Resolution of thiol-containing proteins by sequential-elution covalent chromatography

Journal of Biochemical and Biophysical Methods, 4 (1981) 101--111

101

© Elsevier/North-Holland Biomedical Press

RESOLUTION OF THIOL-CONTAINING PROTEINS BY SEQUENTIALELUTION COVALENT CHROMATOGRAPHY

DAVID A. HILLSON *

Biological Laboratories, University of Kent, Canterbury, CT2 7NJ, Kent, U.K. (Received 5 August 1980; accepted 21 September 1980)

Elution of complex protein mixtures on a matrix containing reactive disulphide bonds (Thiopropyl-Sepharose 6B, Pharmacia) results in immobilisation of thiol-containing molecules. Specific protein fractions can be displaced from the gel using different low-molecular-weight reducing agents. Thus a single sequential elution can separate and resolve thiol-containing proteins in a rapid and convenient step. The method is illustrated with reference to beef liver thiol : disulphide oxidoreductases. Key words: covalent chromatography; Thiopropyl-Sepharose 6B; thiol : disulphide oxidoreductases.

INTRODUCTION

Covalent chromatography involving immobilisation of thiol-containing molecules on a matrix by formation of mixed disulphide bonds, with subsequent elution by reducing agents, has become recognised as a powerful separation technique; the principles of covalent chromatography have recently been discussed [1,2]. The technique has mainly been used to separate or purify thiol-containing proteins, such as papain [ 3 ], urease [ 4--6 ], ficin [ 7 ], type II procollagen [8], type III collagen [9], various human plasma proteins [10,11], ornithine decarboxylase [12], streptolysin O [13] metallothionein [14--16], and 7-glutamyl cyclotransferase [17]. In some of these cases, rapid purification of the protein has been achieved from quite crude sources. Covalent chromatography has also been used to isolate thiol-containing peptides from digests of several proteins [18--20], to purify synthetic thiolcontaining peptides [ 21 ], and to immobilise enzymes [ 22,23 ] or erythrocyte membrane fragments [24].

* Present address: Biophysics Laboratories, Portsmouth Polytechnic, White Swan Road, Portsmouth, P01 2DT, Hampshire, U.K. Abbreviations: PDI, protein disulphide-isomerase (EC 5.3.4.1); GIT, glutathione-insulin transhydrogenase (protein--disulphide oxidoreductase (glutathione), EC 1.8.4.2); DTT, dithiothreitol; SDS--PAGE, sodium dodecyl sulphate--polyacrylamide gel electrophoresis.

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However, in addition to separating thiot-containing molecules from those without this group, the technique can also, in principle, allow resolution and fractionation between different thiol-containing molecules [1]. This could be achieved if elution conditions were such that the reactivity of the thiol group in one class of molecule towards the functional groups of the gel differs significantly from that in another class of molecule. This situation might be expected to arise in enzymes possessing active-site thiols, since the properties of these groups witl be affected by their mieroehemieal environment (i.e. aetive-site structure and surrounding secondary and tertiary structure). Thus different thiol-containing enzymes might bind to the covalent chromatography matrix with different affinities, and require reducing agents of different strengths to displace them from the gel. The principle of this successive elution to fractionate thiol-containing proteins is illustrated diagramatically in Fig. 1, where three such proteins are differentially and specifically displaced by three low-molecular-weight thiolso Obviously the selectivity of such a procedure depends on the difference in reducing strength of the thiols used for each step; one could either employ increasing concentrations of a given thiol, or use different thiols in a discrete step-wise procedure. Previous work in this laboratory has addressed the question of the relationship between two thiol : protein- disulphide oxidoreductase activities, protein disulphide-isomerase (PDI; EC 5.3.4.1) and glutathione-insulin transhydrogenase (GIT; protein--disulphide oxidoreductase (glutathione), EC 1.8.4o2), [25--29,42]° This has been the subject of some controversy [30--35]. Various indirect and circumstantial evidence suggests that the t w o activities are due to distinct proteins, but the sole conclusive criterion is whether or not the activities can be resolved from one another. For both activities, an active-site thiol group has been implicated [36,37]; this raised the possibility that distinct protein species, if present, might be separated by covalent chromatography. In attempting to resolve PDI and GIT activities from partially purified beef liver preparations, a sequential-elution covalent chromatographic protocol was developed, in which the activities are bound to a gel containing reaeo tive disulphide bonds (Thiopropyl-Sepharose 6B, Pharmacia), and successively displaced by a series of reductive elutions with increasing reducingpower. L-Cysteine, reduced glutathione, and dithiothreitol (DTT) were used as the eluants in this procedure; these partieular thiols were also chosen because of the possibility of specific reaction with either protein disulphideisomerase (which acts on cystine linkages in proteins, and is assayed in the presence of DTT) or glutathione-insulin transhydrogenase (for whieh reduced glutathione is a substrate)o The sequential-elution covalent chromatography procedure developed has achieved resolution o.f PDI and GIT activities, thus confirming that the two activities are n o t catalysed by a single enzyme, and demonstrating that the principle of fraetionation of different thiol-containing molecules [ i ] is

103

~'~0 "<-° -~'~'~ PROTEIN ~ ~=Oi~, SAMPLE

was.,

,,o o , i

@,Qo,,~

~,o c>,,~ ../9,,@0,,~ COLUMN ~-'~Y":'5%t ~= ~ G,

* R.zSH

I@-e% l ÷ ~-SS-RI

us

-* R;.SS-R~

+ R~-SS-R, Fig. 1. T h e principle of s e q u e n t i a l - e l u t i o n c o v a l e n t c h r o m a t o g r a p h y . W h e n a m i x t u r e of p r o t e i n s is applied t o a c o l u m n o f T h i o p r o p y l - S e p h a r o s e 6B, o n l y t h o s e p r o t e i n s c o n t a i n ing t h i o l g r o u p s are b o u n d . T h e s e can be selectively displaced f r o m t h e c o l u m n b y a series o f l o w - m o l e c u l a r - w e i g h t thiols, w h o s e r e d u c i n g s t r e n g t h increases R 1 - - S H < R2--SH < R3--SH. A t e a c h e l u t i o n step, t h e eluate c o n t a i n s displaced p r o t e i n a n d t h e oxidised f o r m of the eluant, R--SS--R.

104

indeed practicable. A preliminary account of some of this work has been published elsewhere [27]. MATERIALS

AND

METHODS

Materials Chromatographic gels were obtained from Pharmacia (G.B.) Ltd., randomly reoxidised ribonuclease was from Miles-Seravac Ltd., Slough, Bucks., U.K, highly polymerised yeast RNA was from Koch-Light Laboratories Ltd., Colnbrook, Bucks., U.K., and other biochemicals were from Sigma (London) Chemical Co. Ltd., Poole, Dorset, U.K. Solvents and other chemicals were obtained from Fisons Scientific Apparatus Ltd., Loughborough, Leics., U.K.

Covalent chromatography on Thiopropyl-Sepharose 6B The standard buffer for covalent chromatography contained 50 mM TrisHC1, pH 7.5/25 mM KC1/5 mM MGCI2/1.25 mM E D T A / 0 . ! M NaC1 (TKM/ EDTA/NaC1 buffer). Thiopropyl-Sepharose 6B was used in the disulphide form, in which a matrix-bound thiol is protected by participation in a mixed disulphide with a 2-thiopyridone moiety; this was swollen and washed free of additives in TKM/EDTA/NaC1 buffer (200 ml per g of freeze-dried gel). Protein samples (25--125 mg) were prepared by incubating at 30°C for 30 min at 10 mg/ml in TKM/EDTA/NaC1 buffer, in the presence of 0.1 mM dithiothreito] (DTT). This gentle reduction activates protein-borne thiol groups which may be masked by participation in mixed disulphides, b u t the conditions used are not sufficient to reduce protein disulphide bonds. In some samples, particularly the crude preparations, a small a m o u n t of protein precipitated during the reduction, possibly due to aggregation caused by formation of intermolecular disulphides between activated thiols, or due to the raised temperature. This precipitate was discarded. Reduced protein was separated from DTT and precipitated protein by elution at r o o m temperature from a column of Sephadex G-25 (2 X 25 cm), using TKM/EDTA/NaCI buffer. Two methods of coupling the reduced protein to the gel were investigated, using 30--45 g wet weight of gel. Either the sample was loaded onto a column of Thiopropyl-Sepharose 6B, followed b y incubation at 30°C for 30--60 min, or reduced protein and gel were incubated together at 25°C for 16 h with gentle shaking prior to pouring the column. For both methods, about 60% of the sample was retained b y the gel, b u t 50% of the sample (i.e. 80% of the retained material) was non-covalently b o u n d in the direct-loading method, as compared to 30% of the sample (i.e. 50% of the retained material) in the batch incubation. Thus, batch incubation with shaking results in a higher ievel of coupling of reduced proteins to the gel via formation of prot e i n - g e l mixed disulphides. This is therefore the preferred method for sample loading.

105 After loading, the column was equilibrated to 4°C and washed with TKM/ EDTA/NaC1 buffer to remove u n b o u n d and non-specifically adsorbed prorein. Bound proteins were then displaced from the gel by successive elution with different low-molecular-weight thiol compounds, used in order of increasing reducing power, namely 20 mM L-cysteine, 50 mM glutathione, and 20 mM DTT, each in TKM/EDTA/NaC1 buffer, pH 7.5. For each thiolcontaining step, an aliquot of eluant approximately equal to one void volume was run into the column, which was then incubated at 30°C for 30 min to allow reaction to occur. Elution was then continued at 4°C at flow rates of 2--10 ml/h (controlled b y a Watson-Marlow MHRE 2 peristaltic pump), collecting 5-ml fractions. A wash step using TKM/EDTA/NaC1 buffer alone was included after each thiol-containing step. Eluted fractions were monitored for protein at 280 nm and for displaced 2-thiopyridone at 343 nm. For each step, fractions containing protein were pooled, and solid DTT was added to give a final concentration of 5 mM. The pooled fractions were incubated at 25°C for 30 min to reduce any mixed disulphides formed between protein and eluant, then dialysed extensively against TKM buffer, pH 7.5, at 4°C. The procedure resulted in preparation of four protein fractions, namely u n b o u n d protein washed through the column (fraction W), and material displaced by L-cysteine (fraction I), glutathione (fraction II) and DTT (fraction III) respectively. In some runs, fractions eluted during the L-cysteine step contained a white precipitate of insoluble cystine formed during the displacement reaction, b u t this was removed during the standard treatment with DTT followed by dialysis. Protein fractions were stored at 4°C prior to assays of protein and enzymic activities, or frozen at --20°C for long4erm storage. After use in a n elution, reduced gel w a s e q u i l i b r a t e d for storage with 0.1 M sodium acetate buffer, pH 5, containing 20 mM DTT, and maintained at 4°C to ensure minimal reoxidation of free thiol groups in the matrix.

Regeneration of Thiopropyl-Sepharose 6B The recommended method for regeneration of thiol-binding capacity of covalent chromatographic gels is that of Brocklehurst et al. [3]. This is suitable for matrices with a low level of substitution (1--5 pmol ligand per ml swollen gel), b u t proved inadequate for regeneration of ThiopropylSepharose 6B which is more highly substituted (approx. 20 pmol ligand per ml). A more vigorous method (D. L o w (Pharmacia), pers. comm.) was therefore used. Thiopropyl-Sepharose 6B (50 ml) was completely reduced by incubation with 20 mM DTT at pH 8, 30°C, for 30 min, and then extensively washed at the p u m p with 1 mM HCI (500 ml), followed b y I mM HCI/1 mM EDTA (100 ml), and 0.2 M sodium borate buffer, pH 8/1 mM EDTA (100 ml). The washed gel was then transferred to a r o u n d - b o t t o m e d flask fitted with a reflux condenser, and heated to 80°C in a water bath. A 20 ml aliquot of

106 2,2'-dithiodipyridine (160 mM in absolute ethanol or propan-2-ol) was then added, followed by refluxing at 80°C for 3 h, after which time the water bath was switched off and the mixture allowed to cool overnight. The gel was then washed at the pump with ethanol or propan-2-ol (500 ml), followed by borate/EDTA (I00 ml), and TKM/EDTA/NaCI buffer (250 ml). Regenerated gel was stored at 4°C and used within 2 days.

Preparation of pro rein samples A partially purified sample of protein disulphide-isomerase (PDI) was prepared from beef liver by the standard method [26] up to the elution from DEAE-Sephadex A-50 with 0°1 M Tris-HC1 buffer pH 7.8. This material was freeze-dried; sodium dodecyl sulphate--polyacrylamide gel electrophoresis (SDS--PAGE) showed ten protein bands. Several thiol : disulphide oxidoreductase activities were present, including PDI activity measured with various thiols, and glutathione-insulin transhydrogenase activity [281. A crude preparation of PDI was prepared from beef liver by the method of Carmichael et al. [38], up to the ammonium sulphate fractionation. Protein precipitating between 60 and 85% (NH4)2SO4 saturation was resuspended in 0.I M phosphate buffer pH 7.5 containing 5 mM EDTA; this material showed 15--20 protein bands in SDS--PAGE~ and exhibited several thiol : disulphide oxidoreductase activities. Enzyme and protein assays. Enzyme activities were measured using procedures and units described by Ibbetson and Freedman [25]. Protein disulphide-isomerase activity was measured by the dual-wavelength spectrophotometric method following reactivation of randomly reoxidised ribonuclease towards highly polymerised yeast I%NAo Glutathione-insulin transhydrogenase was assayed by the linked method using glutathione reductase and monitoring oxidation of NADPH. Protein concentration was routinely determined by the Folin--Ciocalteu method [391, using serum albumin (fraction V) as standard. SDS--PAGE was carried out in 1.5 mm slab gels With 7.5% acrylamide concentration, using a modification [40] of the system of Laemmli [41], and staining with Coomassie Brilliant Blue 1%250. RESULTS

Resolution from purified protein mixtures In the standard purification procedure for protein disulphide-isomerase, both PDI and GIT activities behaved as in previous purifications [26]. Both appeared in the same fractions, but did not copurify quantitatively. The active material eluted from DEAE-Sepharose A-50 in 0.1 M Tris-HC1 buffer, pH 7.8, showed protein disulphide-isomerase specific activities of 50--70 units/g and gtutathione-insulin transhydrogenase specific activities of 6--9 units/g.

107

TABLE 1 Sequential-elution covalent chromatography of partially purified material Fraction

Fraction W Fraction I Fraction II

Fraction III

Protein disulphide-isomerase

Glutathione-insulin transhydrogenase

Specific activity (units/g)

Yield a (%)

Specific activity (units/g)

Yield a (%)

35.4 32.5 46.6 57.1 19.5

81.2 82.7 88.1 18.8 17.3

8.0 8.1 1.8 n.d. b n.d.

94.6 91.4 96.6 0 0

14.8 n.d. n.d. n.d.

11.9 0 0 0

n.d. i.I n.d. n.d.

0 0.7 0 0

n.d.

0

7.9

4.7

n.d. n.d.

0 0

1.4 0.5

8.6 3.4

a Yield expressed as percentage of total activity recovered. b n.d., not detectable. This partially purified material was reduced by dithiothreitol, and fractionated by covalent c h r o m a t o g r a p h y on Thiopropyl-Sepharose 6B using the sequential-elution p r o t o c o l with three reducing steps. The procedure resulted in collection o f f our fractions of protein, as described under Methods, namely fractions W, I, II and III. The total yield of protein recovered in unb o u n d and b o u n d fractions was a b o u t 73% of the starting material (range 58--91% in six experiments); losses in protein probably occurred mainly at the r ed u ctio n step, due to a small a m o u n t of precipitation during incubation with DTT, although ot he r small losses are likely when pooling fractions containing protein since it is often uneconomical to include the tail of a peak. The yields o f protein in the prepared fractions were, on average, as follows (expressed as a percentage of total protein recovered): fraction W, 56%; fraction I, 16%; fraction II, 15%; fraction III, 12%. Each fraction was assayed for protein and enzymic activities. The results from three typical experiments are given in Table 1. Both enzymic activities were present in the protein fraction n o t retained by the gel (fraction W), probably as a result of overloading or incomplete reaction. In fact, 80--90% of the recovered PDI activity and 90--95% GIT activity were f o u nd in this fraction (Table I). Other workers have also n o t e d that covalent c h r o m a t o g r a p h y gels exhibited actual binding capacities much lower (by up to 90%) than the theoretical m axi m um , for example with papaln [1], and have implicated steric considerations in preventing formation o f protein--gel mixed disulphides. Despite the low recoveries of e n z y m e activities in b o u n d fractions (10--

108

20% of PDI activity, and 3--10% of GIT activity), resolution was achieved by the sequential-elution method. Thus protein disulphide-isomerase activity was found only in fraction I, with no associated glutathione-insulin transhydrogenase activity, fraction II showed no detectable activities except a smaU a m o u n t of GIT in one run, and fraction IU contained GIT activity but no detectable PDI activity. Although on the whole, the enzyme activities were n o t purified as compared to the starting material, it is nevertheless clear that the measured PDI and GIT activities do n o t copurify, and can be resolved from the partiallypurified preparation by this covalent chromatography procedure.

Resolution from crude protein mixtures Material precipitating between 60 and 85% ( N H 4 ) 2 S O 4 saturation in the procedure of Carmichael et al. [38] was resuspended, and subjected to the sequential-elution covalent chromatography protocol. Paraltel assays of protein disulphide-isomerase and glutathione-insulin transhydrogenase activities were performed on the fractions obtained, and the results from three typical elutions are given in Table 2. As in the covalent chromatography experiments described above, both PDI and GIT activities were present in the u n b o u n d fraction (fraction W), probably as a result of overloading. Protein disulphide-isomerase activity was

TABLE 2 S e q u e n t i a l - e l u t i o n covalent c h r o m a t o g r a p h y of a crude p r e p a r a t i o n Fraction

60--85% (NH4)2SO 4 f r a c t i o n Fraction W

Fraction I

F r a c t i o n II

F r a c t i o n III

P r o t e i n disulphideisomerase

Glutathione-insutin transhydrogenase

Specific activity (units/g)

Yield a (%)

Specific activity (units/g)

Yield a (%)

11.9 0.2 9.9 0.4 23.0 27.9 25.2 4.0 1.1 n.d. 6.8 4.3 0.6

-1.1 6.2 1.1 72.7 92.1 98.7 2.9 0.1 0 23.3 1.6 0.2

0.83 n.d. b 0.19 n.d. n.d. n.d. n.d. n.d. 0.16 n.d. 0.66 0.50 0.81

-0 10.2 0 0 0 0 0 0.8 0 100.0 89.0 100.0

a Yield e x p r e s s e d as p e r c e n t a g e o f total activity recovered. b n.d., n o t d e t e c t a b l e .

109 purified about 2-fold in fraction I, and this fraction did not contain any detectable GIT activity. Conversely, glutathione-insulin transhydrogenase activity appeared mainly in fraction III, with some associated PDI activity. Very low activities were obtained in fraction II. These results are qualitatively similar to the behaviour of PDI and GIT activities during sequentialelution covalent chromatography of the partially purified material (above). However, percentage yields of enzyme activities in b o u n d fractions were much higher in elutions of the crude material (70--98% of PDI activity in fractions I, and 89--100% of GIT activity in fractions III). This may reflect the rapid purification of the crude material, as opposed to the longer preparation time for the partially purified material which could result in partial inactivation. The sequential-elution covalent chromatography procedure described here successfully and reproducibly effects resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase activities from both purifled and more crude starting material, and is a powerful tool for the rapid separation and fractionation of these proteins. DISCUSSION The question of the relationship between protein disulphide-isomerase and glutathione-insulin transhydrogenase activities can be settled by the complete physical resolution of these activities from One another. This has been achieved using the sequential-elution covalent chromatography procedure described in this paper, starting either from material partially purified by the conventional PDI method [26], or from a more crude material obtained by an alternative method [38]. These results both confirm and extend previous circumstantial evidence [25--29], and provide final and conclusive evidence of the existence in beef liver of at least two distinct thiol : disulphide oxidoreductases. Recent work in this laboratory has shown that protein disulphide-isomerase can be rapidly purified from crude wheat germ preparations using the sequential-elution covalent chromatography procedure described in this paper, suggesting that the method is applicable to a variety of systems (L. Roden and R.B. Freedman, unpubl, obs.). The results obtained in resolution of two beef liver thiol : disulphide oxidoreductases clearly demonstrate that the principle of'fractionation of different thiol-eontaining molecules by covalent chromatography [I] is practicable, since sequential elution using reducing agents of increasing strength can effect differential and specific displacement of bound proteins from the gel (Fig. 1). It is also clear that sequential-elution covalent chromatography using matrix-bound disulphides is a very powerful technique for purification of thiol-containing proteins, since it shows absolute specificity for such proteins. Fractionation and resolution can be achieved from quite pure prepara-

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tions, but the method is also applicable to the selective purification of a small number of proteins from relatively crude material. This extends previous work in which proteins have been rapidly purified from crude sources, and allows considerable economies to be made both in time and materials required for a purification procedure. The development of a sequentiM elution protocol considerably increases the range of applicability of covalent chromatography, and should prove useful in both selective purification and resolution of a variety of thiol-containing molecules. SIMPLIFIED DESCRIPTION OF THE METHOD AND ITS APPLICATIONS Application of a mixture of proteins to a column of Thiopropyl-Sepharose 6B results in binding of thiol-containing proteins. These are selectively displaced using a sequential series of eluant buffers containing reducing agents of increasing strength, namely, 20 mM L-cysteine, 50 mM reduced glutathione and 20 mM dithiothreitol. Thus purification and resolution of thiol-containing proteins can be achieved in a single chromatographic step. The method is useful both for rapid isolation of thiol-containing proteins from crude mixtures and for clean separation between different species of these proteins. ACKNOWLEDGEMENTS This work was supported by a Quota Award from the Science Research Council of Great Britain. I wish to thank Dr. Robert Freedman for his help and advice throughout the course of this work, and Drs. Elizabeth Hill and Duncan Low (Pharmacia) for assistance with the regeneration procedure. REFERENCES 1 Brocklehurst, K., Carlsson, J., Kierstan, M.P.J. and Crook, E.M. (1974) Meth. Enzytool. 34B, 531--544 2 Brocklehurst, K. (1979) Int. J. Biochem. 10, 259--274 3 Brocklehurst, K., Carlsson, J., Kierstan, M.P.J. and Crook, E.M. (1973) Biochem. J. 133, 573--584 4 Carlsson, J., Ax@n, R.,'Brocklehurst, K. and Crook, E.M. (1974) Eur. J. Biochem. 44, 189--194 5 Carlsson, J., Olsson, I., Ax@n, R. and Drevin, H. (1976) Acta Chem. Scand. B30, 180--182 6 Norris, R. and Brocklehurst, K. (1976) Biochem. J. 159, 245--257 7 Malthouse, J.P.G. and Brocklehurst, K. (1976) Biochem. J. 159, 221--234 8 Angermann, K. and Barrach, H.-J. (1979) Anal. Biochem. 94, 253--258 9 Sykes, B.C. (1976) FEBS Left. 61,180--185 16 Laurell, C.-B, Thulin, E. and Bywater, R.P. (1977) Anal. Biochem. 81, 336--345 11 Laurell, C.-B. (1978) J. Chromatogr. 159, 25--31 12 Obenrader, M.F. and Prouty, W.F. (1977) J. Biol. Chem. 252, 2860--2865 13 Prigent, D., Geoffroy, C. and Alouf, J.E. (1978) Comptes Rendues D287, 951--954 14 Squibb, K.S. and Cousins, R.J. (1977) Biochem. Biophys. Res. Commun. 75, 806-812 15 Shapiro, S.G., Squibb, K.S.~ Markowitz, L.A. and Cousins, R.J. (1978) Biochem. J. 175,833--840

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