anhydrase,ca. 40 in2)
in a 500-mL crystalhzingdish. The spongewas withdrawn, washedbriefly with
100 mL of 0.01 M phosphatebuffer, pH 7.0, and stirredfor 8 h at 4'C in another
500-mL crystallizingdish containing100mL of 0.1 M acetatebuffer (pH 5.15, 1
M NaCl). The spongewas removedand the original suspension,wash solution,
and eluant assayedfor each of the enzymaticactivities.
Affinity Adsorption of Carbonic Anhydrasefrom a Suspensionof Sonicated
Yeast
was preparedby sonicating20 g of Bakersyeast(SigmaYSC-2) in
A suspension
25OmL of 0.01 Mphosphatebuffer,pH 7.0, and addingca. 80 mg of carbonic
into two 100-mLaliquots,andthe
wasseparated
e (94 U). The suspension
anhydras
carbonicanhydrasewas adsorbedfrom thesewith eitherthe magneticaffinity matrix or the affinity sponge,using the proceduresdescribedpreviously.
Results
Magnetic Affinity and GeneralizedAffinity Matrices
:
Aromatic sulfonamides bind carbonic anhydrase strongly K i
l0-6M-10-8M), are easily manipulatedchemically,and are readily available
(32). The methodsusedto preparemagneticmatricesbasedon this affinity system
are illustratedin Fig. 1.
Sulfanilamidewas coupledto agarosebeadsthrougha diglycidyl etherspacer
arm using a modificationof a literatureprocedure(331 Carbonicanhydrasewas
coupledto BrCN-activatedagaroseby conventionalmethods(34, 35). Thesefunctionalizedmatriceswere made ferrimagneticusing a modificationof a procedure
developedearlierfor the preparationof magneticallyrecoverablecatalystsupports
( 17, I8). A magnetitecolloid was preparedby reactionof a 1 : 2 aqueousmixture
and
of Fe(II) andFe(III) with sodiumhydroxide.Whenthiscolloidwasneutralized
of agarosebeads(eitherfunctionalizedor not),
treatedwith an aqueoussuspension
the agaroseand a part of the magetitebecametightly coupled. The magnetite
in a scanningelectronmicrographof the resultingbrown
particlesareunobservable
compositecan be separatedfrom unconbeads(Fig. 2). The agarose:magnetite
jugated magnetiteby filtration on a coarseglassfrit: the beadsare retained,while
the unconjugatedmagnetitecolloid passesthroughthe filter. We havenot explored
the chemistryof the attachmentof magnetiteto agarose,but it appearsto involve
somecombinationof physicalentrapmentof magnetiteparticlesin the agarosegel
and replacementof magnetitesurfaceoxide groupsby hydroxyl functions(proba-
165
AFFINITY SEPARATIONOF ENZYMES
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Fig. 2. Scanningelectronmicrographsof agarosebeads(top) and Fe:O+on agarose
beads(bottom).The bar labeledI U at thebottomof eachmicrographis a I -pm calibration.
No difference between masnetic and nonmasnetic beads is observed at lower
magnification.
t61
AFFINITY SEPARATIONOF ENZYMES
gel. In any event,the attachmentis firm:
bly chelating)from the polysaccharide
after the initial preparation,very little magnetiteit lost from the beadson subsequentmanipulation.The methodalsoappearsto be sufficientlygentlefor usewith
in carbonicanhydrase
at leastsomebiochemicalsystems:no decrease
activitywas
detectedupondepositionof magnetiteon agarosecontainingimmobihzedcarbonic
anhydrase.
P reliminary Experiments-S eparationof D iamagnetic and F errimagnetic'
AgaroseBeads
We beganour work by examiningthe efficiencyof separationof componentsin a
model biochemicalsystemconsistingof peroxidase(as a representative
soluble
protein contaminant),hexokinasecovalently bonded to agarosebeads (agarose: hexokinase, an insoluble and diamagneticcomponent), and carbonic
anhydrasecovalentlybondedto agarosebeadshavingattachedmagnetiteparticles
(Fe3Oa:agarose:carbonic
anhydrase,an insoluble ferrimagneticcomponent).
Theseenzymeswerechosenfor theiravailabilityandthe easewith which eachcan
be assayedin the presenceof the others:they were intendedprimarily to provide
labelsfor eachof the threephases.Figure 3 summarizesresults.
Magneticfiltrationswerecarriedout usinga magneticfilter constructed
simply
by packing stainlesssteel wool loosely into a l-in diameterglassburette,and
placing the burettein a magneticfield of approximatelyl0 kG (generatedby a
Peroxidose
:Hexokinose
Agorose
e a Q :Ag q l e e s :C o r b o n i cA nhydros
E q u i l i b r o t e I, h
r et o i n e d
HIGH GRADIENl
M A G N E T I CF I L T R A T I O N
possed
retoined
FILTRATION
p os s e d
Activities
Peroxidose
92 "/"
Hexokinose
O "/"
C o rb o n i cA n h y d ro s e O "/"
O "/"
90 %
O "/"
O "/"
5
o/o
t o o%
F i g . 3 . Magnetic separationof soluble. insoluble diamasnetic.and insoluble
ferrimagneticcomponents.
168
HIRSCHBEIN AND WHITESIDES
small electromagnet).The suspensioncontaining the diamagnetic and ferrimagneticagarosebeads(phosphatebuffer, I mM, pH 7) was passedthroughthe
filter rapidly (90 mL min-r), and the materialretainedon the filter washedwith
phosphatebuffer. This magneticallyresponsivematerialwas collectedby turning
off the electromagnetand flushing the filter with additionalphosphatebuffer. The
suspensioncollectedfrom the magneticfilter was assayedfor each of the enzymatic activitiesoriginally present.The numbersin Fig. 3 representrecoveriesof
enzymaticactivity, basedon the activity of the original suspensionbefore filtration. Thus, the recoveryof carbonicanhydraseactivity in the magneticmaterial
retainedin the magneticfilter wasquantitative;the only contaminantwas57oof the
hexokinaseactivity originally present conjugatedto the diamagneticagarose
beads.
The materialthat passedthrough the magneticfilter was subjectedto conventional filtration, and the fractionsretainedon the filter and passedthroughit were
analyzed As expected,the peroxidasewas found in good yield in the solution
(927oof the original activity), and the hexokinasewas found in the material retainedon the filter (90%oof the original activity). There was no contaminationof
fraction by
fraction or the agarose:hexokinase
either the peroxidase-containing
other enzymaticactivities.
Magneticallyresponsive,functionahzedagarosebeadsare thus easilyprepared
and stable,and are separatedrapidly and cleanlyfrom diamagneticinsolubleand
soluble materialsusing a high-gradientmagneticfilter of very simple design.
Magnetic Affinity Adsorption
To demonstratemagnetic affinity adsorption,a suspensionwas preparedcontaining four components:two soluble enzymes (peroxidaseand carbonic anhydrase), the magnetic affinity matrix for carbonic anhydrase (Fe3Oa :
agarose:ArSO2NH2) described previously, and a diamagnetic solid
(agarose:hexokinase,
intendedto modelinsolublecomponentsin a crudebiological preparation).The mixture was allowedto equilibratewith stirringfor I h, then
subjectedto successivemagneticand conventionalfiltrations, using procedures
analogousto thosedescribedin the precedingexperiment(Fig. 4). The major part
of the carbonicanhydrase(837o)was recoveredby elution from the particlesretained in the magneticfilter; 8Vowas found in solutionwith the peroxidase.The
material retained on the conventionalfilter contained93To of the hexokinase
activity.
M agnetic GeneralizedAffinity Adsorption
An example of magnetic generalizedaffinity adsorptionis outlined in Fig. 5.
Hexokinase-sulfonamide
conjugatewas prepared,as reportedpreviously,(26) by
reactionof hexokinasewith the reagentI in the presenceof ADP and glucose(to
protect the enzymeactive site).
r69
AFFINITY SEPARATION OF ENZYMES
--l
lI
I
L
Peroxidos.
C o r b o n i cA n h y d r o s e
I
A e o r o s eH: e x o k i n o s e
I
: rSo.NH.J
t t p o : A g o r o s eA
Equilibrote,
H I GH
GRADIENT
XAGNETIC FILTRATION
I )Wosh
2) Elule
3) Diolyze
FILTRATION
o"h
84 U"
Pcroxidose
Corbonic Anhydrose 8 %
O o/"
Hcxokinoge
Fig. 4.
pH 5.15.)
o o/o
O olo
8 3 oh
o/o
O oh
93
, NaCl,
Model magneticaffinity separation.(Elution with 0 . l M a c e t a t el M
retoined
HIGH GRADIENT
IAGNETIC FILTRATION
rrtoi ncd
FILTRATION
Pcroxidoso
Hsrokinosc
p -eorca oridose
90%
lO %
o%
o%
l) Wosh
2)Etute
o%
57%
98 o/o
O "/"
o "A
Fig. 5. Model magneticgeneralizedaffinity separation.(Elution with 0.1 M acetate,
M N a C l ,p H 5 . 1 5 . )
H I R S C H B E I NA N D W H I T E S I D E S
T7O
Hexokinase* l/-Succinimido- OCOCH,NHCOCH,NHCOPh-SO,NH,
I
H e p e s .p H 7 . 6
2 h. 2-5"C
ffi;
"*.,,_
ADP O.O3M
Hexokinase-ArSOzNH,
The magnetic generalized affinity matrix (Fe3Oa:agarose:carbonic
anhydrase)was equilibratedfor I h with a mixture of the two solubleenzymes
peroxidaseand hexokinase-sulfonamide
conjugate(hexokinase-ArSO2NH2),
(agarose:B-galactosidase).
and a diamagneticsolid
The componentswere separatedby successive
magneticand conventionalfiltrations,as in previousexperiments.Recoveryof the hexokinase-ArSOrNHrwas only modest(57o/o).
We believe this low recoveryresultsfrom problemswith the affinity separationand not
inefficiency of the magneticfiltration, but severalattemptsto improve the efficiency of the separationwere unsuccessful.
Ajjinity and Generctlized
4ff
Sponges
"i,.,^,
In principle,all that is requiredto carry out affinity separations
of proteinsfrom
mixturescontainingsuspended
solidsis an affinity matrixwith physicalproperties
sufficientlydistinctfrom thoseof the suspended
solidsthatthe two typesof solids
can be easilyseparated.
Magneticallyresponsive
affinity matricesprovideone solutionto thisproblem.A secondis an affinity gel connected
to a supporthavingthe
sizeand mechanicalpropertiesrequiredfor it to be convenientlyinsertedinto and
removedfrom the suspension
of interest.The preparation
of affinity gelsof these
types ("affinity sponges")is outlinedin Fig. 6. Thesespongesare preparedby
crosslinkingpoly(acrylamide-co-N-acryloxysuccinimide)
(PAN), as a thin gel
film on a fiberglasscloth support.Affinity ligandsor enzymesare covalentlyattachedthroughthe activeestergroupsof PAN duringthecrosslinkingstep.The gel
is porousto macromolecules
and its capacityfor enzymebindingis not limited by
superficialsurfacen1ga-in principle,the entiregel volume is utilized.
In the preparationof gels basedon PAN, an o,t'l-diamine(commonly triethylenetetramine,
TET) is usedasa crosslinkingagent.We havealsousedTET as
a spacerarm to separatethe afflnity ligandfrorn the polymerbackbone.lncorporation of a spacerarm betweenaffinity ligandandmatrix is generallyconsidered
desirablein affinity chromatography
to relievestericrestrictionsimposedby the matrix. The preparationof ligands incorporatingspacer arms often requires a
synthesisinvolving reactionof the affinity ligand with a large excessof the ct,
ol-difunctionalmoietyintendedto becomethe spacerarm, followedby separation
of the desiredmodifiedaffinity ligandfrom the excessof the spacermoiety. This
procedureis uneconomical
andinconvenient.We havesimplyallowedthe affinity
ligand (herean aryl sulfonamide)to couplewith excessTET, and usedthe crude
reactionmixture,withoutpurification,in thecrosslinkingreactionwhich formsthe
gel. This procedureshouldbe readilygeneralizable
to other affinity systems.In
practice,PAN, TET, and the affinity ligand (or carbonicanhydrase,for enzymecontaininggels)are mixed in aqueoussolution,and then spreadrapidly over the
t7l
A F F I N I T Y S E P A R A T I O NO F E N Z Y M E S
o#o
HZN
NH
/-\a\r-\
NH
NHZ
I ,E-r.
o
H2N02s€ti
HzN NH NH NHC19>SO2NH2
'Fibergloss
///r
r /
F G : P A N :A r S 0 z N H 2
Fig.
6. Preparation of an affinity sponge supported on fiberglass. PAN is
poly(acrylamide-c'o-1/-acryloxysuccinimide),
and the polymer backbonesare represented
schematicallyby curvedlines. TET is triethylenetetramine.
fiberglasscloth. The polymergelsto a soft, resilientfilm over the cloth backing:
the thicknessof the film is ca. 0.5 mm. The gel-coatedfiberglassis then washed
and inserted into the solution or suspensionfrom which the enzyme is to be
adsorbed.
Rate of Adsorption of Proteins by the Affinifi Sponge
The rate of disappearance
of carbonicanhydraseactivity from a solution in the
presenceof the affinity sponge,FG:PAN:ArSO 22NH2, is plotred in Fig. 7.
The relatively slow rate of disappearance
of activity may be due to slow diffusion
of enzymethroughthe gel. This experimentsuggests,
in agreement
with previous
studiesof PAN gels, that the interior of the gel is accessibleto proteinsby diffusion, and emphasizes
a significantdisadvantage
of thesesponges:that is, because
diffusion is a relativelyslow process,enzymeseparations
using thesematerials
take more time to perform than magneticaffinity separations,in which particle
sizesare relatively small and diffusion times fairly short.
Affinity Adsorption of Carbonic Anhydrasewith the Affinity Sponge
The use of the affinity sponge,FG:PAN:ArSO zNHr, for the adsorptionof carbonic anhydrasedirectlyfrom suspension
is summarizedin Fig. 8. A suspension
containing the soluble enzymes carbonic anhydraseand peroxidase,agaroseimmobilized hexokinase (agarose:hexokinase),and the affinity sponge
(FG:PAN:ArSO2NH2)was stirredfor 8 h at4"C. The affinity spongewas then
withdrawn from the suspension,washedwith phosphatebuffer, and storedfor 8 h
172
HIRSCHBEIN AND WHITESIDES
/
in obsence o'
50
A f f i n i f ys p o n s f
c
.9
=
6
a
.= 40
C'
o
o
!t
in presenceof
Affinify Sponge
1
.9
c
o
.o
o
'20
o
o
c,
=
Time (h I
activityfrom solutionin the
anhydrase
of
carbonic
Fig. 7 . Rateof disappearance
sponge.
presence
affinity
of an
at4"C in the stirredelutingbuffer. The resultingsolutionwasdialyzedand assayed
for enzymatic activities. The carbonic anhydrasewas separatedcleanly from
peroxidaseand hexokinase,but its recoverywas only 507o.Attemptsto increase
the fractionof the carbonicanhydraserecoveredby utilizing smalleramountsin the
the possibilitythat the posioriginal suspensionwere unsuccessful.We suspected
tive chargeson the TET spacerarrn were decreasingaffinity of the ligand for carbonic anhydrase.In independentsolutionexperiments,however,the K; of the inhibitor 2 (Fig. 6) was determinedto be 6.9 x l0-7 M. This valueis comparable
itself, 4.6 x l0-7 M Q7). The relativelylow reto that of benzenesulfonamide
covery of carbonicanhydraseby this methodthus remainsunexplained.
n3
AFFINITY SEPARATIONOF ENZYMES
[-aorbonic Anhydrose
I P.ro*ioore
I
OnorosH
e :e x o k i n o s e E q u r l i b r o t e
8 h,4 "C
I
P A N :A T S O z N H 2
Ltn:
Peroxidose
Hexokinose
Seporote
FG:PAN
Wosh
F G :P A N
85 %
7 5 olo
l3 %
Q "/"
lO "/"
O olo
C o r b o n i c A n h y d r o s eZ ? V "
6
El u t e
F G: P A N
o/o
5O o/o
Fig. 8. Adsorptionof carbonicanhydrasefrom a solutioncontainingsuspendedsolids . ( W as hings o l u ti o n0: .0 1 M p h o s p h a tep, H 7 .0; el uti on:0.1 M acetate,
I M N aC l , pH
s .r s . )
GeneralizedAffinity Adsorption of Hexokinase-ArSO2NH2with the
GeneralizedAffinity Sponge
The use of the generahzedaffinity sponge(FG:PAN:carbonic anhydrase)for
the separationof hexokinase-sulfonamide
conjugatedirectly from a suspensionis
illustratedin Fig. 9. The suspension
containedthe solubleenzymesperoxidaseand
hexokinase-ArSOzNHz,as well as suspendedagarose:B-galactosidase.
Separation of hexokinase-ArSO2NH2was accomplished
cleanly.
Separationof Carbonic Anhydrasefrom SonicatedYeastSuspension
To examine the relative efficiency of magnetic affinity matrices and affinity
spongesfor the separationof carbonicanhydrasefrom a mixture that more closely
resembledan actualbiochemicalpreparation,we prepareda suitabletestsystemby
sonicating yeast in phosphate buffer and adding carbonic anhydrase. The
reisolationof carbonicanhydrasefrom this suspension
usingboth the magneticaffinity matrix and the affinity spongeis summarrzedin Figs. l0 and 11, respectively. Magneticaffinity separationresultedin the isolationof 50Voof the original
carbonicanhydraseactivity in the solutionelutedfrom the magneticaffinity matrix. The suspensionretained44Voof its original carbonicanhydraseactivity. The
affinity spongewas slightly lesseffective,with45Toof the carbonicanhydraseactivity found in the solutionelutedfrom the sponge,367oremainingin the suspension. and JVoin the wash solution.
r74
H I R S C H B E I NA N D W H I T E S I D E S
I
-ArSozNHz
Hexotinose
I
I Peroxidose
I
:p -Goloctosidos.
agoror,
I
I
[0,
P A N :C o r b o n i cA n n y d r o s e . ]
Seporote
F GI P A N
Hexokinose
Peroxidose
f
Equrlrbrote
I h,4 "C
I
Wosh
F G IP A N
olo
(s6 %)
- O o l o c t o s i d o s e ( l O O% )
El u t e
F G :P A N
O "/"
70%
(l2 "/"1
o%
(20 %l
o"h
Fig. 9. Adsorptionof hexokinase- ATSO2NH2from a solutioncontainingsuspendedsolids.(Washingsolution:0.01 M phosphate,
pH 7.0; elution:0.1 M acetate,I M
NaCl, pH 5. 15.) *The precisionof theenzymaticassaysis ca. -+_57ofor solubleenzymes
and + l}Vo for immobilizedenzymes.The " l2OC/c"
recoveryof agarose: B-galactosidase
in this experimentmay reflect a systematicerror.
C o r b o n i c A nh y dr o s e
Sonicoted Yeost
F . S % :A g o r o s e: A r SO z NH z
E q u i l i b r o t e I, h
H I GH
GRADIENT
M A G N E T I CF I L T R A T I O N
possed
retoined
l) Wosh
2)Elute
3 ) D i ol y z e
(Yeost Solids )
C o r b o n i cA n h y d r o s e
44"/"
50 "/"
Fig. 10. Magneticaffinity separation
of carbonicanhydrasefrom yeastsuspension.
( E l u t i o n0
: . 1 M a c e t a t eI, M N a C l , p H 5 . 1 5 . )
t75
AFFINITY SEPARATIONOF ENZYMES
F.. icotedYeostI
I C o r b o n i cA n h y d r o sI e
L1,
' pAN:
ArSorNrr_j
Seporote
F G :P A N
C o r b o n i cA n h y d r o s e
Yeost Solids
36 %
Mosf
Equilibrote
8 h,4 "C
Wosh
F G :P A N
7 "/"
Elute
F G: P A N
45%
Some
Fig. I l. Separationof carbonicanhdrasefrom yeast suspensionusing an affinity
pH 7.0; el uti on:0.1 M acetate
sponge.(Washingsolution:0.01 M phosphate,
, I M N aC l ,
pH 5.15.)
Summary
We draw severalconclusionsfrom this work. First, relativelycleanseparation
of
ferrimagneticsolids from biochemicalsystemsis practicalusing high gradient
magneticfiltration. The featuresof this filtration methodthat make it attractivein
biochemicalapplications
arethatit is rapid(especiallyin filtrationof soft or sticky
materialsthat might clog conventionalfilters), it is applicableto relativelysmall
(severalmicrometers)particles,and it is selectivefor one (ferrimagnetic)
solid in
the presenceof other(diamagnetic)
solids.Thus, in principleit might be possible
to use this method for protein isolationdirectly from crude cell homogenates,or
from active fermentations,or to recoverimmobilized enzymesaddedto systems
containingsuspended
solids.
The procedureusedhereto preparethe magneticagarosebeadsis a very simple
one experimentally,and works betterthan might reasonablybe expected.It is certainly lessexpensiveand complexthanproceduresbasedon preformedferrofluids
( I2 , I 5 ), but seemsto yield materialsin which theconnectionbetweenthe agarose
andthe magnetiteis quitedurable.We havenot, however,testedthesesystemsfor
stabilityin thepresence
of strongchelatingagentsor materials(e.g., thiols)having
a high affinity for transitionmetals.We believethatthis techniqueshouldbe applicable to the preparationof other ferrimagneticgel-magnetiteconjugates.
Our comparisonsof magneticand nonmagneticaffinity adsorptionprocedures
suggeststhat they are comparablein their practicality.The magneticmethodsare
somewhatmore complex technically,but becausethe magneticbeadsare small,
the kinetics of adsorptionseembetterthan thosecharacterizingthe macroscopic
gel films.
fiberglass-supported
176
HIRSCHBEINAND WHITESIDES
References
I . Supportedby the National Institutesof Health, Grant GM 24540. BLH held an NIH
( 1 9 8 0 ,T 3 2 C A 0 9 1 l 2 C T ).
t r ainees hip
J ., a n d K e l l a n d ,D . (1975),S ci . A mer.233 (5),46.
2. K olm , H. , O b e rte u ffe r,
(ed.)
(1919),
IndustrialApplicationsof MagneticSeparation,Proceedings
3 . Liu, Y. A.
of an InternationalConferenceat Franklin PierceCollege, Rindge, New Hampshire,
July 30-August 4, lgJS,Institute of Electricaland ElectronicEngineers,New York.
P., a n d C o l l i n s ,D . N . (1 9 1 1 ),Sci ence,C hem.Ind.218-220.
4. P ar s onage,
Methodsof Minand Magnetohydrostatic
5. Andres, U. (1976),Magnetohydrodynamic
eral Separation,Wiley, New York.
6. Oberteuffer,J. (1973), IEEE Trans. MAG 9, 303.
7. K olm , H. H., a n d T ri n d a l e ,S. C . (1 9 7 3 ),IEE E Trans. MA G 9, 310.
B . W a t s o n ,J . H . P . , a n d H o c k i n g ,D . ( 1 9 1 5 ) , | E E E T r a n s .M A G l l , 1 5 8 8 .
G. M. C hemtech.,i n press.
9. Hir s c hbein,B. L ., B ro w n , D . W ., a n d Wh i tesi des,
1 0. Halling, P . J., a n d D u n n i l l , P . (1 9 8 0 ),En z ymeMi crobi al Technol .2,2.
1 1. Dunnill, P . , a n d L i l l y , M. D . (1 9 7 4 ),Bi o te chnolB. i oeng. 16,987.
12. Mosbach,K., and Anderson,L. (1977),Nature 2701259.
13. Robins on,P . J ., D u n n i l l ,P ., a n dL i l l y , M. D. (1973),B i otechnolB. i oeng.14,603.
14. Gellf, G., and Boudrant,J. (1974),Biochem.Biophys.Acta3341 467.
15. A dals t eins s o nO, ., L a mo tte ,A., B a d d o u r,R . F., C ol ton, C . K ., P ol l ak, A ., and
Whitesides,G. M. ( I 979), J . Mol. Catal. 6, 199.
16. vanleemputten,E., and Horisberger,M. (1974),Biotechnol.Bioeng.16, 385.
G. M. (1916),
1 7 . Hill, C. L. , L a m o tte ,A ., Al th o ff, W., B ru n ie,J.-C ., andW hi tesi des,
J. Catal. 43, 53.
1 B. W hit es idesG
, . M ., H i l l , C . L ., a n d Bru n i e ,J.-C . (1976),I& E C P rocessR es.D evelop.15,226.
8, 182.
1 9 . Rem baum ,A ., Ye n , S . P . S ., a n d Vo l k s e n ,W . (1918),C hemtech
2 0. K r onic k , P . L ., C a m p b e l l ,G . L e m ., J o s e p h,K .(1978), S ci ence200,1074.
2 1 . M o l d a y ,R . S . , Y e n , S . P . S . , a n d R e m b a u mA, . ( 1 9 1 7 ) , N a t u r e 2 6 8 1 4 3 1 ,
22 . Guesdon,L L., and Avrameas,S . (1977), Immunochemistry14, 443.
2 3. G ues don,J . L ., D a v i d , B., a n d L a p e y re ,J . (1977),A nn. Immunol .1281799.
M ., M a y e r,M . M ., a n d D e marest,C . (1946),J.l mmunol .52r325.
2 4. Heidelber ge r,
G. M. (1980),
, ., Wa x , M ., Ba u g hn,R . L., andW hi tesi des,
2 5. P ollak ,A . , B l u me n fe l dH
J. Am. Chem. Soc. 102, 6324.
2 6 . H o r o w t i z ,R . , a n d W h i t e s i d e sG, . M . ( 1 9 1 8 ) , J . A m . C h e m .S o c . 1 0 0 , 4 6 3 2 .
2 7. A r m s t r ong,J . M c D ., My e rs , D . V ., Ve rp oorte,J. A ., and E dsal l ,J.T. (1966),J.
Biol. Chem. 241, 5137.
28. Bergmeyer,H. J., Gawehn,K., and Grassl,M. (1974), rn Methodsof Enztmatic
Analysis(Bergmeyer,H. U., ed.), Verlag Chemie,Weinheim & AcademicPress,
New York-London,p. 473.
29. Pi.itter,J. (1974), in Methodsof EnzymaticAnalysis(Bergmeyer,H. U., ed.) Verlag
Chemie, Weinheim & Academic Press,New York-London, p. 685.
3 0 . S t eer sJ, r . , E ., C u a tre c a s aPs .,
, a n dP o l l a rd ,H . B . (1911),J.B i ol . C hem.246,196.
. oc.93,
t , . B . ( 1 9 7 1 ) , J . A m . C h e mS
3 1 . H o w e r ,J . F . , H e n k e n sR, . W . , a n d C h e s t n uD
6665.
3 2 . K r ebs ,H. A . (1 9 4 8 ),B i o c h e m .J . 4 3 ,5 2 5 .
33. Sundberg,L., and Porath,J. (1974\, J. Chromatography90, 87.
s, (1974),A nal . B i ochem.60, 149.
3 4 . M ar c h, S . C., Pa ri k h ,I., a n d C u a tre c a s aP.
35. A x en, R. , P o ra th ,J ., a n d Ern b a c k ,S . (1 9 6 7),N ature2l 4, 1302.