CHROM.

12,045

HIGH-SPEED STERIC EXCLUSION MERS’

C

TIMOTHY

WEEIR

CHROMATOGRAPHY

OF BIOPOLY-

and SETH R ABBOTT

Vkrim Imtrunmt Croup, 2700 MitckeillDrive, Wdmt

Creek, Carif. 94598 (U.S.A.)

SUMMARY

Biopolymerseparationswere studied on Micropak TSK type SW columns, which contain an aqueous compatible steric exclusion support. Columns of two differentpore sizes, designated2CNOSWand 3ClUQSW, were compared for separation of proteinsand nucleicacids covering a molecularweightrange of 13,500 to 340,000 and the effect of molecular shape and denaturationupon elution volume was investigated_ Use of high-speed steric exclusion chromatography as a prefractionation step prior to ion-exchange chromatography iy biopolymer purificationschemes is discussed.

INTRODUmON

The essentialcharacteristicsof stericexclusionsupportsfor use in biopolymer separationsare: (!) A predominantlyhydrophilic character. Hydrophobic interactions can irreversibly adsorb and denatureproteins. (2) A pore size sutiicientto allow permeation by macromolecules.Most protein work requires200-1500 A pore size supports.Permeationby virusesand nucleicacids can requirelarger pore sizes (see Table I). As a general rule, the support pore size should be severaltimes that of the major axis of the macromolecule. (3) An inert surface, to avoid ion exchange adsorption interactions with the macromolecules. (4) Mechanical strength. (5) Geometric insensitivityto changes in mobile phase pH and ionic strength. (6) Stabilityover a widepH range. Classical steric exclusion separationsof biopolymers have utilized relatively soft, hydrophilicgels such as the syntheticpolyacrylamides(e-g_, Bio-Gel P) and the

* Efitar’s note: A similar study was submitted on April 3rd, 1979: S. Rofcushika,T. Ohkawa 176 (1979) 456-461.

andB. Batano,J. Chumtugr.,

CT.~S.RABmxT

454 TABLE I MOJXCULAR

WEIGHT

AND

700 235 150 57 43

FExinogen *pGl0bUh

Albumin Eiemoglohin a23enc.e Jones protein

LQoproteins

300 185

a-lipoprotein /??-Lipoprotfs .?z4rz=rm l%Pb

b=W= RiiSlUdeue

Vii7LWS Bilshy stunt virus To-bacco mosaic virus

MAJOR AXE

84

60 -

250 2700

DIMENSIONS

OF BIOPOLYMERS

34u.ooo 160.000 69.000 68Jmo 35mo 2oo.Qal 1.3oomo 35.oQo 14_?00 12.700 7.6aMxJo 40.000.m

-

po.ysaccharidedextrans(e.g., Sephadex)and agaroses (e.g., Sepharose, Bio-Gel A). Polystyrene-divinylbenzene resins have ken avoided due to irreversibleadsorption and denaturationefficts of the hydrophobicaromaticmatrixand to the relativelybw pore sizesof thesesupports(< 50 A). The polyacrylamideand polysaccharidegels are hydrophilicand can be prepared in large pore sizes_Polyacrylamideand dextransupports have been used for separationof biopolymersof mol_wt_up to 106. Agarose has been used to separate viruss and nucleicacids of mol.wt_up to 1.5 - 108_The extremelyhighexclusionlimits of the agzrose supports@able II) has allowedseparationof subcelhdarparticles’_ Unfortunately, these gels are characterizedby a relativelylow compressive strengthand must be operatedat low pressuresand flow velocities.Thus, equilib‘mtion separationand washingtimes are long, and sample throughputis low. Cross-linked a,oarose,the most mechanic4ly stable of these gels, is operatedat flow velocitiesof 0.002-0.02 cmjsec. This is an order of magnitudeslowerthan typical flow velocities employed in high-speedliquid chromatography(HSLC). The need for a rigid, microparticulatesupportfor biopolymerHSLC has long ‘been evident- Controlled-pore glasses (CPGs) developed by Hallerr in 1965 and available3in pore sizesfrom 40 to I500 A and particlediametersof 5-10 pm, have been usedfor stericexclusionof biopolymersof mol.wt_up to 106_However,both controlled pore gW and silicagel s*urfaces are characterizedby high densitiesof highly polar, weakly acidic silanol (Si-OH) _~oups. Silanols can behave as cation exchange sites and resultin adsorptionand denaturationof biopolymers4m5, especiallyproteinswith isoefectricpoiits above pH 7.5. This problem has seriouslyinhibitedthe use of uncoat& CPG or silicagel for biopolymerHSLC. Coating of CPG and silica gel supports with polyethyleneglycol (PEG) of mol.wt, M 104-105 significantlyreducesthese adsorption e&ct+‘. Although PEGcoated CPG has been used successfullyin the separationof high-molecular-weight

TABLE

CXIROMATOGRAPHY

EXCLUSION

S’lERlC

455

OF BfQPOI.YMJZRS

H

CHARAClERISECS BEOJ?OLYMEECS

Bio-Gel P

OF SUPPORTS FOR STERIC Ex(=LUSEON CHROMATOGRAPHY

(sross-linked poIyacrykmide

OF

2-9

Peptides Proteins Proteins

Sephadex noss-linked dextran

2-12

Peptides, proteins

Eia-GeIA

4-13

Agarose

LiChrospher Sphericalsilica glass 2-S

37-75 75-150

-

5-10

4OthrlI 15ciO

10

100&i-u

5aO,aM-150,000,cm Dextfans 5O,OOO-lSO,CXIO,OOO Dextrails 8000-l,aoo,oclo 8O,ooO-s,ooO,aoo

Dextrans PolyStyrenes

I-SK-SW

Spberi~A, rigid gel, 3-S hydrophilic smionaty pbi?se

10 13

-

20,000and 150,000 @mm

DeXbXllS Dearaas

viruses6,the coating processis a reversibleone and the PEG phaseelutesundercontinuous use’. In addition,adsorptionef$kctsare stillobservedfor certainprofei&. The coating “bleed” problem can be avoided by covalently bonding an inert hydrophilicphase onto CPG or silicagel supports.Regnierand co-workersbonded a “dial” phase (Fig. 1) onto both CPGBand micropaticulatesilicagel*, obtainingHSLC exclusionbonded phaseswith negligibleadsorptionof biopolymers.These phasesare often called ‘cglycoplnses”.Regnierhas also bonded polydextranonto silica through an alkylamine intermediary.However, enzyme recoveries from the dextran-silica supportsweresignificazttly lowerthaa those obtainedfrom a diol-silica support.

Silica

Gel

Fig. i. ‘Dial”

H&C

bonded exclusion phase for bbpolymers.

Tayot et al.” succeededin preparing“bleed-less” ion exchange sz~pportsfor biopolymer HSLC by impregnatingsilica gel wit&cross-linkedDEAE-dextran polymers. This techniquemay also be promisingfor HSLC exclusionof biopolymers,incosporating the separationcharacteristicsof a polysaccbaridewith the me&an&l stre2gtbof a micropticulate silicagel. Another typeof HSLC exclusionsupport,TSK-SW gels,was recentlydeveloped by Toyo-Soda (Tokyo) Japan.The support -a rigid gel fsilicagel) whose surface is

coveredwithhydroxyl groups- has a pH range of 3-g. Part&Ye size is I0~r.n and at a typicalflow velocity of M 0.05 cm/=, eEciermiesof 2O,OOO+O,OQO plates per meter are obtained.Toyo-Soda has demonstratedquantitativerecoveryof proteinsand of enzyme activity from the suppor& The exact structureof this supporthas not been published,Biopolymer separations were obtained on pre-packedVarh M?icroPak TSK-2ooO and 300 SW columns (8 mm x 30 cm). ExP!sRmENFAL

Chromatography was performed on a Varii Model 5020 gradient HPIC systemequippedwitha Varichromvariabiewavelengthabsorbancedetector. The followingproteinand transferRNA standardswereobtained from Sigma (St Louis, MO., U.S.A.) and solutions were preparedin the mobile phase buffer: cytochromec from horse heart, Type VI; human fibrinogen,fraction I; pepsinfrom hog stomach mucosa; ovalbumin; bovine a-globulins, Cohn fractionIV; lactic dehydrogenasefrom rabbitmuscle,Type II; bovine y-globulins, Cohn fractionII; tyrosine specifictransferRNA and N-formyhuethioninespecifictransferRNA from Escherichia culi. Rabbit livertransferRNA, preparedby the method of Anandaraj and Roe= (extractionwith phenol in pH 4.5 acetate buffer followed by chromatography on DEAE-celhrlose) was kindly provided by Dr. R. D. K&ma. Human serum was filteredthrougha O&urn tiherprior to use. RESULTS

AND DISCUSSION

Biopolymerseparationswere studiedon MicroPak TSK 2000 SW and MicroPak TSK 3000 SW cohunns, having exclusionlimitsfor dextranstandardsof 20,000 and I50,OOOrespectively-Exclusionvolume is dependenton hydrodynamicvolume of a solute ratherthan simplemolecularweight.Exclusionlimitswill thus be greaterfor proteins,whichare not as linearas dextmnsand thus penetratethe supportporesmore readily. Fig. 2 shows calibrationplots of a seriesof protein standardsOQ2000 and 3000 SW colts. Exclusion limits for the proteins are seen to be M UlO,OOO and > 350,GQOrespectively. Fig. 3 is a calibrationplot of the same seriesof proteinstandardson a 3QOO SW column with hemoglobin added. Although hemoglobin and albumin have similar moiecularweights(68,000 vs. 69,000) the hemoglobin (57 x 34 A) is more globular than albumin(I 50 x 38 A) and thuspermeatesthe poresto a greaterextent.The larger hemoglobinelutionvolume is characteristic of a more linear(albumin-like)proteinof mol.wt. 28,OQO.For accurate molecular-weightdeterminationof proteins by steric exchrsionchromatography(SEC), one can denaturethe proteinsby additionof guanidiniumhydrochloride or sodiumdodecylsuEate(SDS)L’to the mobile phase. The value of a properchoice of pore size for biopoiymerseparationsis shown in the proteinmixtureseparationsof Fig. 4. The separationsare obtainedin I5 min, at a flow velocity of w 0.04 cm/set. Note that the y-glob&ins (moLwt. M MO,OOO) whicheluteas one peek at the 2ooO SW exclusion volume are split into three peaks on the -3MO SW column. The 3ooOSW column is optimumfor separationof most proteins of m0Lw-c 4O,M,m. The 2009 SW column is optimum for most proteins of : m0l.d K 40,OQO.

2606 10.

4

t s

I d

1 7 -

SW

3000

t * va&su(cd,

1 s

svd

& *I3

I .1

Fig. 2. Calibration curves for MicroRik TSK Type SW gels. Conditions: solvent, 0.067 M KE&FQ~ + 0.1 MKCI + 6 - lo-’ M sodium azide, pH 6.8; flow-rate; 1.0 ml~min; temperature,30”; column dimensions, 30 cm x 7.5 mm. P, Fibrinogen (340,OQO);0 y-globulin (160,000); A, LDH (10!3,OW); A, ovalbumin (43,5GO); 8, pepsin (35,ooO); 0, cytocbrome c (13,500). !lF_

101,

?

e 4 lot,

lax

F% 3. Caliisation curve for l%xoPak TSK-3000. Conditions: solvent, 0.067 MKH,POI 0.1 MN&l; tY.ava& 1.0 mt/min; ahrElu dirimsiw 7.5 mm x 6Oan

(pEi 6.8) f

493

Elution

Volume

i 2 Fi

4. Sp2ration

i Et&ion

(ml)

\-zL-). i s Voiume

10 (ml)

12

of pmteki stadard mixtures. Gxditions as in Fii 2

The 3000 SW column provides the best separation of components in human serum, as shown ti Fig. 5. The first peak probably contains high-mokcular-weight immunoglobulins partially excluded from the gel; the next peak should include y-globulins (160,000 &tons). The Iarge peak at V, M 7 ml matchesthe elution volume of albumin (69,000 d&tons). The last two peaks to elute are unknowns. The hydrodynamicvolume of a protein is a fi.mction of its conformation. Pepsin, a globular protein with unusual stabiity h_ acidic solutions, is denatured at pR values above 5. Consequently, it behaved as a linear molecule under the conditions

2000

SW

3oao O-065

A28w T

1

0.005

4

2

EIution

6

8

Volume

30R

A280

I

12

IL 2

(ml) Pepsin 3000

SW

Stored

4 Efution

24

hours

6 8 10 Volume (ml). at

12

4% il

SW

moo SW

o.aas A

28

t

2

4 El&on

8

10

Voiume

(ml)

6

12

Fig. 5. Separation of human serum. Conditions as in Fig 2_ FL 6. &par&km

of pepsin and degradation pmducts. Conditions as in Fig.2.

used in Fig. 2 (ehent pH 6.8). Upon incubationin the cold, a new peak appeared, correspondingto a specieswith a mo1.w~ of 35,000 daltons (Fig. 6), suggestig either that the denaturedpepsinwas degradedinto fragmentsor underwentpztial renaturation to a more globularconformation. The intiuenceof hydrodynamicvolume upon permeationis also evidentin the elution of transfer RNAs (Fig. 7). These are relativelysmall ribonucleicacids with mokcular weightsin the raqe of 25,000-30,000 dakons, yet they elute from the Zoo0 SW column in the same volume as would a globularproteinwith a mol.wL of GO,0 da&on%This reelectsthe relativelylinear tRNA structure.Note that the two species (tyrosine specific and N-formyhnethionyl speci6c) are partially resolved on the 3OQO SW cohunn. Comparisonof the elutionvolumeswiththose of dextranpolymers” indicatesthat the tRNAs elute as would linear dextranswith mol.wts. of 23,CMMand

2000

O-01

SW

A28*I

0.01

ri

2 Ehth~

4

6

Volume

lSg_ 7_ Separation of tRNA

i

8

Elation

(ml) standank

Gxditions

as in FE

i

6

Volume

k

[ml)

2_

30,OtXIdabons. Separationof a tRNA extract from rabbit liver (Fig. 8) reveaJ.sa major peak correspondingto materialin the range of 24,000 dahons (as determined from a dextrancalibration-curve),another component of about 30,900 daltons and smaller amcunts of material of greater than 100,aoO daltons (possibly ribosomal RNA)_ OrgauicsolventcompatibleStEC is often used in tandemwitha reversed-phase colum& for resolutionof complexmixtures.The StEC acts as a rapid pre-fmctionation and clean-upstep. Aqueous compatib!eStEC on ffie TSK-SW cohrmnshas great potentialfor couplingto a subsequentlarge-poreion-exchangecolumn. One can thus capitalizeon the high speed and etkiency of the StEC cohuxmsalong with the fact that the same mobile phase requiredfor the ion exchangeseparation(typicallya Tris bufferwithNaCl added)can be used for the StEC separation.The futureuse of such a techrnqueshould be enhancedby the ability of microprocessor-controlkdchromatographs to automate such a coupled column-“heartcuttings scheme. Work in our Iaboratory is aimed at developing such HSLC ionexchange columns for coupling to an StEC pre-fractionation.Fig. 9 shows the separationof tRNA standardson an experimental_MicroPakanion exchangebondedphasecohmm; here,the tyrosyltRNA standardwhichelutesas a singIepeak from the stericexclusion column is separatedinto at least three XIV-absorbingspecies. Application of such cohmms for analysis of tRNA preparationsis quite promising,as sug~+ed by the rapid separationof the rabbit livertRNA extractshown in Fig. IO. Use of StEC prefractionationshouldgreatlyenhancethe separatingpowerof the techniqueaud extend ion exchangecoIumnlife by reducingits exposureto extraneousmatrixmaterial_

461

0.006

2 4 6 Elrrtion Volume Fig. 8. Separation

B.

8

(ml)

of rabbit liver tRNA extract. Chditions as in Fii

Tyrosinra

2

tRNA

I

I

‘

2

4 Elution

1

1

6

t

6

IO

Tim.3

L

12

(mica)

Fig_ 9. Separation of transfer RNA on h%icroF& M?AX-SO_ Conditions: solvent A, 0.1 M Tris, pH 6.8; sotventl3.0.1 M Tris. pH as f ZMNaQ;G~~~t:O”~B5mln,ehen(l-450/~Bin~5min: &on* 1.0 ral/min; v 309

462

B

;

6 Elmion

8

10 Cnc

12

14

1s

18

20

jminuter)

Fig 10. Sepamion of rabbit liver tRNA extract on MicroFak MAX-S@. except flow-late is 05 ml~min_

as inF~9

I2EFER.ENcEs 1 2 3 4 5 6 7 8 9 10 11

J. Borovass& P. Ha& asd J. Duchoii, J. Chmnufogr.. W_ Halls, Nature {Lo&m), 206 (1%5) 693.

i34 (1977) 230.

Pierce. Rockford, Ill_, 1978.p. 274. T. Miiti and A. Mizutas& An&. Biochem, 83 (1971) 216. C. W. H&t, A. Shelokov, E 4. Rosenthal and J. M. G&more, J. Cfmmrogr., 56 (1971)362. T. Darling, J. Albert, P. Russell, D. M. Albert and T. W. Reid, J. Chmmuogr.. 131(19x 383. I. Shechter, ti. Sic&m_, 58 (1974) 30. E Regnier and R. Noel, J_ Ckromat~_ Sci., 14 (1976) 316. S. H. Chang, IC_ hi. Goading and F. E. Regnier, J. Chrummgr~. 125 (1976) 10X F. E. Regkr, US_ Pat_ 3983.299 (1976). J. L. Tayot, hf_ Tzxdy, P. G&c!, R Plan arrd M. Roumiantzefi, in R. Epton (Editor), Ciziom~e graphy of Syxtketii ad Bio.bg&d Polymers. VoL 2, Wiley. New York, 1978.p. ‘34. 12 M. P. I_ S. Asmdaraj zud B. k Roe, Ehchetifry, 14 (1975) 506% 13 R J_ Slagrove and M. J. Frenkel, J. Chrommgr., 132 (1977) 399. 14 R. C- Collins and W. HaUer, kz. Biuckem., 54 (1973) 47. 15 SW Type Tdmicai Data, Toyo-Soda Mfg. Co. Ltd., Tokyo, 1978. 16 !Z Johnson, R Gloor and R E. Majors, J. Ch.rommgr_, 149 (1978) 571. Pierce Chemical Company Gztabg,