Further studies on the bioaffinity chromatography of NAD+-dependent dehydrogenases using the locking-on effect

BB

Biochi~ie~a

et Biophysica A~ta

ELSEVIER

Biochimica et BiophysicaActa 1297 (1996) 235-243

Further studies on the bioaffinity chromatography of NAD +-dependent dehydrogenases using the locking-on effect Padraig O'Carra a, Tadhg Griffin a, Martina O'Flaherty b, Niall Kelly a, Patricia Mulcahy b,, a Department of Biochemistry, University College, Galway, Ireland b Department of Applied Biology and Chemistry, Regional Technical College, Carlow, Ireland

Received 30 May 1996; accepted 18 June 1996

Abstract Previous studies have capitalised on ordered kinetic mechanisms in the design of biospecific affinity chromatographic methods for highly efficient purifications and mechanistic studies of enzymes. The most direct tactic has been the use of immobilised analogues of the following, usually enzyme-specific substrates, e.g., lactate/pyruvate in the case of lactate dehydrogenase for which NAD + is the leading substrate. Such immobilised specific substrates are, however, often difficult or impossible to synthesise. The locking-on strategy reverses the tactic by using the more accessible immobilised leading substrate, immobilised NAD +, as adsorbent with soluble analogues of the enzyme-specific ligands (e.g., lactate in the case of lactate dehydrogenase) providing a substantial reinforcement of biospecific adsorption sufficient to effect adsorptive selection of an enzyme from a group of enzymes such as the NAD +-specific enzymes. The value of this approach is demonstrated using model studies with lactate dehydrogenase (LDH, EC 1.1.1.27), alcohol dehydrogenase (ADH, EC 1.1.1.1), glutamate dehydrogenase (GDH, EC 1.4.1.3) and malate dehydrogenase (MDH, EC 1.1.1.37). Purifcation of bovine liver GDH in high yield from crude extrzLcts is described using the tactic. Keywords: Lactate dehydrogenase;Alcohol dehydrogenase;Glutamate dehydrogenase;Malate dehydrogenase;lmmobilised NAD+

1. Introduction The locking-on effect is a tactic intended to strengthen and make more specific the affinity chromatographic adsorption of a range of enzymes having ordered kinetic mechanisms. It was introduced by one of us over a decade ago [1] with experimental 'model' studies indicative of its feasibility, but the tactic hzts been little used in its original chromatographic form during the intervening years, although applied with success to the improvement of the affinity precipitation methods, since introduced by Mosbach, Tipton and co-workers [2-6]. We have recently returned to this field and have been developing and applying the tactic with some success to the chromatographic purification of a range of 'difficult' NAD+-dependent dehydrogenases, aiming at essentially one-step purifications to homogeneity.

Abbreviations: LDH, L-lactate dehydrogenase (EC 1.1.1.27); ADH, L-alcohol dehydrogenase(EC 1.1.1.1); GDH, L-glutamate dehydrogenase (EC 1.4.1.3); MDH, L-malate dehydrogenase(EC 1.1.1.37). * Corresponding author. Fax: +353 503 43787.

We present here some of our developmental (i.e., feasibility) studies and also an actual purification from a crude extract illustrating the practical power of the locking-on tactic. This tactic may also be of some use in enzyme kinetic studies, as illustrated by some of our results. We have, however, encountered some unexplained phenomena and these are also described and discussed.

2. Materials and methods Sepharose 4B, DEAE-Sepharose, Sephadex G-25 and SDS L M W protein standards were obtained from Pharmacia LTD (Central Milton Keynes). Coomassie Protein Assay Reagent (Coomassie brilliant blue G-250 based reagent) was obtained from Pierce (Rockford, IL). All other chemicals, biochemicals and commercial enzyme preparations were supplied by Sigma (Poole, Dorset, England). Commercial enzyme preparations used in this study are as follows: L-glutamate dehydrogenase from bovine liver (Type 11) (GDH, EC 1.4.1.3); L-malate dehydrogenase from porcine heart (mitochondrial) (mMDH, EC 1.1.1.37);

0167-4838/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S01 67-483 8(96)00100-'3

236

P. O'Carra et al. / Biochimica et BiophysicaActa 1297 (1996) 235-243

L-malate dehydrogenase from porcine heart (cytoplasmic) (cMDH, EC 1.1.1.37); L-lactate dehydrogenase from bovine muscle (Type XXV1, LDH 5 (M 4) isoenzyme, EC 1.1.1.27); L-lactate dehydrogenase from bovine heart (Type IX, LDH1 (n 4) isoenzyme, EC 1.1.1.27); L-lactate dehydrogenase from porcine muscle (Type XXX-S, LDH, EC 1.1.1.27); Alcohol dehydrogenase from yeast (ADH, EC

1.1.1.1). 2.1. Synthesis of affinity gels and monitoring of substitution

The agarose matrix (Sepharose 4-B) was first activated with cyanogen bromide and coupled with 1,3-diamino propanol (spacer-arm compound) following the general procedure for coupling of amines described by Cuatrecasas [7]. Synthesis of the p-aminobenzamido alkyl derivative, and attachment of NAD-, NADP + and AMP by the diazo coupling procedure, was achieved using the procedures described by O'Carra and co-workers [8,9]. The point of attachment in these azo-linked derivatives is believed to be through the 8-position of the adenine residue [8]. Substituted gels, after thorough washing, were suspended in a 40% w / v sucrose solution and scanned in a Hitachi U-2000 spectrophotometer. The reference/blank consisted of a suspension containing a balanced quantity of the appropriate Sepharose 4B-spacer arm derivative unsubstituted with ligand. Chemical monitoring of substitution was carried out as described by Barry and O'Carra [8]. The extent of substitution of all the gels used in the affinity chromatographic studies described here was approx. 2 /xmol/ml. 2.2. Enzyme and protein assays

Dehydrogenase activity was measured spectrophotometrically at 30°C, measuring the decrease or increase in absorbance at 340 nm. LDH assays were carried out in the lactate direction with 0.5 mM pyruvate and 0.2 mM NADH as substrates in 50 mM potassium phosphate buffer (pH 7.4). MDH was assayed in 100 mM potassium phosphate buffer (pH 7.4) containing 200 /zM NADH, the reaction being started by addition of 0.1 ml of freshly prepared 6 mM oxalacetate solution (oxalacetate being unstable in aqueous solution). The final volume of the assay mixture was 3 ml. GDH was assayed in 50 mM potassium phosphate buffer (pH 7.4) containing 50 mM ammonium sulfate, 160 p~M NADH and 5 mM a-keto-glutarate in a final volume of 2.5 ml. The reaction was started by addition of an aliquot of the enzyme solution. ADH was assayed in 30 mM sodium pyrophosphate buffer (pH 8.8) containing 0.8 mM NAD + and 0.12 M ethanol; the reaction was started with 0.1 ml of enzyme solution. Alternatively, alcohol dehydrogenase could be

assayed in the opposite direction, using a cocktail of 0.2 mM NADH, 3 mM acetaldehyde in 50 mM potassium phosphate buffer (pH 7.4). The reaction was started by adding an aliquot of the enzyme solution. Protein concentration was determined using the dyebinding procedure described by Bradford [10] and using a commercial preparation of Coomassie brilliant blue G-250 (Pierce). Bovine serum albumin was used as standard. 2.3. Analytical affinity chromatographic methods

When used for analytical purposes, affinity chromatography was carried out in miniature columns (1 ml bed volume) with a hydrostatic head of pressure, adjusted to give a flow rate of one column volume per four minutes. The 'straight through' ('breakthrough') elution volumes were determined by applying 4 mg of glucose, dissolved in 0.5 ml of irrigating buffer, and monitoring the position of emergence in the effluent using the dinitrosalicylic acid reagent. Chromatography was performed at room temperature (l 5 °C) 2.4. Purification of GDH from bovine liver

Fresh bovine liver was obtained from the local abattoir, transported on ice, washed thoroughly and stored frozen at - 2 0 ° C until required. One part weight of tissue was homogenised with 8 parts volume of extracting medium (50 mM potassium phosphate buffer (pH 7.4) containing 0.32 M sucrose) using a Sorval Omnimix. The homogenate was centrifuged at 17300 × g for 1 hour. The resulting supernatants were subjected to an ammonium sulphate fractionation step (between 20% and 70% saturation ammonium sulfate) centrifuging at 17 300 × g for 30 min. The pellet was dissolved in 50 mM potassium phosphate buffer (pH 7.4). Ion-exchange chromatography of bovine liver GDH (the 30-70% saturated ammonium sulphate cut of liver extract) was carried out using DEAE-Sepharose Fast Flow packed columns (2.2 cm by 8.2 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.4). Ammonium sulphate precipitates were desalted on a Sephadex G-25 column equilibrated with the same buffer. The DEAE-Sepharose column was loaded with sample and then washed with two column volumes of 20 mM potassium phosphate buffer (pH 7.4) before applying a 120 ml gradient (0-0.5 M KC1). GDH eluted maximally at 110 mM KCI. 350 mM glutarate was added to the pooled active fractions immediately prior to their application to the immobilised 8'-azo linked NAD ÷ derivative (5 ml bed volume). The column was irrigated with six column volumes of 50 mM potassium phosphate buffer (pH 7.4) containing 350 mM glutarate, before eluting the enzyme by omitting glutarate from the irrigating buffer. Active fractions were dialysed against distilled water and freeze dried in readiness for electrophoretic analysis.

P. O'Carra et al. / Biochimica et Biophysica Acta 1297 (1996) 235-243

2.5. SDS disc gel electrophoresis SDS-PAGE was carried out according to Laemmli [11], using a separating gel of 13% acrylamide. Molecular weight calibration of SDS gels was achieved by using the following protein 'markers': c~..lactalbumin (14 kDa), trypsin inhibitor (20.1 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa) and phosphorylase B (94 kDa) (low molecular weight protein standards for SDS-electrophoresis obtained from Pharmacia). Staining for protein was c~xfied out by first fixing the gels in 12% TCA for 30 min, tollowed by staining with amido black (0.2% in 7% acetic acid) for 3 hours. Stained gels were cleared of residual dye by leaching overnight in 7% v / v acetic acid.

3. Results and discussion

3.1. Basic tactic: theory A range of tactics capitalising on aspects of the kinetic mechanisms of multi-substrate enzymes can improve the applicability and effectiveness of affinity chromatographic techniques [1]. The locking-on effect represents one such tactic, utilising the compulsory order of substrate binding to certain groups of enzymes such as the NAD+-dependent dehydrogenases. The latter group of enzymes serve as illustrative 'test cases' in this study. The mechanism of most, if not all of these dehydrogenases, entails prior binding of NAD + as the leading ligand before the enzyme can bind the second ligand which is generally the specific substrate - - e.g., lactate/pyruvate in the case of lactate dehydrogenase. This compulsory order of binding can be used to design doubly specific affinity systems by immobilising tJae second ligand and adding the (soluble) first ligand to the irrigating buffer to promote adsorptive binding (e.g., [12]). However, this entails considerable and in some cases seemingly insuperable difficulties with the immobilisation chemistry. For example, we have been unable to derive a useful immobilised specific ligand for malate dehydrogenase: all our immobilised derivatives of malate/oxalacetate and their analogues proved entirely ineffective as biospecific adsorbents, presumably owing to steric hi:adrance caused by the immobilisation chemistry. Similar problems have dogged our attempts to devise similar systems for glutamate dehydrogenase and octopine dehydrogenase. Such problems do not apply to the same extent to the immobilisation of the much larger NAD ÷ molecule, whose immobilised derivatives can also be used, in theory, for all of the enzymes of the group, though with a much inferior degree of specificity (group, rather than individual enzyme specificity). The synthesis of a range of such immobilised NAD + derivatives has been described (e.g., [9] and [13]) and some are available commercially. TJaey have been used as effective

237

group-specific steps in purification protocols, but they do not yield the one-step purifications sought as the ideal in affinity chromatographic methodology. The locking-on tactic involves superimposition of individual specificity on this group-specific adsorption. This is achieved by addition of the enzyme-specific second ligand (e.g., a lactate analogue) in soluble, underivatised form to the irrigating buffer. At first sight, this does not seem to be an effective tactic, since the binding of the leading ligand (NAD ÷) would seem to be independent of the following ligand. However, the compulsory order also applies to dissociation, so that when the second ligand binds, it must dissociate before the enzyme can dissociate from the leading ligand. Therefore, at saturating concentrations of the second ligand, dissociation of the first ligand should effectively be blocked. At lower concentrations of the second ligand the adsorption of the enzyme should be reinforced, though not in linear proportion to the concentration of second ligand in the irrigating buffer. This, in a general way, proved to be the case in 'model studies' with lactate dehydrogenase and with several other dehydrogenases, though with as yet unexplained complications, as outlined below.

3.2. Model studies with lactate dehydrogenase: use of oxalate (a competitive inhibitor) in the locking-on mode Lactate dehydrogenase is an enzyme with a compulsory order of substrate addition with NAD ÷ as the leading ligand [12]. It has a poor intrinsic affinity for the 8'-azolinked immobilised NAD ÷ derivative used for the experiment illustrated in Fig. 1. With this immobilised NAD ÷ derivative, the enzyme emerges from the column only marginally retarded beyond the one-column-volume breakthrough position (Fig. la). Oxalate is a competitive analogue of the second substrate, lactate, and as can be seen from Fig. lb, addition of high concentrations of oxalate to the irrigating buffer causes strong reinforcement of the binding of the enzyme, as predicted. That this reinforcement is highly specific is shown by the fact that, firstly, no similar reinforcement is observed when oxalate is replaced by other dicarboxylate ions such as succinate, and secondly by the fact that no reinforcement effect is observed (Fig. lc) when the immobilised NAD + is replaced by the closely analogous NADP ÷ derivative, immobilised in exactly the same way. The affinity chromatographic results with the similarly immobilised AMP analogue were also of mechanistic interest. AMP represents half the NAD ÷ molecule and is a competitive inhibitor against it, but previous studies have indicated that without the nicotinamide half, no binding site for lactate/oxalate is induced [12], and so no lockingon effects should be expected with AMP. The affinity chromatographic results with the AMP analogue (Fig. ld) shows that the barely detectable retardation of LDH by this derivative is not reinforced by the addition of oxalate to

238

P. O'Carra et al. / Biochimica et Biophysica Acta 1297 (1996) 235-243

the irrigating buffer, consistent with a lack of induction of oxalate/lactate binding, and therefore no locking-on effect results. Concentrations of oxalate initially used to achieve this locking-on effect were of the order of 100 mM oxalate. The concentration dependence of this locking-on effect was studied and some results are presented in Fig. 2. It

i l I

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0.4 0.3 "r. 0.2 ~0.1

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0

I

II

+

--NH--CH.--CH--CH~-NH--C~

N NAD

b

4

0.SmMoxalate

~

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g 0.6 -

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8

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m 0.2 gl ,.a 0.1

0

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I

0 --

--

I

~

--

+

--

_..~-.o---~-~--~

io4 f

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,

.

0.2 m M oxalate

&

N.

. - - 9 - ' ~ . ,

I= -~-12 ~- 12 ~100 mM oxalate ~; o.2

6~' ~ ~ =~10Ii 842

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0

,,

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0,1

,.

..

, f

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,

o

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0.6

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;oV A

.~ o.2

~0.1 0.0

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X

N2g-I .-a o ~ . , - #

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. ~

. . . . . . . .

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. . . .

,

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OH

0

I

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[:AO0 mM oxalate

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0

i

i

~ . . . . .

i

. . . .

i

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i

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2 4 6 8 Effluent volume ( column volume units )

i

10

Fig. 1. The locking-on effect experimentally demonstrated by chromatography of porcine heart LDH on the 8'-azo-linked immobilised NAD + derivative in the absence (a) and presence (b) of oxalate. The biospecificity of the effect was checked by chromatographing the enzyme, in the presence of oxalate, on similarly linked immobilised NADP + (c) and AMP (d). The irrigant was 50 mM potassium phosphate buffer (pH 7.4) containing 0.5 M KC1. The presence of oxalate in the irrigating buffer is indicated by the horizontal arrows. Experiments were carried out at room temperature.

4

6

8

10

12

,

14

•

,

16 Effluent volume ( column v o l u m e units )

,

t

18

,

I

20

Fig, 2. Chromatography of porcine heart LDH on an 8'-azo-linked immobilised NAD + derivative in the absence (a) and presence (b to d) of varying concentrations of oxalate. The irrigant was 50 mM potassium phosphate buffer (pH 7.4) containing 0.5 M KC1 and indicated additions. Experiments were carried out at room temperature.

was found that the progressive decrease of the oxalate concentration from the 100 mM originally used produced no apparent weakening of the effect until sub-millimolar concentrations were reached. As can be seen in Fig. 2b, even 0.5 mM oxalate produces strong enhancement of the adsorption, but lower concentrations (e.g., 0.2 mM) promote weaker binding and 0.05 mM oxalate has a negligible effect (Fig. 2c and Fig. 2d, respectively).

3.3. Locking-on with actual substrates by abortive complex formation." model studies with lactate dehydrogenase and pyruvate Competitive inhibitor analogues of a substrate are generally preferable to the substrate itself in affinity chro-

239

P. O'Carra et al. / Biochimica et Biophysica Acta 1297 (1996) 235-243

matography, owing to the problem of catalytic conversion when active substrates are used. However, competitive inhibitor analogues are not always available. With multisubstrate enzymes, one of the active substrates may often be used in affinity chromatography. For example, immobilised N A D ÷, as in studies described above, can be used since catalysis (and hence chromatographic instability) cannot occur in the absence of the second substrate - e.g., lactate in the case of LDH. The use of following substrates (as opposed to their inhibitor analogues) as locking-on ligands might seem precluded, since the combination of such a second substrate with immobilised NAD + would be expected to result in a catalysed reaction. However, in the present study, the use of immobil!ised N A D ÷ in conjunction with the oxidised form of the second substrate was investigated, on the basis of the tendency of many dehydrogenases to form 'abortive' complexes undergoing no catalytic turnover. For example, substrate inhibition of lactate dehydrogenase by high concentrations of pyruvate is well established [14,15] and is generally accepted to be attributable to abortive complex formation on the product side of the kinetic mechanism (as illustrated in Fig. 3). The possibility of exploiting abortive complex formation in the locking-on mode was investigated with model studies of lactate dehydrogenase and pyruvate. All L D H preparations studied here were subject to substrate inhibition by high concentrations of pyruvate, the effect being more pronounced with the H than with the M isoenzymes (this difference has long formed the coruerstone of the most prominent theory concerning differential functions of the two forms [14,15]). As previously shown for porcine L D H (see above), bovine heart or muscle type L D H showed only very raarginal retardation on columns of the immobilised N A D + derivative (e.g., Fig. 4a). Inclusion of 50 m M pyruvate in the irrigant however, resulted in strong adsorption of both types of L D H (e.g., Fig. 4b). However, discontinuation of the locking-on ligand (pyruvate) did not result in auto-elution of LDH. Increasing the ionic strength of the irrigant caused partial elution of LDH, the remainder of the activity being eluted by adding solu-

NADH

E

pyruvate

E NADH

lactate

E pyruvate NaDH ~talysls ~~ ~C, lactate NAD+

lactat~ Abortive complex

NAD +

ENAD+

g

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g NADH

lactate

Abortive complex (non-covalent)

ll OH O ~il l l l ~ ~.llI--NH--ca2"- CH--CH~-NH-- C ~ = N

i

--NAIYr

3.5 o

.~ 3.0 "-- 2.5 =

2.0

~" 1.5 1.0

,=a 0.5 0.0

i

0

2

4

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6

8

10

12

i

i

i

14

16

18

20

Effluent volume ( column volume units )

3.5 _50 mM Pyruvate ~

o

= 3.0 & .~ 2.5

lmM NADH

10 m M 5'-AMP

0.5 M

KCi

2.0

~; .,e e~

].5 1.o

,1 0.5 o.o

, I , I . I . 4

I

I [ . I , 0/*OOO I

8 12 16 20 24 28 32 36 Effluent volume ( column v o l u m e units )

40

Fig. 4. Chromatography of bovine heart LDH on an 8'-azo-linked immobilised NAD+ derivative in the absence (a) and presence (b) of pyruvate. The irrigant was 50 mM potassium phosphate buffer (pH 7.4) and indicated additions. The dashed line represents the point at which irrigation was delayed for 15 min. Experiments were carried out at approx. 15°C.

ble N A D H to the irrigant. Such elution was best achieved by irrigating the column with one column-volume of NADH-containing irrigant and then stopping the flow for 15 min before continuing the irrigation. This apparent 'hysteresis' in the a d s o r p t i o n / d e s o r p t i o n behaviour is considered to be due to formation of a covalent adduct of pyruvate and the immobilised N A D ÷ (such adduct formation has been a much studied phenomenon by Kaplan and his group [14,15]).

F, NAD+ --pyruvate

3.4. Some other dehydrogenases: model studies Abortive complex NA (covalent) gpy~ru~vate

Fig. 3. The reactions catalysed by lactate dehydrogenase showing the formation of the abortive temar~ complexes.(E = lactate dehydrogenase).

3.4.1. Alcohol dehydrogenase with h y d r o x y l a m i n e / pyrazole Previous attempts to purify alcohol dehydrogenase have involved the use of immobilised A M P derivatives to ad-

240

P. O'Carra et al. / Biochimica et Biophysica Acta 1297 (1996) 235-243

I~il

OH

O

~.H--NH--CH2-- ~H--CH~'- NH-- ~ ~ - - - - N a

--NAD+

4

.o &

3

e2

.<

1

e~ .<

b 100 mM 7 hydroxylamine

l m M NAD +

r

6

.=

5 4

.<

3

e~

2 1 0

| , i , o--c~

C

, .\~/~"'o_i i ' ~ I l I , I

100 m M

o=

50

~

l mM NAD +

~

& 4o 30 ,~ =

2o

<

10

°

O

/ ~ I , I ,

2

4

, I

6

8

~o-I,

10

12

14

l~ll

16

I

18

20

Effluent volume ( column volume units )

Fig. 5. Chromatography of yeast alcohol dehydrogenase on an 8'-azo-linked immobilised NAD + derivative in the absence (a) and presence of hydroxylamine (b) or pyrazole (c). The irrigant was 100 mM pyrophosphate buffer (pH 8.8) and indicated additions. Experiments were carried out at approx. 15°C.

sorb the enzyme, followed by elution with NAD +, either alone or together with analogues of the specific substrate (e.g., hydroxylamine, cholic acid and pyrazole; [16,17]). One exception to this approach was the use of isobutyramide [18] to lock horse liver ADH onto an immobilised NADH derivative, elution of the enzyme being achieved by discontinuation of the amide in the buffer. In the present study, the inhibitors hydroxylamine and pyrazole were investigated as potential 'locking-on' ligands for ADH from yeast. Fig. 5 shows that these two inhibitors had the effect of locking the ADH onto an

8-azo-linked immobilised NAD + derivative-gel. Discontinuation of hydroxylamine from the irrigant resulted in elution of the ADH, although the elution was not very sharp (Fig. 5b) and activity continued to elute off over a number of fractions, being completed by adding (soluble) NAD + to the irrigating buffer (Fig. 5b). Where pyrazole was used as locking-on ligand, the activity did not automatically elute from the affinity gel when the pyrazole was discontinued, elution only being achieved by subsequent addition of NAD + to the irrigant (Fig. 5c), as in the case of LDH (above). The cause of this continued adsorption is unclear. The possibility that some form of covalent adduct might have formed between the immobilised NAD + and the locking-on ligand seemed to be ruled out by spectrophotometric examination of the gel which showed no change in UV-absorption spectrum of the immobilised NAD +. A similar problem was encountered with pyruvate-promoted locking-on of LDH (see above).

3.4.2. Malate dehydrogenase with oxalacetate As mentioned above, previous work with malate dehydrogenase (MDH) exemplified the difficulties of developing immobilised specific ligands. In this study, attempts to use soluble inhibitor analogues of malate (e.g., tartronic acid) as locking-on ligands were equally unsuccessful. Locking-on to immobilised NAD + with the substrate oxalacetate in an abortive complex mode proved more successful, as can be seen from Fig. 6b. MDH, like the other dehydrogenases, is only very marginally retarded by the immobilised NAD + derivative, but inclusion of oxalacetate in the irrigant caused adsorption of the mitochondrial enzyme, although some leakage of the enzyme off the column was observed (Fig. 6b). Cytoplasmic MDH, however, was not detectably retarded under similar conditions (Fig. 6b). This is consistent with the observation that the mitochondrial enzyme is much more subject to substrate inhibition by oxalacetate than the cytoplasmic enzyme [19], presumably as a result of a greater tendency to form the abortive complex. 3.4.3. Glutamate dehydrogenase: model studies and puro% cation Glutarate is a competitive inhibitor of bovine glutamate dehydrogenase (GDH) already used in the affinity precipitation mode [3,4]. Model studies (using a commercial preparation of bovine liver GDH) demonstrated partial retardation of the enzyme on the immobilised NAD + derivative when 50 mM glutarate was included in the irrigant (Fig. 7b). Increasing the glutarate concentration to 350 mM converts the relatively poor retardation into a much stronger retardation (Fig. 7c). Discontinuation of the glutarate results in reversion to the original weak affinity and auto-elution of the enzyme (Fig. 7c). This locking-on system is one of the methods we have developed for actual purification, in this case the commercially important glutamate dehydrogenase from bovine liver.

P. 0 'Carra et al. / Biochimica et Biophysica Acta 1297 (1996) 235-243

[~ [ ] i

OH

O

I

II

~" I - - NH -- c a 2 - C H - - CH !,,'-NH-- C ~

~

mo 0.6

o.5 &

0.4

2

=

= 0.3

"~ 0.2 < =

0.1 J

:~ o.o

iI

a

o

CH--CH~'NH--C ~

~-N --NAD+

~0.4 ~" 0.3

21~MID~

DH 0.2 .~. < = 0.1 L~

i~ko'~ . , , , , , . , . , . , . , 4 6 8 10 12 14 16 18 20

0

?"

~i--NH--CHf"

= N --NAD+

241

Effluent volume ( column volume units )

,Om b

~ =

0.7 50 m M " oxalacetate 0.6 ~~

0.4

r2/i

0.5 "-= 0.4

.= 0.3

.......... A Mitoehondrial C3"uP . . . . . . . . . r ~ M D H

0.3 .~

~

1 mM NADH + 0.5 M KCI

0.2 0.1 0.0

";

0.2 0.1

/,

0

. . T~.: . . . . . . . ~...:. , , , , , , , 2 4 6 8 10 12 14 16 Effluent volume ( e o l u m m volume units )

e

~

Fig. 6. Chromatography of cytoplasmic and mitochondrial malate dehydrogenase on an 8'-azo-linked immobilised NAD + derivative in the absence (a) and presence (b) of oxalacetate. The irrigant was 50 mM potassium phosphate buffer (pH 7.4) and indicated additions. Experiments were carried out at approx. 15°C.

350 m M

/

0.6

O.4 <

Initial application of the glutarate locking-on method to extracts from bovine liver were not encouraging, as shown in Fig. 8a. However, this apparent lack of locking-on turned out to be due largely to interference by unidentified factors in the crude extracts. When the extracts were subjected to gel filtration on a Sephadex G-25 column prior to affinity chromatography, however, the locked-on adsorption was greatly increased (Fig. 8b). From this it was concluded that some low molecular weight element(s), present in the crude extract:s, interfered with ligand-promoted adsorption of the enzyme (the most obvious possibilities being NAD +, NADH, ATP ADP, AMP). The viscous nature of the crude extracts also interfered with the chromatography, clogging the columns and also damaging the gel. Inclusion of a preliminary ion-exchange purification

1.0

0.2 O.C

-

0

.

2

,

.

,

.

,

,

,

. ""~--,o---¢......¢

,

.

,

4 6 8 10 12 14 16 18 Effluent volume ( column volume units )

.

,

20

Fig. 7. Chromatography of bovine liver glutamate debydrogenase on an 8'-azo-linked immobilised NAD + derivative in the absence (a) and presence (b and c) of glutarate. The irrigant was 50 mM potassium phosphate buffer (pH 7.4) and indicated additions. Experiments were carried out at approx. 15°C. Before commencement of irrigation, the sample was allowed to equilibrate for 10 minutes at the top of the column.

step not only minimised such interfering factors, but also reduced the protein concentration and the volume of the sample for subsequent application to the affinity gel, all factors resulting in improved capacity and performance, particularly in large-scale purifications.

Table 1 Purification of glutamate dehydrogenase from bovine liver Fraction

% Yield

Specific activity ( ~ m o l / m i n per mg)

Purification factor

Clarified supernatant 20-70% ammonium sulfate cut DEAE-Sepharose Affinity chromatography

100.0 100.7 94.9 71.2

0.03 0.05 0.24 33.70

1.0 1.7 7.6 1053.0

242

P. 0 'Carra et al. / Biochimica et Biophysica Acta 1297 (1996) 235-243

Locking-on affinity chromatography, with glutarate as locking-on ligand, resulted in the purification of GDH (Table 1) as shown by S D S - P A G E of the purified enzyme (Fig. 9). Electrophoretic gels loaded with 2 5 - 5 0 /~g of protein, electrophoresed and stained for protein with amido black, showed one very strong band corresponding to G D H (55 kDa) and two very faint bands of higher molecular weight which appear to correspond to proteins present at high relative levels in the ion-exchange eluate prior to affinity chromatography. These extra bands are only visible when the gels are grossly overloaded and are not visible in the gels shown in Fig. 9. Their persistence in the affinity chromatographic eluate, although in greatly reduced relative concentrations, suggests a p a r t i a l / w e a k retardation by the immobilised N A D + derivative. Loading of SDS gels with greater amounts of protein revealed the presence of three additional minor contaminants with ap-

i lImI OH O I I ~ i - - N H -- CH2==CH--CH ~" NH-- C ~

=N --NAD

6 350 mM glutarate

P1

~3

'breakthrough' peak

"7.

'~2 < 1

0

i

2

4

6

8

10

12

Effluent volume ( column volume units )

5

b

350 mM glutarate ' ~a k t h ro u g h ' peak

~3 "~ 2 1

0

'-

0

-

v

2

4

v'

6

8

3

10

J

12

4

94 0 0 0 ~ 67 000------~"

tb

o

43000------~-

o

30 000------~

20 1 0 0 ~

14 0 0 0 ~

Anode

+

-

g-a

Cathode

Fig. 9. Protein-stained SDS polyacrylamide gel electropherograms of glutamate dehydrogenase (GDH) from bovine liver. Lanes 1 and 3: protein standards. Lanes 2 and 4: GDH purified by ion-exchange chromatography followed by affinity chromatography on an 8'-azo-linked immobilised NAD+ derivative using glutarate as locking-on ligand (Fig. 8b). proximate subunit molecular weights of 4.2 kDa, 47.8 kDa and 51.3 kDa. Thus, although the affinity step results in a preparation very greatly enriched in GDH, the enzyme invariably emerges contaminated with other proteins. This contamination is probably due to the presence of other dehydrogenases which are retarded to varying degrees by the immobilised N A D + derivative, some of which 'dribble' off the affinity gel over the complete elution profile. An outcome of the locking-on approach is that when an enzyme is locked onto an immobilised N A D + derivative, soluble fragments of N A D + (such as 5'-AMP) will not elute the dehydrogenase from the matrix because the N M N is an essential requirement for specific substrate recognition (e.g., LDH [1,12]). The AMP, however, continues to competitively elute other dehydrogenases which are not locked-on, and thus these are effectively 'stripped' off the immobilised N A D + derivative and will not contaminate the subsequently eluted dehydrogenase. This tactic could improve the purification of GDH and other dehydrogenases, and the further development of this approach is part of our on-going research interest.

Effluent volume ( column volume units )

Fig. 8. Chromatography of crude bovine liver glutamate dehydrogenase (the 20-70% saturated ammonium sulfate cut of liver extract) on an 8'-azo-linked immobilised NAD + derivative, in the presence of glutarate, before (a) and after (b) gel filtration chromatography on Sephadex G-25. The irrigant was 50 mM potassium phosphate buffer (pH 7.4) and indicated additions. Experiments were carried out at approx. 15°C. Before commencement of irrigation, the sample was allowed to equilibrate for 10 rain at the top of the column.

References

[1] O'Carra, P. (1978) in Chromatography of Synthetic and Biological Macromolecules (Epton, R., ed.), Chap. 11, pp. 131-158, Ellis Horwood for the Royal Chemical Society, London. [2] Buchanan, M., O'Dea, G., Griffin, T.O. and Tipton, K.F. (1989) Biochem. Soc. Trans. 17, 422.

P. O'Carra et al. / Biochimica et Biophysica Acta 1297 (1996) 235-243

[3] Flygare, S., Griffin, T., Larsson, P.-O. and Mosbach, K. (1983) Anal. Biochem. 133, 409-416. [4] Graham, L.D., Griffin, T., E;eatty, R.E., McCarthy, A.D. and Tipton, K.F. (1985) Biochim. Biophys. Acta 828, 266-269. [5] Larsson, P.-O. and Mosbach, K. (1979) FEBS Lett. 98, 333-338. [6] Irwin, J. and Tipton, K. (19'95) Essays Biochem. 29, 137-155. [7] Cuatrecasas, P. (1970) J. Biol. Chem. 245, 3059-3065. [8] Barry, S. and O'Carra, P. (1973) Biochem. J. 135, 595-607. [9] O'Carra, P., Barry, S. and Griffin, T. (1974) FEBS Lett. 43, 169-175. [10] Bradford, M.B. (1976) Anal. Biochem. 72, 248. [11] Laemmli, U.K. (1970) Nature 227, 680-685. [12] O'Carra, P. and Barry, S. (1972) FEBS Lett. 21,281-285. [13] Lowe, C.R. and Mosbach, K. (1974) Eur. J. Biochem. 49, 511-520.

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[14] Everse, J. and Kaplan, N.O. (1973) in Advances in Enzymology (Meister, A., ed.), Vol. 37, pp. 61-133, Academic Press, New York. [15] Everse, J. and Kaplan, N.O. (1975) in Isozymes 1I - - Physiological Function (Market, C.L., ed.), pp. 29-43, Academic Press, New York. [16] Andersson, L., Jornvall, H., ~,keson, A. and Mosbach, K. (1974) Biochim. Biophys. Acta 364, 1-8. [17] Andersson, L., Jornvall, H. and Mosbach, K. (1975) Anal. Biochem. 69, 401-409. [18] Andersson, L., Larsson, P.-O. and Mosbach, K. (1978) FEBS Lett. 88, 167-171. [19] Delbruck, A., Schimassek, H., Bartsch, K. and Bucher, T. (1959) Biochem. Z. 331,297-304.