ANALYTICAL BIOCHEMISTRY 141, 79-82 (1984)

The Purification of Yeast Glucose g-Phosphate Dehydrogenase by Dye-Ligand Chromatography

EDWARD E. FARMER’ AND JOHN S. EASTERBY

Department of Biochemistry, University of Liverpool, P.O. Box 147. Liverpool, L69 3B.Y. England

Received January 4. 1984

Glucose 6-phosphate dehydrogenase (EC 1.1.1.39) has been purified to homogeneity from baker’s yeast by a simple procedure involving affinity elution from a column of red triazine dye, H-IBN, immobilized to Sepharose 6B. Eight milligrams of homogeneous protein is obtained in 53% yield from 200 g of dried yeast. This represents the first published purification of the enzyme from Saccharomyces Cerevisiae.

KEY WORDS: yeast; glucose 6-phosphate dehydrogenase; purification, dye chromatography.

Glucose 6-phosphate dehydrogenase is widely used in the biochemical and clinical laboratory both for the measurement of hexo- kinase activity in coupled assays and for the estimation of glucose concentration. Yeast is an important commercial source of the NADP specific enzyme but preparations of high spe- cific activity are expensive. The standard pro- cedure for purification is due to Noltmann et al. (1) but is lengthy and requires both silver and solvent precipitation of the enzyme, fol- lowed by crystallization. More recently a number of purification procedures for the en- zyme from other sources have been published (2). One of these, for the isolation of NAD(P) specific glucose 6-phosphate dehydrogenase from Methylomonas M 15, has included affin- ity elution of the enzyme from Blue Dextran- Sepharose CLdB. The baker’s yeast enzyme has also been shown to bind to immobilized Cibacron blue and could be eluted using var- ious dinucleotides (3).

During the course of a study of the inter- action of mammalian hexokinases with the hydrolyzed Procion red H-8BN dye, we no- ticed that this dye was a potent inhibitor of

’ Current address: Biologisches Institut II, Albert Lud- wigs Universitlt, D-7800, Freiburg, West Germany.

glucose (i-phosphate dehydrogenase. Com- mercial yeast enzyme was found to bind to H8BN-Sepharose 6B and could be eluted by low concentrations of NADP. This has formed the basis for the purification procedure de- scribed here for glucose (j-phosphate dehy- drogenase of analytical grade and free from contaminating hexokinase activity. In view of the termination of production of Cibacron blue dye, this method based on the red Procion dye should prove to be of considerable interest.

In addition to the dye chromatography step, the method involves ammonium sulfate frac- tionation, DEAE-cellulose chromatography, and optional gel filtration. The procedure in- volves fewer steps and is cheaper than the pu- rification of the enzyme from brewer’s yeast (1). Surprisingly, although much of the pub- lished data on glucose 6-phosphate dehydro- genase have been obtained using commercial baker’s yeast enzyme, this is the first published purification to homogeneity from Saccharo- myces Cerevisae (4).

MATERIALS AND METHODS

Reagents. Fresh baker’s yeast (A’. Cerevisae) was purchased from United Yeast Company Ltd. (Liverpool, England). Nucleotides were

79 0003-2697184 $3.00 Copyright 0 1984 by Academic Pnss, Inc. All rights of reproduction in any form reserved.

80 FARMER AND EASTERBY

from P-L Biochemicals (Milwaukee, Wiscon- sin). Glucose 6-phosphate was from Boehrin- ger-Mannheim (Mannheim, West Germany). DE-52 was a product of Whatman (Maidstone, England). Sepharose 6B was from Pharmacia (Uppsala, Sweden). Ultrogel AcA34 was from LKB (Bromma, Sweden). Procion brilliant red H-8BN was a product of ICI and a gift of Dr. P. D. G. Dean. All other reagents were from Fisons (Loughborough, England) or British Drug Houses (Poole, England) and were of Analar grade.

Synthesis of H-i?BN-Sepharose 6B. NaCl (2 g) and Na2C03 (1.7 g) were added to a slurry of Sepharose 6B ( 100 ml) and water (40 ml). The mixture was shaken and 0.2 g H- 8BN was added. Coupling was achieved by shaking for 40 h at 45°C. Before and after use the H-8BNSepharose column was thoroughly washed with 5 M urea followed by 2 M KCl.

Enzyme assay. Each assay contained 50 rmol Tris-HCl, pH 7.6, 24 pmol MgCl*, 1.2 pmol glucose 6-phosphate, and 0.37 pmol NADP+ in a total volume of 1 ml. One unit of enzyme activity corresponds to the reduc- tion of 1 pmol NADP per min at 30°C. This is equivalent to an absorbance increase of 6.22 per minute. In general about 0.01 unit of en- zyme was assayed.

The enzyme was dialyzed free of ammo- nium sulfate before assay as the sulfate ion is inhibitory.

Protein estimation. Protein was measured by the microtannin turbidimetric method of Mejbaum-Katzenellenbogen and Dobry- szycka (5).

Electrophoresis. Polyacrylamide electro- phoresis was performed according to Gross- man and Potter (6). SDS*-polyacrylamide gel electrophoresis was performed according to Weber and Osborn (7). Protein was stained with naphthalene black or Coomasie blue.

Ultracentrijkgation. Analytical ultracen- trifugation was performed in a Beckman Model-E machine as described previously (8).

’ Abbreviation used: SDS, sodium dodecyl sulfate.

PuriJication of glucose &phosphate dehy- drogenase. All procedures were carried out at 4°C.

Buffer 1: 0.05 I phosphate, pH 8 (0.0 176 M), containing 0.2 g KH2P04 and 2.7 g K2HP04 per liter; buffer 2: 0.05 I Tris-HCl, PH 7.5 (0.061 M), containing 7.4 g Tris per liter titrated to pH 7.5 with HCl; buffer 3: 0.1 I Tris-HCl, PH 7.5 (0.12 1 M), containing 14.66 g Tris per liter titrated to pH 7.5 with HCl.

All buffers contained 1 mM disodium EDTA and 5 mM &mercaptoethanol. Buffers 2 and 3 additionally contained 5% glycerol.

Lysis of yeast. Yeast cake was air-dried by crumbling onto large sheets of 3 MM filter paper and turning daily for 6 days.

Dried yeast (200 g) was added to 500 ml of 0.2 M Na2HPOa * 12H20 (71.63 g/liter) containing 1 mM EDTA and 5 mM @-mer- captoethanol. The suspension was incubated for 3 h at 37°C and centrifuged at 25,000g for 50 min.

Ammonium Surfate fractionation. The su- pematant from centrifugation (570 ml) was made 48% in (NH&S04 by slow addition of 170 g (298 g/liter) of the salt. After stirring for 30 min it was centrifuged at 25,000g for 45 min. The supematant (600 ml) was made 67% in (NH&SO4 by the addition of a further 88 g (148 g/liter) of the salt and again cen- trifuged after 30 min of stirring. The precip- itate was resuspended in a minimum volume ( 145 ml) of buffer and dialyzed for 36 h against two changes of 4 liters of buffer.

DEAE-cellulose chromatography, pH 8. The dialyzed solution was pumped onto a DEAE- cellulose column (22 X 3.8 cm) equilibrated in buffer 1. Flow rate was 80 ml/h and 1 O-ml fractions were collected. Unbound protein was removed by washing the column with 500 ml buffer 1 and the column was developed with a 2-liter linear salt gradient of O-O.25 M KC1 in buffer. Fractions 73 to 82 after initiation of the gradient were retained.

H-8BN-Sepharose chromatography, pH 7.5. The active fractions from DEAE-cellulose

DYE CHROMATOGRAPHY OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE 81

r-400 3 4 07 .z

=

=300 33 g 2

$200 c

15 pzi b (j:

100 1

0 lo 20 30 40 50 Fraction

FIG. 1. Chromatography of glucose 6-phosphate de- hydrogenase on H-8BN-Sepharose, pH 7.5. The buffer was 0.05 I Tris-HCl, pH 7.5 (0.061 M), containing 1 mM EDTA, 5 mM &mercaptoethanol, and 5% glycerol. Row rate was 20 ml/h and 7.5-ml fractions were collected. Protein (0) and enzyme (0) are shown. The enzyme was eluted by inclusion of 1 mM NADP in the buffer from the fraction indicated by the arrow.

were vacuum-dialyzed overnight against buffer 2 and the concentrated enzyme (10 ml) was pumped onto a column of H-8BN-Sepharose 6B (9 X 2.3 cm) at a flow rate of 20 ml/h. The column was washed with buffer 2 until loosely bound protein no longer eluted. Glu- cose 6-phosphate dehydrogenase was eluted by inclusion of 1 mrvt NADP in the buffer. Fractions (7.5 ml) were collected and fractions 43 to 47 were retained (Fig. 1).

AcA 34 Chromatography, pH 7.5. Enzyme was concentrated by vacuum dialysis against

Buffer 3 to approximately 1.5 ml and applied to a column of AcA 34 (97 X 1.4 cm). Flow rate was 6 ml/h and 5-ml fractions were col- lected. The peak fractions 11 to 13 were pooled.

RESULTS AND DISCUSSION

The results of a typical purification are shown in Table 1. Eight milligrams of enzyme was obtained from 200 g dried yeast and was judged homogeneous by SDS-polyacrylamide electrophoresis, sedimentation velocity anal- ysis (~20,~ = 5.98 S) and sedimentation equi- librium analysis (MZ = 106,000).

The dye-l&and chromatography step de- pends for its success on the degree of substi- tution with dye and size of the column. Too large a column should not be used or frontal elution by NADP no longer occurs and some retardation of the enzyme is experienced. However, this step gives excellent recovery and reproducibility. It is necessary to further purify the affinity-eluted enzyme by the AcA 34 chromatography described and in general only the front-half of the peak is retained. On some occasions there appears to be some minor contamination of the dye column eluant by protease and it is less stable at this stage.

The purified enzyme is at least 94% pure and has a specific activity of 370 u/mg. It contains no detectable hexokinase activity and is suitable for both analytical use and as a coupling enzyme.

TABLE 1

PURIFICATION OFYEAST GLUCOSE ~-PHOSPHATE DEHYDROGENASE

Stage Enzyme (units)

Protein W)

Specific activity (U/w)

Purification (-fold)

Yield (“/I

Lysate 5,590 34,200 0.16 1 100 (NW304 @pt.) 6,470 10,100 0.64 4 116 DEAE-Cellulose eluate 4,684 212 22 138 84 H-8BNSepharose 4,85 1 31.7 153 956 87 AcA 34 2,962 8 370 2,308 53

Note. Dried yeast (200 g) was used.

82 FARMER AND EASTERBY

ACKNOWLEDGMENT

Edward E. Farmer is indebted to the SERC for the award of a research studentship.

REFERENCES

1. Noltmann, E. H., Bugler, C. J., and Kuby, S. A. (1961) J. Biol. Chem. 236, 1225-1230.

2. Steinbach, R. A., Schiitte, H., and Sahm, H. (1982) in Methods in Enzymology (Wood, W. A., ed.), Vol. 89, Academic Press, pp. 271-275, New York.

3. Easterday, R. L., and Easterday, M. (1974) in Im-

mobilized Biochemicals and Affinity Chromatog- raphy (Dunlap, R. B., ed.), pp. 123-133, Plenum, New York.

4. Levy, H. R. (1979) in Advances in Enzymology (Meister, A., ed.), Vol. 48, pp. 97-192, Academic Press, New York.

5. Mejbaum-Katzenellenbogen, W., and Dobryszycka, W. W. (1959) Cfin. Chim. Acta 4, 5 15-522.

6. Grossman, S. H., and Potter, V. R. (1974) Anal. B&hem. 59, 54-62.

7. Weber, K., and Osbom, M. (1969) J. Biol. Chem. 24k4406-4412.

8. Easterby, J. S., and Rosemeyer, M. A. (1972) Eur. J. Biochem. 28,241-252.