Purification of some tricarboxylic acid cycle enzymes from beef heart using affinity elution chromatography

A\/\[

YTI(

AI

114.

I3IOCli~MISTR)’

Purification

of Some

19-27

Tricarboxylic

Using

Ikpartment

Applying enzymes Conditions

(1981)

Affinity

Acid Elution

Cycle

R. DAVIES

o/ Biochmlistr~‘.

La Trobr

University.

Received

October

the

principles

substrate(s). After of these preparations

of affinity

AND

elution

K.

ROBERT

Bumiowa. 27.

step. and activities

chromatography.

in some comparable

Purification of enzymes using affinity elution procedures (I 4) has been shown to be useful for enzymes which have polyanionic substrates. and which are sufficiently basic to adsorb to cation exchangers. Scopes ( 1.2) purified most of the rabbit muscle glycolytic enzymes by this method, successively eluting enzymes from CM-cellulose by stepwise incorporation of substrates and ligands specific for them. Many other single purification procedures have made use of the same principles. Several of the enzymes of the tricarboxylic acid cycle in mammalian mitochondria have properties which appear to make affinity elution procedures suitable for their purification. All have substrates possessing at least two negative charges per molecule, and reported isoelectric points are in many cases over 6.0. It was decided to see how useful affinity elution procedures could be for purifying these enzymes both individually, and for multienLyme purifications from the one extract. Beef heart was chosen as an appropriate tissue: this was homogenized by a procedure designed to disrupt mitochondria. so that all cytoplasmic and mitochondrial matrix proteins were obtained in the one extract. This was preferable to attempting to isolate intact mitochondria first, which results in very poor recoveries. The present paper describes simple affinity elution meth-

Beef

Heart

SCOPES

C’icruria

30X3. .4mtralia

1980 eight

with, the tricarboxylic acid cycle have for adsorbing each enrymc to CM-cellulose.

a gel filtration had specific

from

Chromatography

JEFFREY

of. or associated were found

Enzymes

cases use to the

been

different

mitochondrial

purified from beef he;rrt. and eluting each with its

of an affinity adsorbent. most highest previously reported.

ods that have been found to work successfully for isolating citrate synthetsse (EC 4.1.3.7), aconitase (EC 4.2.1.3). isocitrate dehydrogenase (NADP’) (EC I. I I .42). succinyl-CoA synthetase (GDP-forming) (EC 6.2. I .4), nucleotide diphosphate kinase (EC 2.7.4.6), fumarase (EC 4.2. I .7), malate dehydrogenase (EC 1.1.1.37), and aspartate aminotransferase (EC 2.6. I. 1). The three dehydrogenases for pyruvate. a-ketoglutarate and succinate, being multienzyme complexes of high molecular weigh!. were not amenable to the ion-exchange technique. MATERIALS

AND METHODS

The tricarboxylic acid cycle intermediates cis-aconitic acid, Dl.-isocitric acid (trisodium salt), a-oxoglutaric acid, succinic acid, and oxaloacetic acid were obtained from Sigma Chemical Company, as were ITP. IDP, GTP. dTDP. NADP+, NADH, acctyl-CoA, aspartic acid, 5,5’-dithiobis( 2-nitrobenzoic acid), phenylmethylsulfonyl fluoride. 3’,5’ADP-agarose. and the zwitterionic buffers Mes,’ Mops, Tes, and Tricine. APP and ’ Abbreviations cthancsulfonic

7(N-morpholino)used: Mea, 3-(N-morpholino)proacid; Mops. pancsulfonic acid: Tes, ~-tris(hydruxymeth~l)methyl-~aminoethanesulfonic acid; Tricine. Wtris(hydroxymethyl)methylglycine; SDS. sodium dodecyl fate.

sul-

20

DAVIES

AND

ADP were from Boehringer and Soehne, Mannheim, West Germany. Sephadex G-25, Sephacryl S-200. 2’,5’-ADP-Sepharose and AMP-Sepharose were obtained from Pharmacia, Uppsala, Sweden. CM-cellulose was the microgranular CM-52 form from Whatman, Maidstone, Kent, England. Pyruvate kinase and lactate dehydrogenase were purified by methods described previously ( I ). Beef heart obtained fresh from the local abbatoir was chopped into pieces and homogenized in 2.5 vol of 30 mM K-phosphate buffer, pH 7.0, 0.1% Triton X-100 and I mM phenylmethylsulfonyl fluoride, in a l-liter capacity Waring blender. The Triton was included to ensure disruption of mitochondrial membranes. The homogenate was centrifuged at 5OOOg for 40 min and the insoluble residue was discarded. The pH of the extract was adjusted to 7.0; unused extract could be frozen for several weeks at -30°C. Protein concentration was determined at 205 nm using an extinction coefficient for I mg/ml protein of 31 (5). Enzyme activities were measured by methods based on those described elsewhere, but using common buffers wherever possible. With the exception of the fumarase and succinyl-CoA synthetase assays, the buffers used were 30 mM triethanolamine -chloride, pH 7.5, or 30 mM Tris-chloride, pH 8.0. each containing 3 mM magnesium acetate, 50 mM KCI, 0.2 mM EDTA, and 0.2 mg/ml bovine serum albumin. Citrate synthetase was measured as described (6) using pH 8.0 buffer, 0.1 mM acetyl-CoA, 1 mM oxaloacetate, and 0. I mM 5,5’-dithiobis(2-nitrobenzoic acid). Aconitase was assayed at pH 7.5 by following the disappearance of cis-aconitate (2 mM) at 240 nm in a l-mm pathlength cuvette (7). Pretreatment of aconitase samples was carried out by incubation for 30 min at 37°C with 5 mM dithiothreitol and I mM ferrous ammonium sulfate (8). Isocitrate dehydrogenase was measured at pH 7.5, using 0.2 mM DL-isocitrate and 0.1 mM NADP’. Succinyl-CoA synthetase (GDP

SCOPES

forming) was assayed as described by Cha and Parks (9). A 50 mM Tris succinate buffer contained I mM MgCIZ, IO rnM KCI, 0.5 mM phosphoenolpyruvate, 0. I mM GTP, 0. I mM NADH, and 5 units ml -’ each of pyruvate kinase and lactate dehydrogenase. The mixture was monitored for GTPase activity before adding 0.1 mM CoA to initiate succinyl-CoA synthetase activity. Nucleotide diphosphokinase was assayed at pH 7.5 using 0.5 mM phosphoenolpyruvate, 0.1 mM NADH, 0.1 mM ATP, and 5 units ml ’ each of pyruvate kinase and lactate dehydrogenase. The mixture was monitored for ATPase activity before adding 0.1 rnM dTDP to initiate the NDP kinase activity. Fumarase was assayed BS described by Kanarek et al. ( 10) using 50 mM K-phosphate buffer, and 50 mM L-malate, pH 7.3. Malate dehydrogenase was assayed at pH 7.5 using 0.15 mM NADH and 0.15 mM oxaloacetic acid. Aspartate aminotransferase was also assayed at pH 7.5. using 1 unit ml ’ malate dehydrogenase as coupling enzyme, with 0. I mM NADH, 0.1 mM pyridoxal phosphate. 5 mM Na aspartate, and 2 mM Na CY-OXOglutarate. Enzymic activities are expressed in units of micromole per minute at 25°C. The operating conditions for affinity elution follow those described previously ( I ). Buffers consisted of IO rnM KOH and 0.1 mM EDTA, adjusted to the appropriate pH using Mes (pH 6.0 -6.5), Mops (pH 6.87.2), Tes (pH 7.5), or Tricine (pH 8.0). CMcellulose columns were poured such that the dimensions were height = 2 3 X diameter, using total amounts as appropriate for the protein applied (I cm’/20-30 mg protein). Flow rates were close to 20 cm h ~I. and the columns were run at room temperature (20~ 25°C). Gel filtration was carried out using Sephacryl S-200. applying the protein sample at a concentration of 20 mg ml ’ to 3 column with a volume at least 30 times the volume of the sample. For removing ammonium sulfate. samples were applied to a

TRICARBOXYLIC

ACID

CYC‘t.E

ENZYhlE

AFFINITY

L’LCzTlON

before starting

21

column of !
a little pH 6.5 buffer elution procedure.

the

The crude extract was tirst fractionated with ammonium sulfate in order to remove much unwanted material. With the exception of fumarase, most of each enzyme was precipitated between 55 and 80% saturation of ammoniuln sulfate. This precipitate wtis dissolved in 3 small amount of K-Mes buffer, pH 6.5. and desalted on a column of Seph:tdcx (i-25 preequilibrated with K-Mes buffer. For fumarase, tin ammonium sulfate cut between Ji5 and 60% saturation was more appropriate. On occasions. ;1 different buffer was used: in particular it was necessary to use ;I pH 6.0 buffer for citrate synthetase and fumarase, as these enrymes did not adsorb to CM-cellulose fully at pH 6.5. On the other hand, some of the enzymes were rather unstable if applied at pH 6.0, so it was not desirable to use such ;1 low pH unless really necessary. After the enzyme had adsorbed to the CM-cellulose, the pH was increased until ;I suitable value was reached for affinity elution ( I ). Table I lists the pH’s. and substrates found to be most successful for adsorbing and eluting each enzyme. It is clear that the tricarboxylic acid cycle enzymes have common substrates, resulting in the likelihood of coelution of more than one enrymc. This can be minimized by judicious choice of substrate. for instance by using NADP’ rather than isocitrate for eluting ixocitrate dchydrogenase. Brief descriptions of the overall tnethods used for each enLyme are given below. In each ctlsc (except fumarase) the starting material referred to is the desalted 55 80% ;Immonium sulfate fraction (obtained from I liter extract) in K-Ma buffer. pH 6.5. at ;L protrin concentration of IO 15 mg ml +. With the exception of the method for citrate synthettlsr, this fraction W;IS applied to a column of CM-cellulose, and washed in with

(‘irrarr .vynthrtase. Citrate synthetase has been purified b\; an affinity adsorption procedure using ATP-Sepharose ( I I). It was found that for optimal elution from this adsorbent both oxaloacetate and CoA were needed. In the present procedure, Co.4 did not improve elution compared with oxaloacetate alone. and by itself U’X ineffective, ;1s were 0.2 mM acetyl-CoA or 0.5 mhl citrate. It is probable that CoA was necessary for elution from ATP-Sephtlrose to cause displacement of its binding site on the enLyme from the adsorbent. On an ion-exchange column there is less reason to expect an interaction between adsorbent and the CoA-binding site on the enryrne. It has been suggested that oxaloacetatr binds to the free enzyme and causes a conformational change ( 13, I3 ). Certainly use of oxaloacetate alone in the present procedure is sufficient and economical. To avoid overloading the CM-cellulose with the substantial amount of proteins. especially myoglobin. that are present in the starting material, the following procedure has been used successf’ully. ( I ) CM-cellulose preequilibrated at pH 6.5 was added batchwise to the desalted fraction until the bulk of the tnyoglobin colour had been adsorbed. ‘The CM-cellulose was filtered off, and the pH of the solution lowered to 6.0 using I M Mes. (2) The sample was applied to a column of CM-cellulose at pH 6.0. then the column was washed with at least three column volumes of pH 6.5 buffer. until little protein was being eluted from the column. (3) Citrate synthetase was eluted by inclusion of 0.5 rnkl freshly dissolved oxaloacetate in the pH 6.5 buffer. A representation of the subunit composition of the eluted protein on polyacrylamide SDS gel electrophoresis is shown in Fig. I.

22

DAVIES

AND

SCOPES

TABLE AI I I~ITY

EI LTIOY

PLIKI~IC‘.~TION pH

En7yme Citrate Aconitasc dchy-

FOR Tt+E Eic;tls

EN~YMFS

D~,XRIRW

for

Substrates

adsorption

synthetasc

lsocitrate

SC.HEMES

1

Elution

ubed

for

elution

pH

6.0

6.5

0.5

mM

Oxaioacetate

6.5

1.5

0.5

mxl

Aconitate

6.5

1.5

0.2

mM NADP’

6.5

6.8

0.2

mM

drogenase (NADP) Succinyl-CoA synthetasc (GDP

t 0.2 + 0.1

forming)

Nucleotide phosphate

diki-

6.5

Succinate mu mM

GTP CoA

0.1 mM ATP

6.8

+ 0.1

mM

ADP

nase Fumarase Malate

6.0 dehydro-

6.8

6.0-7.0

8.0

gcnase Aspartate

amino-

The column

+20

mu

was brought

to the elution

pH

by a prcelution

FIG. I. Diagrammatic representation amide-dodecyl sulfate gels of purified rified cn7ymes. I. Citrate synthetase.

of polyacryland partially pu2, Aconitase, af-

finity eluted fraction. 3, lsocitrate dehydrogenase, affinity cluted fraction. 4, NDP kinase, affinity eluted fraction. 5. NDP kinase, final purified ervyme. 6, Fumarase. after tw’o affinity elution steps. 7, malate dehydrogenase. affinity eluted fraction. 8. Malate dehydrogenase. after gel filtration. 9, Aspartate amino transferase. affinity IO. Aspartate

amino

transferase,

after

mM

Malatc

0.2

mu

NADH

KCI

8.0

The enzyme had few impurities and has had specific activities of up to 180 units mg-’ (cf. 16 I for pig heart enzyme ( 14).)

eluted fraction. gel filtration.

mM

6.0-7.0

transfcrase Foote.

+I0

0.2

I! mM Aspartate t 2 mM a-OXOglutaratc

KCI

wash

with

buffer

before

applying

the substrates.

Aconitase. Aconitase has previously been purified to a specific activity of 10.2 units rng-’ ( 15). Its activity is highly unstable, but can be recovered by a preincubation procedure involving thiols and Fe’+ ions. A simple procedure for obtaining enzyme of specific activity of over IO units mg -’ is described below. After applying the sample to the CM-cellulose column, 2- 3 vol of K-Tes buffer, pH 7.5, were passed through until the bulk of the myoglobin had been eluted; 0.5 column volumes of pH 7.5 buffer containing 0. I IIIM NADP+ was then applied, followed by a further similar amount of pH 7.5 buffer without NADP’: this was to elute any isocitrate dehydrogenase that may have survived the treatment in the absence of glycerol (see below). One column volume of pH 7.5 buffer containing 0.5 mM cis-aconitate was then applied, and the aconitase was eluted by its substrate.

TRICARBOXYLIC

ACID

CYCLE

Most of the impurities that coeluted could be removed ,by gel filtration. The best specific activity obtained has been 18 units mg-‘. The gel electrophoretic diagram of the affinity-eluted enzyme is shown in Fig. 1; subunit size was estimated to be 8 1,000. Isocitrate dehydrogenase. The method for this enzyme is exactly as described above for aconitase, except that it is necessary to include at least 20% glycerol in the column buffers to stabilize the enzyme. Even so, the recovery depends on speed, and is lower in larger-scale preparations and in the multienzyme preparation described later, when it spends more time adsorbed to the column. Reducing the temperature did not improve the recovery, and although higher concentrations of glycerol were beneficial, the final product was then more difficult to concentrate before storage. or gel filtration to remove impurities. For the best purity the NADP’-eluted enzyme was concentrated by ultrafiltration, and applied to a column of Sephacryl S-200 in a K-Tes buffer (as above) containing 20% glycerol. Specific activities of up to 42 units mg-’ have been obtained (cf. 39 ( 16)) which showed a single band on electrophoresis at M, 44,000. Figure 1 illustrates the purity before gel filtration. Succinyl-Co.4 synthetase. This is a complex enzyme reaction, with six substrates/ products, all of which are negatively charged and so might be used for affinity elution. Not all possible combinations have been tried, and although succinyl-CoA was not available, it was presumably synthesized on the enzyme by the combination of substrates that was found to be most effective, i.e., succinate + CoA -t- GTP. However, the eluted enzyme was far from pure, due to the combination of substrates and the many possibilities for coelution. The specific activity of the eluted enzyme was 6 units mg-‘; a 17fold improvement over the applied sample, but far less than the value of 110 (at 30°C) reported by Cha ( 17). Gel filtration on Sephacryl S-200 took the value to 25; affinity adsorption chromatography using 3’5’-ADP-

ENZYME

AFFINITY

ELUTION

23

Sepharose did not greatly improve on this. The best preparations showed several subunit bands on gel electrophoresis, indicating heterogeneity. The procedure used was as follows. ( 1) After applying the sample to the CMcellulose, two column volumes of K-Mops, pH 6.8, were passed through. (2) The enzyme was eluted with K-Mops, pH 6.8, buffer containing 0.1 tn~ CoA, 0.2 mM GTP, and 0.2 mM succinate. (3) After ultrafiltration, the enzyme was applied to a column of Sephacryl S-200 in K-Mops buffer, and the eluted activity was collected as final preparation, specific activity 25 units mg-‘. Nucleoside diphosphate kinase. This enzyme exists in a variety of isozymic forms; moreover the bulk of the activity in heart tissue is cytoplasmic. It is reported that the mitochondrial enzyme is more acid than the cytoplasmic (18) and so the preparation described here is almost certainly cytoplasmically derived. NDP kinase has low specificity for nucleotides, consequently almost any nucleotide di- or triphosphate should be able to elute the enzyme from CM-cellulose, and it is a matter of choosing nucleotides which cause the least coelution of other enzymes. ITP, ATP, or ADP all eluted most of the adsorbed NDP kinase; the method chosen used only 0.1 mM of each of ATP and ADP. A further sevenfold purification was obtained by using gel filtration and affinity adsorption chromatography on AMPSepharose, and the method is summarized below. (I ) After applying the sample to the CMcellulose column two column volumes of pH 6.8 buffer were passed through, followed by one column volume of the same buffer containing 0.1 mM each of ADP and ATP. The enzyme was eluted by the nucleotide-containing buffer. (2) The fraction was concentrated by ultrafiltration and subjected to gel filtration on Sephacryl S-200 in the same K-Mops buffer.

24

DAVIES

AND

(3) The enzyme was adsorbed on a IOcm’ column of AMP-Sepharose. and eluted with 0.1 mM ADP + 0. I mM ATP. The purified enzyme showed a single band on polyacrylamide gel electrophoresis corresponding to a molecular weight of 16,500 (Fig. I ). This value agrees with the value reported (181, the native enzyme being a hexamer. The specific activity of the final preparation was 1060 units mg-‘; it is difficult to compare this with other preparations because of the wide variety of assay methods used. This specific activity was obtained using only 0. I mM of each substrate, ATP and dTDP. At 2 mM of each substrate the specitic activity of our purified NDP kinase was 4300 units mg-‘. Thus NDP kinase is ;i very active enzyme, its value of k,,,/K,,, (K,,, for ATP) approached IO8 M- ’ s- ‘, in the class of diffusion-controlled enzyme reaction rates [ 191. Fumarasr. Fumarase has a lower solubility in ammonium sulfate than the other enzymes described here, and is best purified from the fraction precipitating between 45 and 60% saturation. The sample was applied, at pH 6.0, and after passing two column volumes of pH 6.8 buffer through the column, fumarase could be eluted by inclusion of 0.2 mM malate in the buffer. Although this was effective, recovery was poor (30%) probably due to dissociation and inactivation in the low-ionic-strength conditions (20). A second affinity elution (after gel filtration) resulted in a nearly pure enzyme of specific activity 300 units mg-‘, but overall recovery was low. It is probable that preparation of this enzyme would benefit by an affinity elution procedure, if buffer conditions can be found that protect the enzyme’s activity. The purified enzyme showed only one band on polyacrylamide -SDS gel electrophoresis, at 3 molecular weight of 49,000 in confirmation of earlier reports (IO) (Fig. I ). Malate drhydrogenase. Mitochondrial malate dehydrogenase is a basic enzyme and so is readily separated from the more acidic

SCOPES

cytoplasmic form (which does not adsorb to CM-cellulose even at pH 5.5). As with lactate dehydrogenase, a substantial conformational change on binding NADH causes a much reduced affinity for the adsorbent (4) with rapid elution if the pH is right. Above pH 8 the enzyme becomes unstable. At pH 8 it is readily eluted if its adsorption to the column is weakened by inclusion of B little salt. The affinity eluted enzyme contains glyceraldehyde phosphate dehydrogenase which is coeluted by NADH, but this can be removed by gel filtration. The procedure used was as follows. ( 1) After applying the sample to the CMcellulose column, 2 -3 vol of K-Tricine buffer, pH 8.0, containing IO mM KCI, was passed through until no more protein was eluted. (2) The enzyme was eluted with 0.2 mM NADH in K-Tricine-KC1 buffer. (3) The eluted enzyme was concentrated by ultrafiltration, and further purified by gel filtration on Sephacryl S-200. The purified enzyme had a specific activity close to 1000 units mg- ‘. and a subunit molecular weight of 32,000 (Fig. 1). Aspartate aminotransferase. This enzyme is closely linked with the tricarboxylic acid cycle, being involved in the malate/aspartate shuttle for transferring reducing equivalents across the mitochondrial membrane. Like malate dehydrogenase, mitochondrial aspartate amino transferase is very basic, whereas the cytoplasmic isoenzyme is acidic. An affinity elution procedure has been developed which is not entirely specific, since the substrate concentrations needed increase the ionic strength enough to coelute other proteins (4). However, after gel filtration the preparation compares favorably with other purified preparations of this enzyme. The procedure used was 3s follows. ( 1) After applying the sample to the CMcellulose column, two to three column volumes of K-Tricine buffer, pH 8.0. containing 20 mM KCI, were passed through until no more protein was eluted.

TRICARBOXYLIC

ACID

CYCLE

(2) The ‘enzyme was eluted by inclusion of 2 rllM Nn-aspartate + I mM Na-ru-oxoglutarate in the K-Tricine KCI buffer. ( 3) After concentration by ultratiltration, the enzyme was further purified by gel filtration on :Sephacryl S-200. The purified enlyme had a specific activity of 230 units mg ‘. The purity of the affinity etuted fraction is illustrated in Fig. I,

Since so many of the methods described above are similar. on most occasions more than one of the enzymes was purified from the extract. The following describes a procedure which. results in six of these enzymes being obtained from the one column. The fraction was desalted into pH 6.5 buffer containing 20% glycerol v/v. At this pH citrate sy,qthetase is only partly adsorbed on CM-cellulose: to prevent it from adsorbing at all. 0.5 mM oxaloacetate was included in the sample when running on to the column. The nonadsorbed fraction was retained for citrate synthetase purification later. Two column volumes of pH 6.8 buffer (+209# glycerol) were passed through, and then one column volunte of this buffer containing 0.2 171~ADP. This eluted NDP kinase with little or no succinyl-CoA synthetase. The buffer next contain4 0.2 rnbf GTP, 0.2 rnra succinate. and 0.1 mM CoA, which eluted the succinyl-CoA synthetase uncontaminated by NDP kinuse. However, both enzymes obtained as described were contaminated by myoglobin which by now could be seen emerging from the column. The buffer was next changed to K-Tes. pH 7.5 (+L!O”tj v/v glycerol). and all the myoglobin ws quickly moved out of the column. Gel electrophoresis indicated this to be ;I nearly pure preparation of myoglobin; 0.5 column volumes of 0.1 mhl NADP’ in the pH 7.5 buffer caused elution of isocitrate dehydrogenase, after which it was convenient to omit the glycerol from the buffers

ENZYME

AFFINI’I-Y

ELI’TION

25

since it was not needed for the stability of the subsequent enzymes. Aconitase was eluted using 0.5 column volumes of pH 7.5 buffer containing 0.5 mw c.is-aconitate, then the column WBS equilibrated to pH X.0, using 2 3 vol of K-Tricine IO rnM KCI buffer. After eluting malate dehydrogenase with 0.2 mM NADH. the KC1 content of the buffer was increased to 20 KIM before adding the substrates for aspartate aminotransferase. After elution of this entyme there was little protein left on the column. although a pink band of cytochrome 1’ could usually be observed. A diagram of the elution schetne i< shown in Fig. 2. The nonadsorbed fraction containing citrate synthetase and oxsloacetate was adjusted to pH 6.0 using I ,V Mes. However, citrate synthetase would not adsorb to CMcellulose at this pH in the presence of oxaloacetate, and denatured on the column a~ lower pH’s. To overcome this problem. 0.5 mM NADH was added to the solution. Cytoplasmic malate dehydrogenssc present rapidly reduced the osaloacetate to malate, which does not bind to citrate s!,nthetase. The enzyme then adsorbed to another CMcellulose column at pH 6.0. and ~;IS eluted 3s described above, at pH 6.5 with oxaloacetate. A further benefit was that fumarase, which would normally adsorb at pH 6.0.

26

DAVIES

AND

passed through the column in the presence of the malate. Under the conditions for elution of citrate synthetase, some fumarase may otherwise be eluted also. All of the enzymes (except citrate synthetase) affinity eluted in this multienzyme procedure required subsequent treatments for further purification, as described previously. Details of the specific activities, recoveries, and estimated purities of the enzymes from the multienzyme scheme are given in Table 2. DISCUSSION The experiments described here confirm the principle and usefulness of ionic affinity elution whereby an enzyme may be eluted specifically by a charged ligand from an ion exchanger of the same charge as the ligand. The procedure is largely restricted to using a cation exchanger, since few ligands (other than metal ions) are positively charged. It also follows that, for adsorption to a cation exchanger, the enzyme must have an isoelectric point at least as high as 6.0 (unless

TABLE DFTAILS

or TtiE STAGES

lh

PURIFICATION

Specific

activities,

SCOPES

it is particularly stable at low pH’s and the ligand will bind to it at low pH). These conditions apply for many mitochondrial matrix enzymes, and we have been able to use affinity elution from CM-cellulose as a step in the purification of eight different enzymes. Because many of the ligands used are common to several enzymes present, the eluted samples have usually not been pure, but have required at least a gel filtration step to approach the specific activities of the best preparations reported previously. Although affinity adsorbents such as AMP-Sepharose, ATP-Sepharose, 2’,S’-ADP-Sepharose, and 3’,5’-ADP-Sepharose can be used successfully for purifying these enzymes ( l4), their adsorbent capacity is limited, so it is preferable to use them at a late stage in a purification procedure. Affinity elution from CM-cellulose can be used earlier, since ion exchangers, apart from being much cheaper, have high capacities which more than makes up for their lack of specificity. The principles of affinity elution have been examined (4,21) and it is effective both because of charge cancellation on the enzyme when binding

2 IN TIIF

units

MLILTIEULYMF

mg ’ --~

EnLyme

in order

of elution

N DP kinase Succinyl-CoA synthetase lsocitrate dehydrogenase Aconitase Malate dehydrogenase Aspartate aminotransferase Citrate synthetase

Crude extract

Desalted ammonium sulfate fraction

1.2 0.13 0.22 0.17 28 I .3 0.85

4.7 0.37 0.75 0.64 85 4.2 3.2

SciiFMi

Affinity eluted fraction I50 6.0 ‘5 12 680 I50 180

Recover) artcr affinity elution (%I 50 40 30 55 80” 70” 50

Estimated ptlrity after affinity clution (%) I4 7 60 60 75 65 95

Note. Percentage recovery after affinity elution relates to the activity present in the crude extract. The degree of purity at this stage was estimated from gel electrophoretic patterns. and from the specifc activity compared with the maximum reported values. “ Recovery assumes that 40%# (malate dehydrogenase) and 50% (aspartate aminotransferaxe) of the activity in the crude extract was mitochondrial enzyme.

TRICARBOXYLIC

ACID

0’CL.E

substrate and because of conformational changes. In many cases the enzyme goes from fully adsorbed to fully desorbed by introducing small concentrations of its substrate, without any change in pH or ionic strength. Moreover it is possible to isolate large quantil.ies of enzyme on quite modest sixd columns, eluted at 3 concentration high enough to enable the enzyme to be salted out or used directly in ;I subsequent purification step. ACKNOWLEDGMENT

REFERENCES I. Swpcs. R. K. (1977~1) 7. Scopes. R. h.. (1977b) 3. Von der Haar. F. (1973) (Jakoby. .tdcmic 4.

Scopes.

S Scopes.

Hio~~hrrrr J. 161, 153-263. B/ochrnr. J. 161. 265-277. irr Methods in Enzymology

W. B.. cd.), Vol. Presh. New York.

34.

pp.

R. K. ( 198 I ) .4nu/.

Biochrm

R. K. (1974)

BiocAvw.

.3nul.

6. Srcrc. P. A. (1969) irl Methods ILowenstsin, .I. M.. cd.). Vol. ,idemlc Prss. NW York.

163 114,

171.

ENZYME

Racker. 214.

8.

Fanslur. B.. and Lowenstein. J. M. ( 1969) ods in Enzymology (l.owcnstcin, J. v..

9. Chs.

E. ( 1950)

Bi,whiv~.

Bio,~~~~\.

13. pp. 26-30. Academic Press. S., and Park>. R. F. ( 1964)

10.

239, Kanarck,

Il.

239, 4201- 1706. Mukhcrjce. A.. and

12.

C’hcm 251, 1376 Srerc. P. A. ( 1965)

1961 1967. I... and Hill.

204. P. A

R. L. (196-I) Srcrc.

P. A.

IGO. .-lrch

Hruc,hr,rtt

J. H/o/.

C’htJnt.

(1976)

I.w.

16.

MncFarlane.

17.

(1977) t‘lrr. J. Blochenr. Cha. S.. Cha. C.-J. M.. and

1X.

Rid Colomb.

19.

( 1972 Bioc~hrnmrry II, 3370-337x Fersht. A. (,1977) irr Enrymc Structure

and Johanswn. 77, 40- IO’.

N..

(‘hem. 242, 2577 M. G.. Chkru).

anihm.

p. 129,

20.

Tcipcl. 246.

J W.. and 1X59-3865.

71.

Swpc~. 106.

K. 239

in

I< and 24,.

B.,

Alpr.

J

Bicd

2 I j7- 2 I65

( 1977)

and

110.

.-lw/

Dalriel.

K

74. FT.3 5.59. Parks, R. E. (1967) ?SX I. A., and

Freeman. Hill.

241.

C.-J.

Mathe~h.

Chrn~

Hi+,,\

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( 1966)

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J. Hit~i.

I-t.

c‘ -Y.. Brr,c~hrr,r

..I[‘~u 4, 2 l I -

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looSrerc.

8-1 X

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27

El.UTION

7.

.Ac-

59, Z77-281.

AFFINITY

Vipnai\.

Reading. R. I

(197

and

J. P. V.

Llrch-

I .K. I ) J. H/o/

E. (1979)

FEHS

(‘lrrut. /XI!.