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Multiple Enzyme Purifications from Muscle Extracts by Using Affinity-Elution- Chromatographic Procedures

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Multiple Enzyme Purifications from Muscle Extracts by Using Affinity-Elution- Chromatographic Procedures Biochem. J. (1977) 161, 265-277 265 Printed in Great Britain Multiple Enzyme Purifications from Muscle Extracts by using Affinity-Elution- Chromatographic Procedures By ROBERT K. SCOPES Department ofBiochemistry, La Trobe University, Bundoora, Vic. 3083, Australia (Received 20 July 1976) 1. Starting with (NH4)2SO4 fractions of muscle extracts, procedures for purifying four to six separate enzymes from each fraction by using affinity-elution-chromatographic techniques are described. 2. Schemes for purifying 12 separate enzymes from rabbit muscle, and eight from chicken muscle extracts, are included. In nearly all cases the overall procedure involves three steps: the initial (NH4)2SO4 fractionation, the ion- exchange chromatography with affinity elution of the enzyme, and gel filtration. The specific activities of the enzymes so purified are comparable with the highest values in the literature. 3. The five schemes described include illustrations of affinity elution of the separate enzymes at different pH values, at different ionic strengths and in combination with conventional gradient elution. They also include stepwise adsorption on columns at different pH values. 4. Separation of two electrophoretically differing forms of phospho- glycerate kinase was achieved by gradient affinity elution from CM-cellulose. The lower-pI form was eluted by a lower concentration of substrate than the higher-pI form. Methods for purifying individual glycolytic and of extract (approx. 350g of muscle), and the columns related enzymes from rabbit muscle by using affinity- used were always 16cm2 cross-section and close to elution techniques were described in the preceding 5cm tall unless otherwise indicated. The buffers used paper (Scopes, 1977). Although most investigators in chromatography were 10mM of base (Tris, KOH, are interested in only one or perhaps two different NaOH) adjusted to the required pH with a-picolinic enzymes, it may be desirable to purify others to use, acid, Mes,* Mops or Tricine as appropriate. The for example, in assaying the enzymes or the substrates buffers also contained 0.2mM-EDTA. being investigated. In this laboratory all glycolytic enzymes are required in large quantities both for Results assaying purposes and for metabolic reconstitution studies (Scopes, 1973). Use ofthe affinity-elution pro- Five schemes are presented here (three for rabbit cedurehas beenwidely adopted by us (Scopes & Fifis, enzymes, two for chicken) which cover 12 different 1975; Stewart & Scopes, 1975; Chappel et al., 1976; enzymes. Phosphorylase (EC2.4.1.1), phosphofructo- Scopes, 1977), and the present paper describes kinase (EC 2.7.1.11), glycerol phosphate dehydrogen- procedures for purifying several different enzymes ase (EC 1.1.1.8) and glyceraldehyde phosphate simultaneously from extracts of rabbit muscle. dehydrogenase (EC 1.2.1.12) are not included; In addition, methods for purifying some of the alternative techniques are more convenient for these corresponding chicken muscle enzymes by affinity enzymes (Scopes, 1973). In particular, phospho- elution are described. fructokinase is mostly destroyed by (NH4)2SO4 fractionation at pH6.0; phosphorylase and glycer- aldehyde phosphate dehydrogenase form the major Materials and Methods portions of fractions A and E respectively, and each Materials and basic methods were as described in can be crystallized directly after suitable treatments. the preceding paper (Scopes, 1977). In addition, chicken muscle from freshly killed hens was obtained Scheme (a): purification of pyruvate kinase (EC and extracted exactly as therabbit muscle. (NH4)2SO4 2.7.1.40), lactate dehydrogenase (EC 1.1.1.27), fractions obtained as described were dissolved in a aldolase (EC 4.1.2.13) and phosphoglucomutase (EC small volume of Tris/HCl buffer, pH 8.0, and the pH 2.7.5.1)from the 45-60%-satd. (NH4)2S04fraction was adjusted to 8.0 with 1M-Tris before desalting. Fraction BC contains between 70 and 80 % of each The fractions were often stored as a slurry at 4°C of the aldolase, lactate dehydrogenase and pyruvate before desalting, since it was not possible to work up * Abbreviations: Mes, 2-(N-morpholino)ethanesul- more than one fraction at a time. phonic acid; Mops, 3-(N-morpholino)propanesulphonic In all cases procedures relate to material from 1 litre acid; Tricine, N-tris(hydroxymethyl)methylglycine. Vol. 161 266 R. K. SCOPES kinase present in the extract, these enzymes being the major protein components of the fraction. Proteins in smaller amounts include creatine kinase (EC 00 2.7.3.2), phosphoglucomutase, phosphoglycerate e4 - mutase( EC 2.7.5.3), phosphoglucose isomerase C ._ UE (EC 5.3.1.9) and enolase (EC 4.2.1.11); some of these (A are more concentrated in other fractions. The pro- cedure described here gives four of the main components, together with some others isolated in a) lesser overall yield. cd Fraction BC from 1 litre of extract was first de- salted, then adjusted to pH6.5 with Tris/Mes buffer, and diluted to a protein concentration of between 10 200 250 300 350 and 15mg/ml (approx. 400ml). A CM-cellulose Eluate volume (ml) column (of dimensions 16cm2x 10cm) in Tris/Mes buffer was used first, and the sample run on under Fig. 2. Separation ofimpuritiesfrom pyravate kinase bki gel gravity (300-400ml/h). The sample was washed in filtration with buffer, and KCI was added to the buffer to a The fraction applied to the Sephadex G-150 column (8cmnx90cm) was the phosphoenolpyruvate-eluted concentration of 20mM. Approx. 300ml of this was fraction in Fig. 3, after concentration by ultrafiltra- pumped in (300ml/h), and a protein peak, which tion to 25ml. , Pyruvate kinase. - , Percent- included most of the phosphoglucose isomerase in age transmission at 280nm. the fraction, was obtained. This isomerase could be further purified by affinity elution from phospho- cellulose as described in the preceding paper (Scopes, 1977). 1 2 3 4 5 6 7 8 9 After the column had been equilibrated with Tris/ Mes/KCl buffer, 0.5mM-phosphoenolpyruvate was 100 e added to the buffer [a 'dummy-substrate' wash 80 (Scopes, 1977) was not necessary at this higher ionic - PGM __ strength], which specifically eluted pyruvate kinase, 60 m - -_ _/ PK \ PGI -9 somewhat contaminated with enolase, phospho- C PGK glycerate mutase and phosphoglycerate kinase. These ,EN u --CK 40 ALD contaminants are clearly identifiable by their subunit 0 - LDH sizes on sodium dodecyl sulphate/polyacrylamide-gel x 30 _~~~~~~~ = - PGAM electrophoresis (Fig. 1). They were present because 0 during the affinity-elution step the enolase and mutase combined to convert the added phosphoenol- 20 pyruvate through 2-phosphoglycerate into 3-phos- phoglycerate, so causing affinity elution of all four enzymes; however, pyruvate kinase was the major Fig. 1. Diagram of a sodiwn dodecyl sulphate/polyacryl- component, and being a much larger molecule than amide-gel-electrophoresis slab containing samples relevant the other enzymes was subsequently completely to Schemes (a) and (b) separated from the others by gel filtration on Sepha- Sample 1: fraction BC complete; 2: fraction BC not dex G-150 (Fig. 2). adsorbed on CM-cellulose at pH 6.5; 3: phospho- enolpyruvate-eluted pyruvate kinase, Scheme (a); The column now contained only lactate dehydro- 4: pyruvate kinase after gel filtration (Fig. 2); 5: genase and aldolase in significant amounts. The buffer lactate dehydrogenase eluted by NADH, Schemes was changed to KOH/Mops, pH7.2, containing (a) and (b); 6: aldolase eluted by fructose 1,6- 10mM-KCl, and after 200ml of this had been passed bisphosphate, Schemes (a) and (b); 7: phosphogluco- into the column 0.2mM-NADH was added to it mutase eluted by glucose phosphates, Scheme (a); (150ml), specifically eluting lactate dehydrogenase. 8: phosphoglucose isomerase eluted by glucose 6- The column was next washed with KOH/Mops phosphate, Scheme (b); 9: phosphoglycerate mutase buffer, pH7.2, containing 20mM-KCI, followed by Scheme eluted by 2,3-bisphosphoglycerate, (b). the same 0.2mM-fructose Abbreviations: PGM, phosphoglucomutase; PK, buffer containing 1,6- pyruvate kinase; PGT, phosphoglucose isomerase; bisphosphate (200ml), which eluted the aldolase. PGK, phosphoglycerate kinase; EN, enolase; CK, The purities of these preparations can bejudged from creatine kinase; ALD, aldolase; LDH, lactate de- Fig. 1. The complete elution diagram from this hydrogenase; PGAM, phosphoglycerate mutase. column is shown in Fig. 3. All elution diagrams in this 1977 MULTIPLE ENZYME PURIFICATIONS BY AFFINITY ELUTION 267 pH 6.5 pH 7.2 Buffer pH: D. 20 mM-KCI 10 mM-KCI Extra ionic _ strength: I la nn Pbospbhenol- 0.2me- 0.2 Imm- I pyruvate NAOH fructose 1,6-bisphosph-ate I I I I~~~~~ 13 wC 2?.a \J t - .1 \,! 0 400 600 800 1000 1200 Eluate volume (ml) Fig. 3. Elution diagram for rabbit muscle fraction BC adsorbed to a CM-celildose column atpH6.5 The enzymes obtained by affinity elution were pyruvate kinase (---- ), lactate dehydrogenase ( ) and aldolase (- .-.). - , Percentage transmission at 280nm in 3mm-path-length cell. For details of procedure, see the text. pH 6.0 Buffer pH: 0.5 mm-Glucose I-phosphate 0.5 rmm-EDTA + 0.I mm-glucose .6bisphosphate Added ligand: a 25 ce Ce aS 1500 -;- 'a 4 Ce .-f, C2 1000 P. §75 4) Oo 500 w 500 600 Eluate volume (ml) Fig. 4. Affinity elutdon ofphosphoglucomutase from a CM-cellulose column atpH6.0 - The fraction applied was that protein not adsorbed at pH6.5 (see Fig. 3). , Phosphoglucomutase. , Percentage transmission at 280nm. For details of procedure, see the text. Vol. 161 268 R. K. SCOPES paper are typical profiles selected from several through the two columns, which were then washed separate experiments. with a little buffer. Of the five enzymes noted, only The final step in this scheme was to take the protein phosphoglucomutase was not adsorbed on either that had not adsorbed on the CM-cellulose column, column, and could subsequently be purified on CM- lower its pH to 6.0 with 1 M-Mes, and run it on to an- cellulose at pH6.0 as described in Scheme (a).
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