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Partial purification of bacterial aspartase by starch electrophoresis
ARCHIVES
OF
Partial
BIOCHEMISTRY
AXI)
Purification JACK Prom
RIOPHYSICS
of Bacterial 8. M’ILKINSOK
the Deptrrtrrlext
93, 80-84 (1061)
Aspartase AiYD
by Starch
VIl1GINIA
Electrophoresis
I<. WILLIAP\lS
of Agl-icrtltuml Chemistry cud Riochcnzistry, Cxivmsity, Baton Houge, Louisianrc &xxi\-et1
Septcmbcr
Louisiana
State
21, 1960
The aspartasc of Bwlcri~n~ cutZuvur.is has been separated from fumnrnse, succinic dehydrogennse, aspsraginase, and sdcnosine deaminnse by clectrophoresis on granular starch. The chnracteristivs of the purified enzyme ha\-c been compared with those of the crude preparation. Electrophorctic purification resulted in a l&fold increase in specific activity and a demonstrable stimulation of activity on addition of certain metal ions. The preparation of fumarasc-free aspartasc permittted a direct thermodgnamic analysis of thr deamination reaction. IKTBODUCTIOK
Ccl1 debris was rcmo\-ed by centrifugntion at 18,000 X q for 30 min. at 4°C. To each milliliter of clear supermitant ccl1 extract, 2 ml. of 0.2”% protamine sulfate solution (pH 7.0) was added. The solulion was allowed to stand at 4°C. for 30 min. and was then crnlrifuged at 18,000 X g for 30 min. at the same temperature. The supernstant solution was c*:rrefully decanted into chilled flasks and lyophilixed until dry. The crude dry preparation was thtn siorcd at -40°C. until needed.
In the attempt to resolve conflicting opinion regarding the activation of aspartase by various organic compounds and metal ions, a number of techniques have been used to obtain enzyme extracts of enhanced activity and purity. Salt fractionation, protaminc fractionation, and ion-exchange chromatography have all been utilized to this end (l-5). This report describes the preparation of a highly active, fumarasefree aspartase by the technique of zone electrophoresis on starch.
ASPARTASE
METHODS CULTVRAL
METHODS
Unc(el-i/rn~ cntloz;ciG (apparently identical with N. cadnur~ris Ai.T.C.C. 0760) was grown aerobically for 40 hr. at 30°C. on a medium consisting of 1% yeast extract, 1% tryptone, and 0.5% monopotassium phosphate. CRUDE
ENZYME
ASSAY
E’or routine aspnrtasc determinations the following system was [Ised: 1.0 ml. of 0.10 M phosphate buffrr (pII 7.01, 0.2 ml. of 0.10 M potassium Laspartate (pII 7.0), 0.2 ml. of enzyme preparation, 0.4 ml. of water and,/or additions, and 0.2 ml. of 25% trichloroncrtio acid added at the beginning fol blanks and nt the cncl for all other tubes. The incubation trmpernturc~ JV:ISortlinarily 38°C. and the incu1)ation pcriotl. 15 min. Precil)itatctl protein was rcmo\-et1 t,y wnlrifuginp, and the nmmonin Ilrotluc~~l w:~s dclcrminrcl by nfwlcrization of a 1 .O-ml. alicluot.
PREPARATIONS OTHER
The 40-hr. cultmea were harvested in a rcfrigerand the cells were washed once atcd centrifuge. with 0.08 df potassium chloride. Twenty milliliters of ~ashc~l ~11s was snqxmded in an equal volume of cold 0.1 111 phosphate buffer, pH 7.0: 2 ml. of 0.01 ;lI sodium formate (l>H 7.0) was :rclcled; and the resulting suspension ww treated for 30 min. in :I g-kc. Rnqihron sonic oscillator chilled with cirwl:iting iw mater.
ASSAYS
Protein was dotcrmincd tither by the sulfosalicylic acid method of Weston (6) or by the biurct mptlro(l. Fumaranr w-ns deic>rmincd by the method of Rwkrr (T), succinic dehydrogennse was determinccl by the method of Hcpaskc (S), and adenosine dc:tminasc wad tlctcrmincd by the method of Williams ant1 McIntyre (9). hsparaginasc was detrrmincd by adding 0.5 ml. of chute from n 0.5~cm. 80
BACTERIAL
ASPARTASE
XI
t.h:m is u~udly otkained for fresh sonic lgsatcs (RI. 1200) nncl reflects some loss of :Ictivity on lyophilization. The optimum tcmlwrature was found to bc 3%39°C.; the optiilnllll t)H in 0.05 ;I1 phosphate tdfcr, 7.2; und t.licb -tlichacli5 constant, 1.7 X IO “. I)ifY(~l~(~iiti:tl centrifugation of il huffrw(t solution of criidc cnzymc in a Spinco rvfrigc~r:ltcd ultraccntrifuge for 2 hr. showed no ~~tliilic~nt~~tic,n of t,tlc cnzymc :d. 114,400 x g. ‘1’11(,(~rii(l(~ prq):lr:Ltions showed no stimuI:ltion or inllihition from tlic following c~o~~~t~rintl~in 0.001 dl final concentration: ~lllcow, flYl~‘tON’! :ictcninc sulfate, adenylics :Icicl. glut:~thionc, vysteinc liydrochlolrdc, s:tnt IGnc,. :md inohinc. No dimulation was ol)t:rincvl on atl(lition of ljiotin. The addition of :I iiuint~1r of t)ivnlent nlctal salts in final lld:~r corlc,c,tlt,i.:ltion of 0.0001 produced no ~tiinul:rt~ion ; tlo\vc,vc,r, clialyzctl crude (anzytiio wv:l:: signifirantly ~t,iiiiul:ttcd by 0.0001 .\I (‘0 + ‘-. Alg i - , AIn+-+ , and %n 1-+. ‘1’11t~ Illo\Tlll(‘llt~ of t11cxellzymc 011 t1w darctt t)l:itct :LS :L funrtion of pH was dctcxrlrlinvcl :it pH’* 5.8, 6.3, 6.7, and 7.3. For tllc 1%hr. t~vriotl, tllcb c~lcc*trophoretic moljility t,on.artl t,tlc anotlc increased with pH. 7’htw rc~r~lt~ :lrc’ xtlown in Fig. 1. Extrapol:ition of tllc>c cl:lt:l to zero mobility gave an isoc~loctric point of 4.8 which agrees \vith tl1:1t rc~pol’tcvl t’s’ Ellfolk (1).
Since one of the major objectives of this work was to prepare a fumarase-free aspartase, the position of fumarasc was detcrmined along with those of certain other enzymes of interest in relationship to aspartase : succinic dchydrogcnase, aeparaginase, and adenosine deaminase. Fumarasc and nspartase were cleanly separated from each other at all of the four pH values given above. The only pH, however, which separated aspartnse from all of the cnzymcs identified was pH 7.3; results of t,his experiment arc presented in Fig. 2. The purified enzyme displayed the following characteristics: optimum temperature, 38-39°C.; optimum p1-I in 0.05 M phosphate buffer, 7.2 ; Ptlichaelis constant, 1.7 X lo-“; specific activity of the best preparation, 8200. Since the dialyzed crude preparation was stimulatrd by t,hc addition of certain bivalcnt metal salts, the sensitivity of the tlectrophoretically purified cnzymc was tested. Results 1)rescntcd in Table I sho~ t,hat, like the diulyzcd crude enzyme, the purified enzyme responds to 0.0001 M Co++, Mgf-+, Bin++, and Zn++, the most effective metal activator being &In+ +. The successful preparation of fulnaraseA 6
ASPARAGINASE FUMARASE
C
SUCCINIC OEHYDROGENASE
D ADENOSINE
DEAMINASE
E ASPARTASE pH AB
C
7.3 E
! CENTIMETERS
frclc :rsl)artaso l)ormittcd a direct cletormination of tlicl apparent ccluilibrium constant ant1 :I stutlp of the tllc~rmodgii:lInics of the sY5tcw. L41)l):li’cnt c~cluilibriuni ctonstants &YX~ clctcrlninetl at 29” and 39°C. and A.N” wah c~valllatctl t)y the vanY Hoff cquat,ion. Froiri tlic apparclnt equilibrium constant, at 39”C’.. IF” was c*alculated. AS” was c~alculatc~tt for 39°C. from tlic: familiar rcIatioiisliil): AF” = 11-I” - Y&S”. As an additional check, tllc apparent equilibrium constants at tlic two tcmpc~ratures wcr(’ also clctc~rirkic~cl starting with a potassiul~i fuiliaratc~ and ai~iinoniuni sulfate system. 31-I” for the amination reaction calculated from tllcw constants w-as -15,500 cal. per molt. Thcsc results arc shown in Table II. X sclcond cl~eck \vas matlc by (Ailnating AII” of tlc:nnination from bond cncrgics as gircn by Forgudon ( 11 1, This calculat~ion gives :I val~ie of + 19,000 CA./ riiolc for the cntlialpy Yh:qc.
MOVED
FIG. 2. Position of aspartase and other enzymes on starch plate nt pH 7.3 after 18 hours of electropl~oreuis.
A ~oiqarkon of the cliaractcktics of tllcl ~udc and purified enzyme rcrenls that tllc princil,al changes effected by elcctrol~llorc~,+isn-cre c~nhancetl activity and sensitivity to stiniulat~ion by metal ions, part~ic~ulnrly AIn* + I :md the removal of fumaraw. .I specific activity of Cu. 8200 for tllc most actirc purified preparation compares favoraljly with activities rcportctl by Scott (4) for preparations purified on ion-exchange resins, but is lower t,han the specific (rcl. 10,300 ) obtained by Smith nctivitics (3 I for ll~r plot:uninc-frnctionatccl ext,racts.
BSCTERIAL
SSPSRTASE
84
WILKINSON
is attributahlc to two fact,ors: (a) small analytical differences result in larger differenccs in the condtxnts nncl (,b) wcn in dilutcl solutions cquilibriuln is approached slowly and not complctcly attained in incubaiion periods as long as 18 hr. dttempts to d&ermine equilibriuni const,ants in solutions of lo- or IOO-fold higher conrcnt~rnt#ion w;cre nbandoncd bccnusc the purified cnzymc extracts were not sufficiently eonccntrated to permit equilibrium to he appronchcd in 3 rcnsonahle timc. For lwtll forward and ~CWTSCdir&ions a tempernturc rise of 10” approximately douhlcd the cquilihrium constant. Tlic resulting agrccnlent in 311” is quite snt’isfactory. An analysis of the tllcriiiotlynarnics of thv dcamination of aspartic acid rcvcals t,llat, the reaction is both cndothernlic and rnclcrgonir. Ikq)ite tlic large increnw in entropy gained in the conversion of aspartat,c t,o fumxratc> and ammonium ion, the standard frccvncrgy change shows tllct rca&ion to bc nonq)ont~ancow in the dcamination dircvtion Iwcause of the large incrrasc in bond cncrgics as rcrcalcd by t,he enthnll)y cl1ange.
AND
WILLIAMS
OF
Partial
BIOCHEMISTRY
AXI)
Purification JACK Prom
RIOPHYSICS
of Bacterial 8. M’ILKINSOK
the Deptrrtrrlext
93, 80-84 (1061)
Aspartase AiYD
by Starch
VIl1GINIA
Electrophoresis
I<. WILLIAP\lS
of Agl-icrtltuml Chemistry cud Riochcnzistry, Cxivmsity, Baton Houge, Louisianrc &xxi\-et1
Septcmbcr
Louisiana
State
21, 1960
The aspartasc of Bwlcri~n~ cutZuvur.is has been separated from fumnrnse, succinic dehydrogennse, aspsraginase, and sdcnosine deaminnse by clectrophoresis on granular starch. The chnracteristivs of the purified enzyme ha\-c been compared with those of the crude preparation. Electrophorctic purification resulted in a l&fold increase in specific activity and a demonstrable stimulation of activity on addition of certain metal ions. The preparation of fumarasc-free aspartasc permittted a direct thermodgnamic analysis of thr deamination reaction. IKTBODUCTIOK
Ccl1 debris was rcmo\-ed by centrifugntion at 18,000 X q for 30 min. at 4°C. To each milliliter of clear supermitant ccl1 extract, 2 ml. of 0.2”% protamine sulfate solution (pH 7.0) was added. The solulion was allowed to stand at 4°C. for 30 min. and was then crnlrifuged at 18,000 X g for 30 min. at the same temperature. The supernstant solution was c*:rrefully decanted into chilled flasks and lyophilixed until dry. The crude dry preparation was thtn siorcd at -40°C. until needed.
In the attempt to resolve conflicting opinion regarding the activation of aspartase by various organic compounds and metal ions, a number of techniques have been used to obtain enzyme extracts of enhanced activity and purity. Salt fractionation, protaminc fractionation, and ion-exchange chromatography have all been utilized to this end (l-5). This report describes the preparation of a highly active, fumarasefree aspartase by the technique of zone electrophoresis on starch.
ASPARTASE
METHODS CULTVRAL
METHODS
Unc(el-i/rn~ cntloz;ciG (apparently identical with N. cadnur~ris Ai.T.C.C. 0760) was grown aerobically for 40 hr. at 30°C. on a medium consisting of 1% yeast extract, 1% tryptone, and 0.5% monopotassium phosphate. CRUDE
ENZYME
ASSAY
E’or routine aspnrtasc determinations the following system was [Ised: 1.0 ml. of 0.10 M phosphate buffrr (pII 7.01, 0.2 ml. of 0.10 M potassium Laspartate (pII 7.0), 0.2 ml. of enzyme preparation, 0.4 ml. of water and,/or additions, and 0.2 ml. of 25% trichloroncrtio acid added at the beginning fol blanks and nt the cncl for all other tubes. The incubation trmpernturc~ JV:ISortlinarily 38°C. and the incu1)ation pcriotl. 15 min. Precil)itatctl protein was rcmo\-et1 t,y wnlrifuginp, and the nmmonin Ilrotluc~~l w:~s dclcrminrcl by nfwlcrization of a 1 .O-ml. alicluot.
PREPARATIONS OTHER
The 40-hr. cultmea were harvested in a rcfrigerand the cells were washed once atcd centrifuge. with 0.08 df potassium chloride. Twenty milliliters of ~ashc~l ~11s was snqxmded in an equal volume of cold 0.1 111 phosphate buffer, pH 7.0: 2 ml. of 0.01 ;lI sodium formate (l>H 7.0) was :rclcled; and the resulting suspension ww treated for 30 min. in :I g-kc. Rnqihron sonic oscillator chilled with cirwl:iting iw mater.
ASSAYS
Protein was dotcrmincd tither by the sulfosalicylic acid method of Weston (6) or by the biurct mptlro(l. Fumaranr w-ns deic>rmincd by the method of Rwkrr (T), succinic dehydrogennse was determinccl by the method of Hcpaskc (S), and adenosine dc:tminasc wad tlctcrmincd by the method of Williams ant1 McIntyre (9). hsparaginasc was detrrmincd by adding 0.5 ml. of chute from n 0.5~cm. 80
BACTERIAL
ASPARTASE
XI
t.h:m is u~udly otkained for fresh sonic lgsatcs (RI. 1200) nncl reflects some loss of :Ictivity on lyophilization. The optimum tcmlwrature was found to bc 3%39°C.; the optiilnllll t)H in 0.05 ;I1 phosphate tdfcr, 7.2; und t.licb -tlichacli5 constant, 1.7 X IO “. I)ifY(~l~(~iiti:tl centrifugation of il huffrw(t solution of criidc cnzymc in a Spinco rvfrigc~r:ltcd ultraccntrifuge for 2 hr. showed no ~~tliilic~nt~~tic,n of t,tlc cnzymc :d. 114,400 x g. ‘1’11(,(~rii(l(~ prq):lr:Ltions showed no stimuI:ltion or inllihition from tlic following c~o~~~t~rintl~in 0.001 dl final concentration: ~lllcow, flYl~‘tON’! :ictcninc sulfate, adenylics :Icicl. glut:~thionc, vysteinc liydrochlolrdc, s:tnt IGnc,. :md inohinc. No dimulation was ol)t:rincvl on atl(lition of ljiotin. The addition of :I iiuint~1r of t)ivnlent nlctal salts in final lld:~r corlc,c,tlt,i.:ltion of 0.0001 produced no ~tiinul:rt~ion ; tlo\vc,vc,r, clialyzctl crude (anzytiio wv:l:: signifirantly ~t,iiiiul:ttcd by 0.0001 .\I (‘0 + ‘-. Alg i - , AIn+-+ , and %n 1-+. ‘1’11t~ Illo\Tlll(‘llt~ of t11cxellzymc 011 t1w darctt t)l:itct :LS :L funrtion of pH was dctcxrlrlinvcl :it pH’* 5.8, 6.3, 6.7, and 7.3. For tllc 1%hr. t~vriotl, tllcb c~lcc*trophoretic moljility t,on.artl t,tlc anotlc increased with pH. 7’htw rc~r~lt~ :lrc’ xtlown in Fig. 1. Extrapol:ition of tllc>c cl:lt:l to zero mobility gave an isoc~loctric point of 4.8 which agrees \vith tl1:1t rc~pol’tcvl t’s’ Ellfolk (1).
Since one of the major objectives of this work was to prepare a fumarase-free aspartase, the position of fumarasc was detcrmined along with those of certain other enzymes of interest in relationship to aspartase : succinic dchydrogcnase, aeparaginase, and adenosine deaminase. Fumarasc and nspartase were cleanly separated from each other at all of the four pH values given above. The only pH, however, which separated aspartnse from all of the cnzymcs identified was pH 7.3; results of t,his experiment arc presented in Fig. 2. The purified enzyme displayed the following characteristics: optimum temperature, 38-39°C.; optimum p1-I in 0.05 M phosphate buffer, 7.2 ; Ptlichaelis constant, 1.7 X lo-“; specific activity of the best preparation, 8200. Since the dialyzed crude preparation was stimulatrd by t,hc addition of certain bivalcnt metal salts, the sensitivity of the tlectrophoretically purified cnzymc was tested. Results 1)rescntcd in Table I sho~ t,hat, like the diulyzcd crude enzyme, the purified enzyme responds to 0.0001 M Co++, Mgf-+, Bin++, and Zn++, the most effective metal activator being &In+ +. The successful preparation of fulnaraseA 6
ASPARAGINASE FUMARASE
C
SUCCINIC OEHYDROGENASE
D ADENOSINE
DEAMINASE
E ASPARTASE pH AB
C
7.3 E
! CENTIMETERS
frclc :rsl)artaso l)ormittcd a direct cletormination of tlicl apparent ccluilibrium constant ant1 :I stutlp of the tllc~rmodgii:lInics of the sY5tcw. L41)l):li’cnt c~cluilibriuni ctonstants &YX~ clctcrlninetl at 29” and 39°C. and A.N” wah c~valllatctl t)y the vanY Hoff cquat,ion. Froiri tlic apparclnt equilibrium constant, at 39”C’.. IF” was c*alculated. AS” was c~alculatc~tt for 39°C. from tlic: familiar rcIatioiisliil): AF” = 11-I” - Y&S”. As an additional check, tllc apparent equilibrium constants at tlic two tcmpc~ratures wcr(’ also clctc~rirkic~cl starting with a potassiul~i fuiliaratc~ and ai~iinoniuni sulfate system. 31-I” for the amination reaction calculated from tllcw constants w-as -15,500 cal. per molt. Thcsc results arc shown in Table II. X sclcond cl~eck \vas matlc by (Ailnating AII” of tlc:nnination from bond cncrgics as gircn by Forgudon ( 11 1, This calculat~ion gives :I val~ie of + 19,000 CA./ riiolc for the cntlialpy Yh:qc.
MOVED
FIG. 2. Position of aspartase and other enzymes on starch plate nt pH 7.3 after 18 hours of electropl~oreuis.
A ~oiqarkon of the cliaractcktics of tllcl ~udc and purified enzyme rcrenls that tllc princil,al changes effected by elcctrol~llorc~,+isn-cre c~nhancetl activity and sensitivity to stiniulat~ion by metal ions, part~ic~ulnrly AIn* + I :md the removal of fumaraw. .I specific activity of Cu. 8200 for tllc most actirc purified preparation compares favoraljly with activities rcportctl by Scott (4) for preparations purified on ion-exchange resins, but is lower t,han the specific (rcl. 10,300 ) obtained by Smith nctivitics (3 I for ll~r plot:uninc-frnctionatccl ext,racts.
BSCTERIAL
SSPSRTASE
84
WILKINSON
is attributahlc to two fact,ors: (a) small analytical differences result in larger differenccs in the condtxnts nncl (,b) wcn in dilutcl solutions cquilibriuln is approached slowly and not complctcly attained in incubaiion periods as long as 18 hr. dttempts to d&ermine equilibriuni const,ants in solutions of lo- or IOO-fold higher conrcnt~rnt#ion w;cre nbandoncd bccnusc the purified cnzymc extracts were not sufficiently eonccntrated to permit equilibrium to he appronchcd in 3 rcnsonahle timc. For lwtll forward and ~CWTSCdir&ions a tempernturc rise of 10” approximately douhlcd the cquilihrium constant. Tlic resulting agrccnlent in 311” is quite snt’isfactory. An analysis of the tllcriiiotlynarnics of thv dcamination of aspartic acid rcvcals t,llat, the reaction is both cndothernlic and rnclcrgonir. Ikq)ite tlic large increnw in entropy gained in the conversion of aspartat,c t,o fumxratc> and ammonium ion, the standard frccvncrgy change shows tllct rca&ion to bc nonq)ont~ancow in the dcamination dircvtion Iwcause of the large incrrasc in bond cncrgics as rcrcalcd by t,he enthnll)y cl1ange.
AND
WILLIAMS