versatile partial purification of glucose-6-phosphate dehydrogenase by ion-exchange chromatography

2
Versatile Partial Purification of Glucose·6.Phosphate Dehydrogenase by lon.Exchange Chromatography Jom W. KeRer. Matthew K. WIN, and Rebe<:ca A. Reynokls U1 ....... sl1y 01 Alaska F,lr!WIks. Fair1Wlkl, IV< 99175 01 340 om. The molor ot.o,ptivity of NA DPH .t thit w.v._ I.ngth io 6.22 m),-1-\ em-I. A Varian Mod.1 6OOCl!io.uid ch", ... ""rop!l <quipped wi,h. 7·"L UV de"""" ... <11«1. A BioRad TSK <01""'" (1).15 X <JIll ",!b IU"'d <oIu.. n ..... <qwlib•• ted with depoood ro ",lot '" NADPIl NADP' + 2{NH. 1 D-Glurooe-'-Ioctone_ 6·phooph.te D-Glucooe--6- phooph... H'Jb· EnzjffltlAssay Th. >lod ,ubot .. te ooIution oonlainod 3ll "'lI D'IILl<OOoO-4-pboo- pIIoto, "'lI NADP' ('110 "",tonlOoI 0 .... ""i,:hed viol. 8i,:ma z.oo-3O,SJ. and ro ",L ro mM "iethanolami"" pH 1.4. A pw'ic dio_blo <o .... t .. (1·<m poOh lonrth) """toini", 2.00 ",L <Iht-o<k .ubo"'" ooIution .... O'luilibrated at :n ·c in tho,,,,,,,, .. tod ",U hoIde,. an a1iquo< of. ''''YOlO ooIution (5-00 "LI "' dd<d ar.d mi .... , and 'ho .boorbo"", .. 3010 nm ... N><OI'd<d fun<tion of ,iIM. A the ............. ",II Oolder io <lOt -.ry"IonI_ the ,oom tempor.. "", 10 """lan' .,...., tho period of the .. porime", and tho ",110 .r.d ,lOCk ooIution .re "'luilibn..... t mom ,".. po.. tu,". 'Tho .. in ... h -" (,omol min-') - ,lope (AU mL-' min-') X 0.1606 "mol AU-' X (2.00 + y) mL. .. ho.. y io '110 vnlwno of. onl.)'"'" addod '" tho uoay. 0". ""I, (u) of ><Ii";ly oqoalo 1 "fL>OI NADpH prodoc<d ",In-'. Aosa:y Apro<eu.-oontaini", .. mplo (o-w .. r. r.-loo "L) .... piPO'ted in'" a pwtir dia_h1o 'u...... to, 2_00 mL of dyo rear<n' (1$) ... added ",ith a repeati", d "pame" ar.d the .boo, bo:><.o .. b96 0 ... m",",<d. 'The aboorbo:><.o ol>ouId bo road 10 min. o<herwiot the proteln-<l,.. oompleo "'in procipitoto. Amounta of. pmtoin ""to determi nod ffODl a cu..... ,tandardiUld wi'h o-w ... of. bovi De oorum albumin. Yo.. , enzym. COD«ntrate (8i,: .... YU3.I) .... dWoJ ...... in burr.r (flO mM trio'lw>olamine pH 7.•• .j(I _L ..,..1..1. 28 ..... ml.-' io detenainod by protein uoay), ",n'rifure<! 10 min in. m",,-n_ and filtered ,hrouj:h. IIP!.C filter_ AU oth" <h.mi. ",,10 ""'" obtained from s;,m. Chtmical Co. En:.!ymo purifleotl<m continu... to be an important tool in bi""hemi'lry. Biologiaolly 101;'". prouino .an no", be duo_ ""IOfT"ph.d OIlth, prepor.live..,.,1e using column, paded with .moll ripd I"'rtid... "'h""" PO'" a,. large enough for penOl:rat.i"" of Wil proUin molewl ... These m.l<tial .,., ideal for ohrom.lOcuphy in the in.tru<;tiOTUlllobo'atory due to t.hei' .Ipid buffer robu.t bi<>eom- I"'libl. ,urf.<:eo, Iud tolerance fo' flow rou., Pharm.- oil" Fat! Prouin Liquid Ch,omato«r.p!ly (PPLC) «>lorn... "'ere the fi .. t of llIi. kind to be IntroduO<ld and they "moin Ih. best known. Other "'"leria!o that hov. limil., P'OPC'- \i ... luch u the Toyo"".r1 TSK. Wale.. Acull, fullnin Hy- dropor •• and nth......., 01.., .,".iloohl •. Thi... periment dlmOJUlI,.teo the ..... of tiro Toyopt,rl DEAE ion-ox.honge column »IIckinp for puriflC!ltion of ye.. t gluoooe·6·phoophate dehydrog.n_: an HPLC ool_ umn packed with to'pm partid.. (1. Z) .nd a .m.lI. gravily. J1owcolumn packed with partiel.. (3). Hirh recov- ery of enzymatic activity and purific.tion facto .. rangi", ft-om 15 to 30 are routinely obteined with th... columna Thi. experiment aloo f .. lur .. a comm.rcial """1"<'0 of the crude protein .nd a convenient and quantitaU"" .pec"o, photom." ic assay for d.hydrog.n... acti,;ty. Lindblom ha. described 0 .imnor exl>Orlment with FPLC oolumn. but did not report enzymo attivity m... urem.n'" (4). Enzyme pu,ifiC!ltion •• perimon.... re particulorly .ppro- pri.te fo' biochemiatry laboratory ainee they apply ..""r.l fundam.ntal te<hnlqu.. induding NADPH·linked .n.yme kinelic .soayo•• n<yme activity ... Iculatio.... protein con"",· troUon d... rmination •• nd cbromatographic manipulot.ion. 5i nC!l I970 only two .uch .. perim.n.... puri nC!ltion of bovine lactate d.hydrog...... uoing dyoligond chromatogr.phy Or AMP.oephar<- affinity chromotogrophy (6). ha"" been defCl'ibed in lhi. Journal. Moot current biochomiatry lob- oratory telto. with at I ... t One ••ception (7). indude on enzym. purincotion experim.nt. 1'ypieal •• perim.n... in· dude DF.AE·oeph.d•• purincolion. of in'..,ttaee (8) galoctooid (9) •• " ... rov. purific.tion of wh.OI rerm .cid phooph (10). ond another AMP'Mpho""", omnily chromotography of 10elate debydrog.na.. (11). Only the loete.. d.hyd'og...... purincation. (5. 6. I I) incorporate 0 rapid chromotography with high .nX)'m. recovery and a preci... pectrophotom.tric 'OMY. G luoooe·6· phoo pbale d.b.\-d togen... catolyuo con"".. ion of D-gluoooe-6-phoopll.ote to !>.glucono·.·lacton.·6-phoo- phate with accompanying conv.raiDn of NADP' 10 NADPH (eq I. R• ribooyl.ADP). In yo.. t thi. _ction oe,_ ... major """rce of reducing equival.nto fot v.riouo biooynth.t- ic patbwayo. Early ...... -ch on the enzyme b.. been re- viewed (lZ). Tb. yeolen.ymo ia a dim.. of r.o KDo .ub- unito; il it oel""'ive fo, tho NADP' .ubatra .. over NAD': lhe Michaeli. conotan'" fo' NADP' and glucooo·6·phoo- pha" .re 7.5 .nd 51 "M. respectively, Rocenl work h.. included .tudi.. on .ctiv. lite (/3) .nd tranoition .tote (14) .lrucIur The kinetic assay for glucooe--6-phoopn.te deny. dtogen "''''ureo lhe rate of fOT"""i"" of NADPH by monilo,i", the in...... in .t.orhance of th•• ooay mi>ture VOIutroo 88 !'IutrCet 3 Mar"" '991 255

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Versatile Partial Purification of Glucose·6.PhosphateDehydrogenase by lon.Exchange Chromatography

Jom W. KeRer. Matthew K. WIN, and Rebe<:ca A. ReynoklsU1.......sl1y 01 Alaska F,lr!WIks. Fair1Wlkl, IV< 99175

01 340 om. The molor ot.o,ptivity of NA DPH .t thit w.v._I.ngth io 6.22 m),-1-\ em-I.

~,

A Varian Mod.1 6OOCl!io.uid ch",...""rop!l <quipped wi,h. 7·"LUV de"""" ... <11«1. A BioRad TSK DEAE·~PW <01""'" (1).15 X7~ <JIll ",!b IU"'d <oIu..n ..... <qwlib••ted with depoood ro ",lot

'"NADPIl

NADP'

+ 2{NH.

1

D-Glurooe-'-Ioctone_6·phooph.te

D-Glucooe--6­phooph...

H'Jb·

EnzjffltlAssayTh. >lod ,ubot..te ooIution oonlainod 3ll "'lI D'IILl<OOoO-4-pboo­

pIIoto, ~ "'lI NADP' ('110 "",tonlOoI 0 ~"'I' ....""i,:hed viol. 8i,:maz.oo-3O,SJ. and ro ",L ro mM "iethanolami"" pH 1.4. A pw'icdio_blo <o....t.. (1·<m poOh lonrth) """toini", 2.00 ",L <Iht-o<k.ubo"'" ooIution .... O'luilibrated at :n ·c in • tho,,,,,,,,..tod ",UhoIde,. an a1iquo< of. ''''YOlO ooIution (5-00 "LI "' dd<d ar.dmi...., and 'ho .boorbo"", .. 3010 nm ... N><OI'd<d fun<tion of,iIM. A the............. ",II Oolder io <lOt -.ry"IonI_ the ,oomtempor.."", 10 """lan' .,...., tho period of the ..porime", and tho",110 .r.d ,lOCk ooIution .re "'luilibn.....t mom ,"..po..tu,". 'Tho.n.~mo ..tivi'~ in ...h -" (,omol min-') - ,lope (AU mL-'min-') X 0.1606 "mol AU-' X (2.00 +y) mL. ..ho.. y io '110 vnlwnoof. onl.)'"'" addod '" tho uoay. 0". ""I, (u) of ><Ii";ly oqoalo 1 "fL>OINADpH prodoc<d ",In-'.

""'~ Aosa:yApro<eu.-oontaini", ..mplo (o-w ..r. r.-loo "L) .... piPO'ted

in'" a pwtir dia_h1o 'u......to, 2_00 mL of dyo rear<n' (1$) ...added ",ith a repeati", d"pame" ar.d the .boo,bo:><.o .. b96 0 ...m",",<d. 'The aboorbo:><.o ol>ouId bo road ~illUn 10 min. o<herwiotthe proteln-<l,.. oompleo "'in procipitoto. Amounta of. pmtoin ""todetermi nod ffODl a cu..... ,tandardiUld wi'h o-w ... of. bovi De oorumalbumin.

.~-Yo.., enzym. COD«ntrate (8i,:.... YU3.I) .... dWoJ...... in burr.r

(flO mM trio'lw>olamine pH 7.•• .j(I~mL_L ..,..1..1. 28 ..... ml.-'io detenainod by protein uoay), ",n'rifure<! 10 min in. m",,-n_trif~.and filtered ,hrouj:h. o.~"m IIP!.C filter_ AU oth" <h.mi.",,10 ""'" obtained from s;,m. Chtmical Co.

En:.!ymo purifleotl<m continu... to be an important tool inbi""hemi'lry. Biologiaolly 101;'". prouino .an no", be duo_""IOfT"ph.d OIlth, prepor.live..,.,1e using column, padedwith .moll ripd I"'rtid... "'h""" PO'" a,. large enough forpenOl:rat.i"" of Wil proUin molewl... These m.l<tial• .,.,ideal for ohrom.lOcuphy in the in.tru<;tiOTUlllobo'atory dueto t.hei' .Ipid buffer ~uilibr'li<m. robu.t .trnctu~,bi<>eom­I"'libl. ,urf.<:eo, Iud tolerance fo' h~h flow rou., Pharm.­oil" Fat! Prouin Liquid Ch,omato«r.p!ly (PPLC) «>lorn..."'ere the fi ..t of llIi. kind to be IntroduO<ld and they "moinIh. best known. Other "'"leria!o that hov. limil., P'OPC'­\i... luch u the Toyo"".r1 TSK. Wale.. Acull, fullnin Hy­dropor•• and nth......., 01.., .,".iloohl•.

Thi...periment dlmOJUlI,.teo the ..... of tiro Toyopt,rlDEAE ion-ox.honge column »IIckinp for puriflC!ltion ofye..t gluoooe·6·phoophate dehydrog.n_: an HPLC ool_umn packed with to'pm partid.. (1. Z) .nd a .m.lI. gravily.J1owcolumn packed with ro-6O·~mpartiel.. (3). Hirh recov­ery of enzymatic activity and purific.tion facto.. rangi",ft-om 15 to 30 are routinely obteined with th... columnaThi. experiment aloo f..lur.. a comm.rcial """1"<'0 of thecrude protein .nd a convenient and quantitaU"" .pec"o,photom." ic assay for d.hydrog.n... acti,;ty. Lindblom ha.described 0 .imnor exl>Orlment with FPLC oolumn. but didnot report enzymo attivity m...urem.n'" (4).

Enzyme pu,ifiC!ltion ••perimon....re particulorly .ppro­pri.te fo' biochemiatry laboratory ainee they apply ..""r.lfundam.ntal te<hnlqu.. induding NADPH·linked .n.ymekinelic .soayo••n<yme activity ...Iculatio.... protein con"",·troUon d...rmination••nd cbromatographic manipulot.ion.5i nC!l I970 only two .uch ..perim.n.... puri nC!ltion of bovinelactate d.hydrog...... uoing dyoligond chromatogr.phy (~)

Or AMP.oephar<- affinity chromotogrophy (6). ha"" beendefCl'ibed in lhi. Journal. Moot current biochomiatry lob­oratory telto. with at I...t One ••ception (7). indude onenzym. purincotion experim.nt. 1'ypieal ••perim.n... in·dude DF.AE·oeph.d•• purincolion. of in'..,ttaee (8) and~.galoctooid (9) ••" ...rov. purific.tion of wh.OI rerm .cidphooph (10). ond another AMP'Mpho""", omnilychromotography of 10elate debydrog.na.. (11). Only theloete.. d.hyd'og...... purincation. (5. 6. I I) incorporate 0rapid chromotography with high .nX)'m. recovery and apreci...pectrophotom.tric 'OMY.

G luoooe·6· phoopbale d.b.\-d togen... catolyuo con""..ionof D-gluoooe-6-phoopll.ote to !>.glucono·.·lacton.·6-phoo­phate with accompanying conv.raiDn of NADP' 10 NADPH(eq I. R • ribooyl.ADP). In yo..t thi. _ction oe,_ ...major """rce of reducing equival.nto fot v.riouo biooynth.t­ic patbwayo. Early ......-ch on the enzyme b.. been re­viewed (lZ). Tb. yeolen.ymo ia a dim.. of r.o KDo .ub­unito; il it oel""'ive fo, tho NADP' .ubatra.. over NAD':lhe Michaeli. conotan'" fo' NADP' and glucooo·6·phoo­pha" .re 7.5 .nd 51 "M. respectively, Rocenl work h..included .tudi.. on .ctiv. lite (/3) .nd tranoition .tote (14).lrucIur The kinetic assay for glucooe--6-phoopn.te deny.dtogen "''''ureo lhe rate of fOT"""i"" of NADPH bymonilo,i", the in...... in .t.orhance of th••ooay mi>ture

VOIutroo 88 !'IutrCet 3 Mar"" '991 255

", - ' __ _--_____".1$)< • .1 __.<.0....... _..... I pH 1.4 U~ II ll.A().aL __ d ___ _;,,;.n.l" l.o.a. ...-' ..... 1 ioio<-_ p-...... _I"OlolN.a.50.lt ' ,_idl1 _ .......~e-...... _ " "'____u-LfIr'__rl' '_..,.4_..--.., .;.~-

SnlIIf t:JU,£ T", = 1 c::..um"1Oo00L porUoedD£AE.~"N C~ _ ....

=:~i...s=:::,,;,:~~=':Z:=dU ..I, .,..-'__....... by oUa<biJ>c. -.II _ 010.1....,ld. pIMti< wbi,. 10 "'"-.. .....utI 10 _ "'" ........!lJ"droolot;c hoood. A \.0-..1. _pIo at ,... ••....,. ..... _.101"""""' 1••' .... by ... '.0-",1• ....- of WIl., 0.0, 0.10,o.'lCl. &..30. 0..0, 0.... 0.60 M I. NoCI...pecti,...,. PToai<.- (l,0 ..1.)..... ....1_ .n<l _yod ~ p.,Wn ......_ octi';'y.

UloroluN CItedI, ......,__ .,........Mf'\C__I'...~..-.y,_ M I TJ,-, ,~"-'Y._K." I TJ" I__ ......~'''-II,J.'' __....~ _ .."'(:Iooo,D.J _.l_.._.T_....-..&J _,_.._,_.&,_'" .. t .... e .....-.

~-• _.&L.-......_ ...._. I ,_-'

_Y.... - ...-..--.... ..__ ...._-.-...._-."""'..-... e.-,T .........·• ,,__y..........__" _.&"''-''-'" I' ..__T__....-11 _....__ ' ,.............._ ,._........ .-._---... -. _..-._,-" ...-.._...-. ' ---......._-y--..-'"

rad"nl 0/ ;nc:teM.........iu... chIorida ......,.nl.nltiDn. AbDul_·haIf the proIein in the 28-111C _Ie. inchod.... tloaC ~phnapb·tade~_..... mitialIy bnund II>tloa ",.~ aluwoIlota .. .-o.llol 1UCl ......0... rnonicm .. purirIed l$·r.... ..,d I.....! abouI~0/ tloa ....,- .etrriI,.~ to tbt n. ",.~purifieatioeo fader • ponbably """ to tha IimiwiDea inl·s-ed by tloa Iarpr paniela 0/ dot DUET-'....lM.ai ucI tloa p padioool. peoood......",. Pf'Il'C'!d d-ribed r dot i ••

u.- or s.- ia Ioio tho:Ir<luP __ 01 tloa tloaDry '"""pnn;e. or icm....~ purifiratiDoo 0/ ptCIUi... (11). Inponi<\du. tloo .- 01. trial .........;... bWfor_~bWf.rine ..padl,. .hi,," ",ini..ili.. ..,.... iDnie 0U0tIJIh,.. tloo uieclwlolami. """,_I .llIlCharpd.~ to.....li '.. bindine or tloa deoUed p~in. it ill deoir.bl. that...ithe. burr•• """por>"nt inta.od with the.harp<! rMid_oCthtcolumn.

This .operim.nt is convent-nL The ctUdo ye..~ ..tract.nd ...y material••n ,eadily .v.il.obl, nd in...,. ...i"..,and tho prote'n ond «tlvity • ...,.. .... pteeiN yet tequi",UtU. P'pettinl. Th. HI'I,c .,.rotloa .equi,.... 30 min in·dud..... columa .....hinc and , ....otion: the DEAE-Toyu-peatl ch1<>aIatoerophy req" bout:.o min. It tho DUE.1'oyopeu1 c:o/umDo ... .-d. tho wt.ole ..peri....al. Ir>clud·i""~phy. proIein~ and ac:tivity _ """be ..mod ouc in _ S-<l " Iobontooy period if tloa bWf__ ...."tiooo,cd--. proIeUo _y~ in.--.m.:"Mol ....~ If _ HPLC imtnament"avnilnble, ttptooD. .....pIoo """ be ..... iII.-1ob periocI witIlItaldeaIO ott.rnotinJ btt_ tho HPLC and -rinI their....-.

n.e... ...._,.......tinno~bk (I) 1lled.- """ *'"dJtl"_t elution t whido • _ if tho HPLC padi-ttlt blo. (2) ",. __ <:aD be.' 0<0-paplord Ihe _ """'- ot 0dlIT....... pH (4) Of witlo •.... uduoioA "o'''..n '"'" oetivof~"- tho tlntehtonoo"'l"OPhy .... be _bt ........ntrntad with A",.....<OMrif""bIo "hnlilta.. (Centri<oll--30l. (3) Tho prowinfroniono .... be "hKbd rot pu.ity b:r SIllS pol)...yloalidopi.~ (4) Tho ....,..... c:ould be ..,,~ di·......ly r."." boll.,.. )IUIt. (5) 1'u.i1ied ,..., 111>COM~phoe­phoUl d.hydrocen_ lSiJ"'. G 1750 OJ" G 6378) c:ould bechronuo!OC.ophed on lho i(on·uch...... <:oIu",n to ....,po..pu.i (y .nd chroma!OC••phic proP" rti.. with tho "rud, mota·rial.

1: >

•z,•• ~•,,• >-• •,.. •..•. • •..

...... >,,• •

• '0 "r,...'I.~ No.•

u •!., ..

• \ i

•.. .. ,.of tU ':,

• .. : ;;,• . ,".• , .: : : .! •• '. . :J..• ••.• .J•• ..

IIt",llf W'Id~Tho lpecir", oclivity ot ,1.--4,p!>oophlte dehyd"'lla·

_ in the """'. )"UlIOl\.raet WM 0.20 u .....-', FoIIowi.., ion••~ HPLC, one fractiool h8d ••poociflC Ooni'rily ol6.7 "...-'•• 33·fold pwificoti<>o (r'P"!). n.- ....Ita o...pha.....WI. typ;eal du'omo~...."...purif_liooo_..........~_ to tho~ aliIl_-nheIoot..oipW-... pmif........ l1li&)' _. I.. IhiI~ tho-.. iIVocted ..to the HPLC -.Wiaed 11.2 ..... J _"udtho~1'no<tioII wiLblho~_

citk..,;.my """'....... """'IUS .. 01 prutein :ret maitIod1ft, of the loud ..,.yme 0IdMt,.. n- t1'llo 01 the owt.i"I""'""" _ ofi...........teeLcoodcI be ciiocardtd. '""...wi.ry iD tt 13-16..aboul 1.2$ ~.-..,.... KIiYit,.lD.- iQjeded ....pIe; l!Uo lU3' be -...d by.-••_,01 0/,,4lidecttitled inhibiliDJ comp)UDl/oo dun""~pby.",. .....UDEAET~ cdum.... eluted with.sup