Quantitative studies of ion-exchange and affinity elution chromatography of enzymes

4YAI

Yll(‘,\l.

114, 8-18

HIOCH~MISTRY

Quantitative

(1981)

Studies of Ion-Exchange and Chromatography of Enzymes K.

ROBERT Department

of Biochemistry.

Affinity

Elution

SCOPES

La Trohe

University.

Received

October

Bundoora.

Victoria

3083.

Austrulia

27. 1980

Quantitative studies on the binding of a variety of enzymes to CM-cellulose have been carried out. and the magnitude of the affinity elution effect in the presence of substrates of the enzymes has been determined. In most cases the weakening of binding in the presence of substrate corresponded closely to the amount expected as a result of the overall charge change, but in a few examples the effect was greater. Some calculations have been made demonstrating the range of strengths of interactions between enzyme and adsorbent. and the energy involved per charge on the protein molecule.

Perhaps the most widely used method in enzyme purification during the past 20 years has been ion-exchange chromatography. Developed originally for simple low-molecular-weight compounds, ion-exchange techniques were first successfully used in protein purification after Peterson and Sober (I) produced exchangers based on porous biological matrices, which had sufficiently high adsorptive capacity for large molecules. The three principal charged substituents used today are, for anion exchangers diethylamino ethyl (DEAE-), and for cation exchangers carboxymethyl (CM-) and phospho-. The original matrix of fibrous cellulose has largely been replaced by microgranular cellulose powders, dextrans, agaroses, and spherical cellulose particles (2). Although the basic principles of ion-exchange chromatography are readily appreciated, and detailed mathematical calculations have been made concerning the process for small molecules, few quantitative calculations, or investigations have been carried out for macromolecules such as proteins (3). For such molecules there are many complications; unknown features of charge distribution and molecular configuration make any accurate mathematical predictions of 8

0003-2697,‘Sl/O90008-11$02.00/O C upyrqht

c I I)8 I by Zc.,dcm,c

ion-exchange behavior impossible. Nevertheless it is possible to obtain generalized pictures of protein behavior by making approximations and simplifying assumptions, given some quantitative data. The present paper provides some data for purified proteins adsorbing to CM-cellulose, and attempts to describe their behavior in a semimathematical way, according to simplified formulas presented previously (4). Affinity elution from ion-exchange columns involves the elution of enzymes (or other ligand-binding proteins) by inclusion of a substrate or specific ligand in the eluting buffer (5,6). In the simplest terms, a charged ligand, on binding to the protein molecule. reduces the overall charge and so decreases the strength of interaction with the ion exchanger. Thus the ligand must have the same charge as the ion exchanger, opposite to that of the protein it binds to. As most ligands are negatively charged, affinity elution is virtually restricted to cation exchangers, which require the binding proteins to be positively charged. i.e., to have an isoelectric point higher than the operating pH. Despite this restriction, the technique has found widespread application, particularly with animal enzymes which tend to be less acidic

Pie\\.

lnc

IOk-EXCHANGE

AND

AFFINITY

ELUTION

than those from other sources (7-9). More complex explanations of the affinity elution effect include conformational changes in the protein molecule on binding of the ligand and displacement of an active site adsorbent interaction (4,5, IO). The present experiments describe the extent of the affinity elution effect with a range of enzymes, and give ;I qualitative indication of a variety of other features of enzyme -1igand interaction such 3s conformational changes, dissociation constants, and order of substrate binding. An account of l.he detailed affinity elution behavior of yeAst phosphoglycerate kinase has been published ( IO).

CHROMATOGRAPHY

OF

ENZYMES

9

species changed from Taps (pH 9 -8.5) or Tricine (pH 8.5 8.0) to Tes (pH 8.0-7.2) and Mes (pH 7.2 6.3) and finally picolinic acid to get to lower pH’s. without the ionic strength changing. Starting buffers consisted of 10 rnM KOH adjusted to the appropriate pH using Taps, Tricine, or Tes; thus the ionic strength remained at 0.01. The effective liquid volume was determined using bovine serum albumin; it was found that 80% of the settled volume of the CM-cellulose was free liquid; thus the total volume was 14.4 ml of liquid. Using these figures it was possible to determine a partition coefficient (a) describing the proportion of protein absorbed to the CM-cellulose within the settled MATERIALS, METHODS, AND THEORY volume of 8 cm’; the concentration of nonThe sources of biochemical reagents, de- adsorbed protein within the adsorbent volume wx assumedto be the same as that in tails of enzyme assay methods, and purification procedures for enzymes were as de- the free liquid phase outside the adsorbent. scribed in previous publications [ 6,7,9]. The The sampled liquid was mixed with appropriate reagents using the proportioning pump, ion exchanger used was Whatman CM-celto measure protein (Lowry method) or enlulose CMS:!. Whatman. Maidstone. Kent, England. The experiments to determine par- zymic activity (single-point determination tition coefficients were carried out as follows. 2 3 min after mixing reagents), and the Eight cubic centimeters settled volume of mixture was passedthrough a flow cell. The CM-cellulose preequilibrated in a K-Tricine protein/enzyme concentration was continuor K-Taps’ buffer pH 8 9 (see below) was ously recorded, as was the pH ( 10). There was a lug in responseto sudden changes such added to 3 small beaker with magnetic stirrer. and ;1 further 8 ml of buffer. including that it took 1 min to respond 90%. mainly protein and (when appropriate) ligand. was due to the dead volume in the filter system. The marginal inaccuracy this caused could added. The liquid phasewas sampled through a small dead volume filter system consisting be allowed for when reading off the concentration and pH values from the chart. Tiof ;I sintered polyethylene disk 2 mm in ditration rates were between 0.02 and 0.05 pH ameter, with two layers of Mirracloth (Chicopee Mills Inc., Milltown, N. J.) on the units min- ‘. When the pH approached 6.0, outside as an additional filter. The sample a significant proportion of carboxyl groups began to be protonated, releasing K’ ions was taken up at a rate of 0.1 ml min ’ with the aid of a ‘Technicon auto analyser pro- and so increasing the ionic strength, Thus portioning pump. The pH of the slurry was figures below 6.0 rnust be regarded as relating to an increased ionic strength. For this adjusted simultaneously by titrating at 0.1 ml min ’ with 0.05 to 0.1 v zwitterionic reason in reverse, the method was not applicable to DEAEkellulose except in the buffer (acid form). In this way the buffering very low pH range. ;IS the DEAE groups used: Taps, 3-[tris(hydroxq’ Abbreviation:, titrate from pH 6.0 up (2.3). Also. phosmethql)meth~l]an~inopropancsulfonic acid: Tes. JYphocellulose titrates in the critical pH range [rih(h~dro~ymethvI)meth~i-~-~~linoetnic acid: of 6.0 7.0. Only by doing individual batchMcs. ‘-(~~-morph(,lino)ethllnrsulf~nic acid: Tricine. .Vwise adsorption experiments can comparable t.ris;( hydrouymethql )methylglycine.

10

ROBERT

results be obtained with these exchangers. QAE-SephadexandSP-Sephadexhavebeen used successfully, but all experiments described in the present manuscript refer to CM-cellulose. Adsorbent capacity was determined by taking a known amount of CMcellulose equilibrated in pH 6.0 K-Mes buffer, and adding batches of enzyme in pH 6.0 buffer until no more would adsorb; protein remaining in the supernatant after letting the adsorbent settle was measured. Since the capacity increased with time, the amount adsorbing in IO-I 5 min has been recorded. All experiments were carried out at room temperature unless otherwise indicated. Theorerical. If the interaction between protein and adsorbent is assumed to be an equilibrium binding reaction, then it can be shown that the partition coefficient a as described above is the solution of the equation P@’ - (p, + nz, + K,)a + m, = 0, where p, = total protein (adsorbed + nonadsorbed) per cubic centimeter of the packed adsorbent, NZ, = concentration of binding sites for the protein (adsorbent capacity), and K, is the average dissociation constant for protein adsorbent interactions under the specified conditions (4). If p, 6 m,, a value for K, can be calculated from the measured a using: K, = m,( I /a - 1). The energy of interaction per charge on the protein molecule can be calculated by plotting log K, against number of charges. The latter was calculated from an actual titration curve of the protein ( phosphoglycerate kinase and aldolase) or from published amino acid compositions using average pK, values for His. 6.2; Cys, 8.0; and Lys, 10.0. Varying these pK,‘s by up to 0.3 unit made no substantial difference to the final result. Since AGo = RT In K,, then the energy of interaction per charge is dAG” d==

-7 jRT L.

d(‘w

6) dr

The slope of the graph of log K, against number of charges is d(log K,)/d;, hence the value for AC” per charge can be calculated.

K. SCOPES

L

40

L--L 45

50

55

log MW

FIN;. I. Capacity of Whatman CM-S2 CM-cellulose for enzymes of various sizes. The enzymes were from rabbit muscle unless otherwise indicated. Buffer was KMes. pH 6.0; for details see Materials, Methods. and Theory. I, Hen egg-white lysozyme; 2. AMP kinase; 3, phosphoglycerate kinase (both rabbit muscle and yeast preparations gave the same capacity): 4. phosphoglycerate mutase; 5, creatine kinase (at 0°C); 6, enolase; 7. lactate dehydrogenase; 8, glyceraldehyde phosphate dehydrogenase; 9, aldolase; IO, pyruvate kinase.

RESULTS Deferminafion

of Adsorpfive

Capacities

Using CM-cellulose at pH 6.0, the capacity of I cm3 of packed exchanger for several pure enzymes which are strongly adsorbed at that pH has been determined using the method described in the previous section. Although some 80% of the available sites are immediately taken up, the ultimate adsorptive capacity may not be reached for 30 min to I h, consequently the capacity reported is to some extent a time-dependent value. The basic features of chromatographic behavior occur on time scales of less than an hour. and the values shown in Fig. 1 are those attained within IO -15 min, which in most cases were 90 -95%, of the “ultimate capacity.” For comparison, the capacity of DEAE-cellulose (Whatman DE-52) for bovine serum albumin was determined to be 105 mg cmm3 (imidazole -chloride buffer, pH 7.0, I = 0.01) by this method. It will be noted that the capacities. especially when expressed in molar terms, varied considerably despite the partition coefficients being very

ION-EXCHANGE

AND TABLE

pH

V.41 I ts

4~

E\/YMI

S C’h

AHSI.IL<

1. ZND

Rabbit

muscle

CY = 0.5

CM-Ct.1

l.lll~OS~.

FOR

I = 0.01.

kinase

0.1

phosphoenolpyruvate

mM

+ 0. I mM

ose

6.86 6.80

1.6-bisphosphate

fructose

I .6-bisphosphate

Rabbit

muscle

20 mw 0.2 0.2 Bovine

creatine

6.30 I,&bisphosphate

6.20

kinase

6.46

crcatinc

mhi m\l

ADP ATP

(No (No

6.44 6.10

Mg) Mg)

6. IO

heart mitochondrial amino transferax

2.5 mu 10 mhl

woxoglutarate KCI

2.5

w-oxoglutarate

mu

aspartatc 8.84 8.76 8.50 + 5 InM

aspartatc Rabbit 0.2 Rabbit

8.30

muscle enolasc mM pho\phoenolpyruvate muscle

6.84 6.56

glycersldehydc

phosphate

dchydrogenase 0. I mcl I ,3-bi:;phosphoglyccrate Rabbit

muscle

phosphoglycerate

0. I m%f 3-phosphoglycerate 0.1 m\i 2.3-bisphosphoglyccrate Yeast 0.1

6.68 6.45

m’vc phosphoenolpyruvate + 0.1 mM fructose

phosphogl)cerate mlvl 3-phosphoglycerate

8.10 7.74 mutase

7.00 6.24 6.12

mutase

CHROMATOGRAPHY

(2) are comparable in Fig. 1.

OF

ENZYMES

to the results

11

presented

11\1 TtIF, LIGANDS

6.82

Ycaat pyruvate kinasc 0. I mM phosphocnolpyruvate 0.1

OF

7.04 7.04

I mu Mg acetate 0. I rnbt phoaphoenolpyruvate ADP frucl

A VARI~.,TY

OF VARIOCIS

PRFSFNCt

0. I mu 0. I mM

ELUTION

I

UHIC‘II

pyruvatc

AFFINITY

6.50 5.90

close to unity under the conditions used. The larger proteins bound less, and a plot of capacity against. log molecular weight was nearly linear (Fig. 1 ) indicating that the exchangers behave like gel-exclusion materials, in this case with an exclusion molecular weight (by extrapolation of Fig. I ) of about 500,000. DEAE-Sepharose and DEAE-Sephacel are reported to have exclusion molecular weights of about IOh (2), and other capacity values reported in this publication

Variation of Partition with pH

Coefficients

In these experiments, enzymes purified in the laboratory. often by empirically determined affinity elution procedures, were used to determine the variation of the proportion adsorbed to CM-cellulose (cu) with pH. The enzyme concentration in solution was determined by the Lowry method in most cases. Starting at a high pH where no enzyme adsorbed, the pH was lowered by titration with the acid zwitterions (Tes, Mes, picolinic acid) at a rate of between 0.02 and 0.05 pH units per min. Both pH and protein/enzyme concentration were recorded continuously. After making corrections for the amount of protein removed, and the dilution by the titrant. the value of the partition coefficient LYcould be calculated for each pH. As there was a continuum of results from the recorder trace, no experimental points have been placed in the figures below. The experiment was then repeated in the presence of ligands appropriate to the enzyme in question. Results for yeast phosphoglycerate kinase have been presented previously ( 10). Figures 24 and Table I show results for a variety of other enzymes, which are described individually below. /a) Lactute dehydrogenase (muscle isoc)n:~,~~j. The rabbit muscle enzyme has an isoelectric point of 8.5 (1 1). and began to adsorb to CM-cellulose at about this pH, as would be expected of a simple charge interaction between enzyme and adsorbent, regardless of charge distribution (Fig. 2). The partition coefficient reached 0.99 at around pH 7.0 under these conditions; the adsorbent was only 6%) saturated compared with its maximum capacity of 21 mg cm-’ (Fig. I ). In the presence of 1 mM Na pyruvate no change in the o/pH curve was detected; pyruvate does not bind to lactate dehydrogenase in the absence of nicotinamide nucleo-

12

ROBERT

tide ( 12). In the presence of 0. I mM NAD’, the curve was substantially shifted; oxidized NAD 3t this pH has one negative charge; despite the enLyme being able to bind four NAD molecules, the shift of the curve was more than would be expected if the only effect were to decrease the positive charge on the enr.yme by 4 units. In the presence of NADH (two negative charges per molecule), the curve was even more shifted, and assumed a very steep aspect rising from a = 0 at pH 7.2, to N = 0.9 at pH 6.7. This curve was nearly duplicated by NAD’ plus pyruvate, indicating that the abortive complex is very similar in charge and conformation to the enzyme- NADH complex. Clearly an affinity elution procedure can be used in which the enzyme is adsorbed at pH 7.0 or less, and eluted with NADH. or with NAD’ plus pyruvate, at a pH of 7.5. This is very close to the procedure worked out empirically (6); the original method has now been modified slightly, and works excellently under the conditions BS above. Titration of the enzymes CM-cellulose complex at pH 7.4 with NADH caused a rapid elution above 2 FM (the concentration of the enzyme in the mixture), complete by 9 PM (Fig. 2, inset). The dissociation constant of NADH under these conditions is 3.5 PM ( 13); The rapidity of response to the changing [NADH] is no doubt due to the

PH

FI(, 2. CM-cellulose binding curvch for rabbit muscle lactate dehydrogenltss. Concentrations of ligands were: pyruvu~. I mM; NAD’, 0.1 mM: NADH. 0.1 mhi. Maximum occupancy of binding sitea was 6%. Inset: elution of bound en;lymc at pH 7.4 by titration with NADH.

K. SCOPES

5.5 FIN,. 3. CM-cellulose aldolase

in

the

presence

blnding

curves

and

absence

for rabbit of

Concentration of fructose I .&bisphosphatc 0.2 mhl. Maximum occupancy of binding Inset: elutiun with FBP.

of bound

enzyme

at pH

30

muscle mlvl

KCI.

(FBP) was sites was S1. 7.2

by titration

binding of up to four molecules per molecule of enzyme, resulting in a fourth-power effect. Thus IO PM NADH would be sufficient to cause elution of lactate dehydrogenase from a CM-cellulose column at pH 7.4; however, since the concentration of enzyme on a column could be up to 0.6 mM of subunits (21 mg cm- ‘) it is more effective to use an NADH concentration of at least 0.2 mM, or ;I short pulse of even higher concentration, thereby eluting all the enzyme in a smaller vofume.

(b) Musde fructose bisphosphatr

aldol-

nsr. Rabbit muscle aldolase has an isoelectric point above 8 ( I I ), and is known to adsorb to cation exchangers such as CMcellulose, phosphocellulose, and also to muscle myofibrillar components which, at neutral pH, are also highly negatively charged ( 14). Fructose I ,6-bisphosphate binds tightly to aldolase (and is split to triose phosphates), and is very effective in eluting the enzyme; the tetramer acquires 16 negative charges when the substrate saturates it. The shift of 1.6 pH units for the midpoint of the curves shown in Fig. 3 corresponds to a titration of between 15 and 20 charged groups on aldoluse in this pH range, so the affinity elulion effect is mainly due to the direct charge cancellation by the substrate. Figure 3 also illustrates the curves in the presence of 30 tnM KCI (ionic strength four times higher than the standard value), for which each was

ION-EXCHANGE

AND

AFFINITY

ELUTION

PH

FK,. 4. CM-ccllulase binding curves for rabbit muscle AMP kinase (myokinase). Concentrations of ligands were: AMP, 0.2 mv: ADP, 0.2 !llM (+O.l rn~ EDTA): ATP. 0.2 mv (-to.1 IBM EDTA): MgATP: 0.2 mM ATP + 0.5 mM Mg acetate: A,,
shifted acid-wards. but remained the same shape. The curves in the presence of substrate were :steeper, indicative of a higher energy of interaction per charge (see later). The inset in Fig. 3 contains the results on the titration of aldolase bound to CM-cellulose at pH 7.2 with fructose bisphosphate; complete elution was achieved by 25 pM substrate. approximately twice the amount of enzyme subunits present. Further results with rabbit muscle aldolase are presented in a later section. (c) Rabbit muscle nrjwkinase. Myokinase is capable of binding adenine nucleotides in all forms, complexed with magnesium or not. With ADP the enzyme dismutates the ligand unless all traces of divalent metals are removed. Figure 4 shows the results for the rabbit enzyme. Compared with the other enzymes, the adsorption curve was very shallow; this is to be expected, since this small protein has very few groups titrating in the relevant pH range. AMP shifted the curve only by the amount expected for two negative charges. ADP ( -+ magnesium) more than doubled the shift: this would also be expected as there are two binding sites for ADP. However, ATP moved the curve further than ADP, and MgATP further still despite having only two charges compared with free ATPs. three (at pH < 6.5). This is

CHROMATOGRAPHY

OF

ENZt’MES

13

indicative of either a conformation change. or ;1 displacement of specific binding between CM-cellulose and the active site of the enzyme. The curve with MgATP was shifted by nearly 1 pH unit more than with AMP. With P’,P’-di(adenosine-5’)pentaphosphate. ;1 potent inhibitor of myokinase, little adsorption occurred to CM-cellulose even at pH 5.8. This inhibitor has been used for affinity elution purification of the human muscle enLyme ( 15 ). ADP. at pH 6.5- 6.6. appears to be ;1 satisfactory ligand for affinity elution, and in fact methods developed empirically (6,8) used exactly these conditions.

cd) Pyruvatr kinases fi-onz rabbit muscle nndfi-on? ~~a.ct. Rabbit muscle pyruvute kinase has been demonstrated to show several components isoelectric in the pH range 7.8 8.6 (I 1); our preparation adsorbed to CMcellulose below pH 7.5. Both its substrates phosphoenolpyruvate and ADP possess 3 negative charges, since it is believed that the ADP substrate is not complexed to mngnesium (16). The curves in the presence and absence of substrates were determined. and the pH’s at which a was 0.5 are presented in Table I The presence of I mM Mg acetate did not affect the basic curve. With the substrates, there was little difference between ADP and phosphoenolpyruvate; the shift in the presence of either was somewhat less that the expected value for the addition of 12 negative charges per tetramer, but the substrates were not fully saturating at the concentrations used. Although the rabbit enLyme is not kinetically affected by fructose 1,6-bisphosphate. this ligand also binds to the enzyme. since it had about 3s much effect on shifting the curve acid-wards as did phosphoenolpyruvate. Moreover the phosphoenolpyruvate and fructose bisphosphate effects were additive. Although it would appear from these results that purification of rabbit muscle pyruvate kinase by affinity elution would best be achieved with a combination of phosphoenolpyruvate and fructose bisphosphate ;it about pH 7, this is not so. since columns inevitably also contain

14

ROBERT

large amounts of aldolase, which is slowly eluted by the fructose bisphosphate (see section (b)). Phosphoenolpyruvate alone at a pH just above 7.0 has been the most successful procedure (6). The yeast enzyme has an isoelectric point of 6.7 (17); it behaved in an ideal fashion, adsorbing to CM-cellulose below this pH. As with the rabbit enzyme, the presence of ligands shifted the curves as much as expected for the charge additions, fructose bisphosphate and phosphoenolpyruvate being additive (Table I ). In the case of yeast, these two ligands together make an excellent method for affinity elution; yeast aldolase is too acidic to adsorb to CM-cellulose.

(e) Rabbit

muscle creatine

kinase.

Be-

cause this enzyme is unstable under mildly acid conditions (IS), and slowly denatures on CM-cellulose at pH 6.0 at room temperature. the experiments with this enzyme were carried out at 0 -1“C. Although its isoelectric point is 6. I at I “C ( 19). the enzyme was almost fully adsorbed to CM-cellulose at this PH. The presence of 20 mM creatine, the uncharged substrate, made practically no difference to the curve, but either ATP or ADP (0.2 mM) shifted the curve some 0.35 pH unit (Table 1). The identity of the shifts may be coincidental, since ATP (three negative charges at pH 6) would not have been saturating, whereas ADP (two charges) binds more tightly to the enzyme. However, the magnitude of the shift is greater than expected for two charges, suggesting conformational or active site interaction displacement effects.

K. SCOPES

sorption curve (Table 1). Aspartate. with only one negative charge, was shown to have more effect than the comparable amount of KU, and a combination of cu-oxoglutarate and aspartate is moderately effective in eluting the enzyme from the adsorbent. However, this must be regarded as mainly an ionic strength effect, since several millimolars of each is needed. The method has been employed successfully, at a slightly lower pH but in the presence of 20 mM KCI, to purify the enzyme from beef heart extracts (9). (g) Some other enzymes. Listed in Table 1 are results obtained with some other enzymes using the same system. Phosphoenolpyruvate can be used for affinity elution of muscle enolase (6), the shift at cy = 0.5 was nearly 0.3 pH units using 0.2 mM substrate. Glyceraldehyde phosphate dehydrogenase was displaced by 1,3-bisphosphoglycerate, though the effect was not large. Phosphoglycerate mutases from both rabbit muscle and yeast were markedly affected by the presence of substrates; unfortunately affinity, or even salt elution of this enzyme from CM-cellulose gave very poor recoveries.

Comparison of Partition Coej’cient/pH Curves ut Different Protein Concentrations

It is anticipated that an ion exchanger will have a range of adsorption sites for a particular protein, from very strongly attracting areas which would be occupied first, to relatively weak sites which would result in more partitioning into solution (3). Consequently the partition coefficient at a given pH is ex(f) Beef heart mitochorldrial aspartate amino transferase. This enzyme has a very pected to be lower as the total protein preshigh isoelectric point, and began to adsorb ent increases and the proportion of sites occupied rises. Some experiments were carried to CM-cellulose at pH 9.4: the partition out with rabbit muscle aldolase to test this coefficient reached 0.9 by pH 8.5. Although the K,,, values for the keto acids are quite proposition; these results are presented in low. with a ping-pong mechanism these do Fig. 5. As expected, the partition coefficient not relate directly to dissociation constants; decreased with increasing protein concentrasmall concentrations of oxaloacetate or LY- tion. At any given pH the “average dissooxoglutarate made little impact on the ad- ciation constant,” K,, for the enzyme-CM-

IOkEXCHANGE

AND

AFFINITY

ELUTION

P”

FK;

5. CM-c~~llulose

bindingcurvcs

for rabbit

muscle

,ildolasc with different percentage occupancy of binding sites. From right to left, the percentage occupancies at LY =

1.O were:

0.8,

2.5.

8.3.

25.

and

10048.

respectively.

cellulose interaction increased also, and these have been calculated and plotted in Fig. 6 against the percentage of binding sites (as determined at pH 6.0) occupied at each point. It will be noted that the average dissociation constant appeared to increase more rapidly with site saturation when the interaction was weaker, although this is probably vrtifactual since fewer sites are likely to be effective at higher pH’s.

Elution

Most resu1l.sreported above used buffers with an ionic strength of 0.01. At higher ionic strengths the question arises as to whether affinity elution can be so effective, since the electrostatic interaction between protein and e.uchanger is weakened and intercepted by the buffer ions. Yeast phosphoglycerate ltinase at pH 6.0 required an ionic strength of 0.075 to make a = 0.5. and the presence of 3-phosphoglycerate only reduced N to 0.35. Thus affinity elution at this pH would be Ineffective. However. with aldoluse (see Fig. 3). the effect of substrate was so great that even in the presence of 30 mM KC1 affinity elution is very effective. With aldolasr: and some further results with apartate amino transferase. the shapes of the a/pH curves in the presenceof various levels of salt were very similar if plotted against net protein charge instead of pH.

CHROMATOGRAPHY

OF

f-.1(;. 6. Variation dissociation constant pancy culated pll

of the average protein-adsorbent with the percentage of site

for rabbit muscle from the curves 8.0;

A, at pH

15

ENZYMES

7.8;

aldolase in Fig. &

at pH

on CM-cellulose. 5, 0, at pH 8.2:

occuCal0. at

7.5.

This suggests that salt displacement of proteins from CM-cellulose is very similar to pH displacement in terms of sharpness of response, but in some CBXS high salt concentrations may not sllow affinity elution to work so efficiently. Energ! of Interaction Ion- Eschangr Matris

of

Proteins

MYth

As indicated under Material. Methods, and Theory, the energy of interaction of the protein molecule per unit charge can be calculated from the partition coefticient/pH curves. In each case the condition p,
16

ROBERT

K. SCOPES

TABLE CHANGI,

IY 1.~1. E~~RGII

2

s ot IYT~RACTION OF ENTVMW WITH PosIuvt. CHARGE ON THL ENYYM~ X7/A;. mol ’ for

Enzyme Yeast phosphoglycerate AMP kinase Creatine kinase Muscle aldolase Muscle pyruvate kinase Yeast pyruvate kinase

kJ free

Note. Since some enzymes charge on the enzymes.

overall

negatively

hcient to establish whether an affinity elution procedure will work, provided that appropriate conditions of ionic strength and pH are tried. The present quantitative experiments have shown the extent of the affinity elution effect with a number of proteins which have negatively charged ligands. In some cases the partition coefficient was reduced from 0.99 to 0 with very low concentrations of ligands ( 10e5 to 10eJ M). In other cases the effects were not so dramatic, and a reduction of (Y from around 0.9 to 0.2 was the best that could be achieved without a significant rise in ionic strength. A lesser effect than this is unlikely to result in easily reproducible column purification procedures. In the presence of ligand, a range of different states of the enzyme is possible, especially if it has multiple binding sites. At the pH’s where (Y = 0.5 (e.g. in Table l), the bound proportion may be enzyme with few if any ligand molecules attached. Alternatively it may be saturated with ligand, yet still attracted to the adsorbent. If the latter, then we can compare the pH shift in terms of total charge on the saturated enzyme-ligand complex with the charge on the free enzyme. But if some enzyme molecules, particularly the adsorbed ones, were not totally

for

+ ligand

I .3 (Bisphosphoglycerate) 5.8 (ADP) 2.0 (ATP) I .2 (Fructose bisphosphate) I. I (Phosphoenol pyruvate) I .6 (Fructose bisphosphate + phosphoenol pyruvate) I.8 (NADH)

0.7 0.9 bind even when

kJ mol-’

enzyme

2.0 4.2 I.5 0.9 I .o I.7

Lactate dehydrogenase Aspartate amino transferase

by OSE FOR EActi

AG”/A:.

enzyme kinase

CM-CELI

charged,

these do not relate

directly

to the foral

complexed with ligand, then the pH shift would be expected to be less than in the fully saturated case. In most of the examples described the ligand concentration chosen was 5-20 times its known dissociation constant, and the pH shift was at least equal to that expected for total saturation of all molecules. Indeed in some cases the pH shift was even larger than this. Thus with phosphoglycerate kinase ( IO), lactate dehydrogenase (Fig. 2). and AMP kinase (Fig. 4) certain ligands caused large shifts in the curves. But with aldolase (Fig. 3), pyruvate kinases, and several others (Table 1) the ligands caused shifts corresponding approximately to the value expected if the only effect (at a given pH) was a reduction in net charge. In the large-effect cases, the additional shift must have been due to one or both of the alternatives described for phosphoglycerate kinase ( IO). namely, a masking of a localized positive-charge cluster at the active site, or an overall conformational change which lessened the protein’s adsorption tendencies. There has been no evidence so far of a conformational change which works the other way. In the case of creatine on creatine kinase, no significant shift was observed; the ligand is uncharged, and presumably neither

ION-EXCHANGE

AND

AFFINITY

ELUTION

masked a specific interaction between the enzyme and CM-cellulose, nor caused a conformational change which altered the interaction. With lactate dehydrogenase. pyruvate alone had no effect as it was not expected to bind to the free enzyme ( 12), but NAD’ caused a shift larger than the charge alone would. This is to be expected, since it is the conformational change of the enLyme on binding NAD( H) that allows it to bind the other substrate. NADH caused a larger shift than NAD’, comparable to that of the abortive enzyme-NAD+-pyruvate complex, which suggestsan assymmetry in the conformational changes during the progress of the lactate dehydrogenase reaction. Very low levels of NADH were able to elute 1actal:edehydrogenase from CM-cellulose at pH 7.4, confirming a very low dissociation constant. Similarly with fructose bisphosphate eluting aldolase; an order-ofmagnitude estimate of the dissociation constant can be obtained. In the simpler cases of single-binding-site enzymes, an accurate measure of the dissociation constant can be obtained using this CM-cellulose binding technique. Figure 5 shows that the actual partition coefficient depends on total protein concentration -this would be predicted even if all binding sites ‘were equivalent. However, on calculating the average value of K, from these results, and plotting them against the total number of sites actually occupied at that particular point (Fig. 6). it is seen that the average site becomesweaker as more are occupied; the Imore strongly attracting sites are occupied iirst. Arbitrary straight lines have been drawn through these points. It will be noted that under conditions where the protein binds tightly (such as would be used during adsorptlon and preelution washeson ion-exchange columns), the value varies over a factor of about 5 for the first 50% of the sites. With weaker binding it appeared to vary more quickly. but this may have been due to an overestimate of the total sites available under these conditions. Thus the ex-

CHROMATOGRAPHY

OF

ENZYMES

17

trapolation from the experimental conditions of low site saturation in Figs. 2- 4 and Table 1, to the “real” situation on top of a column where all sites may become occupied is not substantially incorrect. Indeed a theoretical allowance of a downward shift of 0.2 -0.4 pH units for high protein concentration appears to be too much in view of the ideal pH values deduced directly from the curves agreeing so well with those found empirically. Some compensating factor, such as an error introduced in measuring the pH of a thick slurry of ion exchanger. may be operating here. The thermodynamic calculations presented in Table 2 must be considered very approximate in view of the fact that during the titrations equilibrium was never really attained, since there are sites which take tens of minutes to be occupied due to diffusional barriers. Nevertheless the curves were reproducible, even at lower titration rates. No distinct trend can be detected, reflecting the variety of different types, shapes. and sizes of enzymes used in these experiments. However, it is apparent that the smallest enzymes, AMP kinase and phosphoglycerate kinase. have the strongest interactions per charge, reflecting both the tighter packing into the pores of the adsorbent, and the shorter average distance between a charged residue in the protein and the charged carboxylates of the CM-cellulose. A typical value of AC” per charge for a protein of molecular weight around I O5is 1.5 kJ mot-‘, and in itself represents a very weak interaction; a K, value of lo-’ (a of approximately 0.9) represents an interaction energy of 30 kJ mot-‘. Thus in a simple situation where all charges can be considered equivalent, about 20 positive charges are required on the enzyme to interact with the adsorbent to give LY= 0.9. Because of the very broad assumptions being made, further interpretation is not justified. ACKNOWLEDGMENT This work was supported Grants Committee, Grant

by the Australian D27815338R.

Research

18

ROBERT

REFERENCES I. Peterson, E. A., and Sober, H. A. (1956) J. Amer. Chem. Sot 78, 75 I-755. 2. Ion Exchange Chromatography, Principles and Methods, Pharmacia Fine Chemicals, Uppsala. Sweden ( 1980). 3. Peterson, E. A. (I 970) Cellulosic Ion Exchanges in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S., and Work, E.. eds.), Vol. 2. Part 2, North-Holland, Amsterdam. 4. Scopes, R. K. (1978) in Techniques in Protein and Enzyme Biochemistry BIOI (Kornberg, H. L., Metcalfe. J. C., Northcote, D. H.. Pogson, C. I. and Tipton, K. F., eds.). Elsevier/North-Holland. Amsterdam/New York. 5. Von der Haar. F. (1974) in Methods in Enzymology (Jakoby. W. B.. ed.) Vol. 34, pp. 1633171. Academic Press, New York. 6. Scopes, R. K. (1977) Biochem. J. 161, 253-263. 7. Scopes, R. K. (1978) Biochem. J. 161, 265-277. 8. Noda. L., Schulz. G. E., and von Zabern. I. (1975) Eur. J. Biochem. 51, 229-235.

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I I. Susor. W. A., Kochman, M.. and Rutter. W. J. ( 1969) Srience 165, 1260-I 262. 12. Holbrook, J. J.. and Gutfreund, H. (1973) FEBS Left. 31, 157-169. 13. Stinson. R. A.. and Holbrook. J. J. ( 1973) Biochem. J. 131, 719-728. 14. Clarke, F. M.. Masters. C. J., and Winzor, D. J. (1974) Biochem. J. 139, 785-788. 15. Feldhaus, P.. Frohlich. T.. Goody. R. S.. Isakov. M.. and Schirmer, R. H. ( 1975) Eur. J. Biochem. 51, 197-204. 16. Ainsworth, S.. and MacFarlane. N. (1973) Biochem. J. 131, 223-236. 17. Aust, A. E., and Suelter, C. H. (1978) J. Bid. Chem. 253, 7508-75 12. 18. Scopes. R. K. ( 1965) Arch. Biochem. Eiophys. 110, 320-324.

19. Kuby. S. A., Noda, L.. and Lardy, J. Biol. Chem. 209, 203-210.

H. A. (1954)