THE PURIFICATION OF COENZYME A BY ION EXCHANGE
            CHROMATOGRAPHY

       BY E. R. STADTMAN AND ARTHUR KORNBERG
(From the Section on Cellular Physiology, National Heart Institute, and the National
Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United
        States Public Health Service, Bethesda, Maryland)

         (Received for publication, December 22, 1952)

With the increasing demand for coenzyme A (CoA) to be used as a sub-
strate in enzyme studies, a need has developed for additional methods of
isolating this substance from natural sources.  Two methods have been
published whereby CoA of reasonably high purity can be prepared (1, 2).
Both of these procedures are lengthy and the over-all yields are relatively
poor (10 to 30 per cent).  One of these methods is relatively expensive,
since large amounts of glutathione are required (2).

In the present communication a method is described for the preparation
of CoA of 50 to 65 per cent purity from crude yeast extract in fair yield
(50 to 55 per cent) by a relatively simple two-step chromatographic pro-
cedure.  In this procedure the CoA is adsorbed on charcoal and is con-
centrated by selective elution with ammoniacal acetone.  The CoA con-
centrate thus obtained is adsorbed on a Dowex 1 resin and is isolated as a
distinct fraction by elution with strong buffer.  The final product contains
200 to 270 units (1) of CoA per mg.

Materials and Methods'

Preparation of Charcoal-Acid-treated, degassed charcoal is prepared by
suspending about 400 gm. of unground Nuchar C charcoal in 4 liters of
6 N HCl.  The suspension, in two 4 liter flasks, is kept under a vacuum
overnight during which time the charcoal settles, leaving a clear super-
natant solution.  Sufficient charcoal is transferred to a large glass column
(10 cm. in diameter) to give a bed height of 29 cm.  The glass column is
fitted at the bottom with a stainless steel screen (80 mesh) which is packed
around the edges with glass wool.  The charcoal is washed with distilled
water until the pH of the effluent is 3.5 to 4.0.

Preparation of Resin-Dowex 1  X 2 (2 per cent cross-linked resin)2

1 All operations are carried out at room temperature unless otherwise indicated.
2 Dowex 1 X 10 (10 per cent cross-linked resin) was previously tested by us and
found unsatisfactory for CoA isolation.  We are indebted to Dr. Waldo Cohn, who
called our attention to the availability of Dowex resins with low cross-linkage (low

divinylbenzene content).

                          47

48 PURIFICATION OF COENZYME A

(200 to 400 mesh) is washed by suspending 5 pounds in 30 liters of 3 N
I-ICl.  The suspension is stirred mechanically for 8 hours and then allowed
to settle for 2 days and the supernatant solution is decaiited.  This step
is repeatcd three to four times until the ultraviolet absorption of the
supernatant solution at 260 mp is 0.080 or less in a 1 cm. cell.  The resin
is washed with 30 liter batches of water (two to three times) until the test
for chloride ion in the wash water is faint.  The resin is transferred to a
glass column fitted with a porous sintered glass base and is washed with 3
M sodium formate until the chloride ion test on the efiluent is faint.  Fi-
nally, it is washed with water until the effluent is practically salt-free (20
parts per million on Barnstead purity meter, referred to NaC1).

Other anion exchange resins tested, including Duolite A-3 chloride,
Dowex 2 X 10 formate, and Dowex 1 X 10 chloride and formate proved
unsatisfactory.
CoA Assay-All purification steps are followed by direct CoA analysis
by means of the phosphotransacetylase assay system (3) with a slight
modification to permit more rapid determinations.  The method is as fol-
lows: Water to give a final volume of 1.0 ml.; tris(hydroxymethy1)amino-
methane hydrochloride buffer (1 M, pH 8.0), 0.1 ml.; dilithium acetyl
phosphate, 6 PM; cysteine hydrochloride, 10 p~; an aliquot of the test
solution containing 0.5 to 3.0 units of CoA; and phosphotransacetylase (4),
8 units, are added in the indicated order.  The reaction mixture is incu-
bated at 28' for 5 minutes and then 0.1 ml. of potassium arsenate (0.5 M,
pH 8.0) is added.  After 10 minutes, the residual acetyl phosphate is
estimated by the hydroxamic acid method (5).  Under these conditions
the amount of acetyl phosphate decomposed is proportional to the amount
of CoA present in the reaction mixture.  Up to twelve samples can be
conveniently examined in a single assay.  For reference, a standard tube
containing 2 to 2.5 units of CoA and a control sample containing no CoA
are included in each assay.  All CoA samples must be adjusted to pH 7.5
to 8.0 prior to testing.

The reference standard used in this study was an acetyl CoA sample
prepared by the enzymatic acetylation of CoA and isolated by paper
chromatography (6).  The acetyl-adenine ratio of this preparation was
0.97 and it was assumed to contain 316 units of CoA per PM (1).

Preparation of Yeast Extract-3 kilos of dried yeast (Anheuser-Busch ,
strain G) are dropped into 15 liters of boiling water.  The suspension is
stirred and boiled vigorously for 5 minutes.  25 pounds of cracked ice are
added and the cold suspension is centrifuged at about 2000 X g for 30
minutes,  The slightly turbid supernatant solution (19 liters) should con-
tain 100,000 f 30,000 units of CoA.

E. R. STADTMAN AND A. KORNBERG

49

Charcoal Chromatography of Yeast Extract3-19 liters of yeast extract are
adjusted to pH 3.0 with 6 N HCl (200 to 250 ml.).  The extract (which
becomes turbid upon acidification) is passed through a charcoal column
(10 cm. X 29 cm.) at a rate of 1 liter per 3 minutes.  The milky white
effluent is discarded.  The charcoal is washed with 10 to 15 liters of dis-
tilled water, 2 to 4 liters of 40 per cent aqueous acetone, and then with
40 per cent aqueous acetone containing 1.0 ml. of concentrated ammonium
hydroxide solution (28 per cent) per liter.  The eluate is collected in 2
liter fractions.  The pH of successive fractions increases gradually from
3.3 to 5.0 and then increases sharply to pH 7.5 to 9.0.  In most runs the
CoA is concentrated in the eluates having a pH of 3.8 to 7.0; occasionally,
however, the fraction on either side of these limits will contain some CoA

             TABL~ I
Charcoal Chromatography of CoA in Crude Yeast Extract

Fraction No. (2 liters)

Color

Straw, turbid

(( clear

f6

46 ft

Yellow, ((
Deep amber

Amber

(4 (4

Total.. . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . .

3.4
3.6
3.9
4.0
4.2
5.0.
8.5
9.4

"A, per cent of initial

0
0
7
26
28
26
7
0

94

also.  Data from a typical experiment showing the distribution of CoA in
the eluates from a charcoal column are given in Table I.

The fractions containing CoA are pooled, the pH is adjusted to 1.7 with
6 N HCl (any precipitate which forms is removed by filtration through a
fluted Schleicher and Schull No. 588 filter paper), and the CoA is precipi-
tated by the addition of 5.5 volumes of cold (2") acetone.  After 1 to 2
hours the precipitate is recovered by filtration through a Buchner funnel
(10 cm. in diameter).  The precipitate is washed with acetone and with
ether and dried in a vacuum desiccator over PzOs.  5 to 6.5 gm. of dry
acetone powder are obtained containing 10 to 18 units of CoA per mg.
The over-all recovery based on the boiled yeast extract is about 80 to 95
per cent.

Chromatography on Doiuez I Resin-15 gm. of acetone powder are dis-
3 This charcoal step is a modification of a method developed by M. Soodak and

F. Lipmann (private communication).

50 PURIFICATION OF COENZYME A

solved in water and the pH is adjusted to 8.1 with 2 N KOH and the
volume to 200 ml.  Any material which does not dissolve is removed by
centrifugation.

The crude CoA solution is placed on a Dowex 1 X 2 formate column
(13 cm. X 15 cm. square).  Invariably the colored front progresses un-
evenly, due presumably to severe shrinkage of the resin caused by the high

        TABLE I1
Ion Exchange Chromatography of CoA

Fraction No. (500 ml.)

ot

1
2
3
4
f,
6
7
8
9
10
11
12
13
14
15

Total recovered.. .........

initial

I<

..............

Adenine'

PM

165
9,900
8,500
1,500

660
435
520
490
275
151
113
123
117
107

85
69

23,210
25,900

CoAt

PM
0
0
0
0
6
5
0
0
0
25
75
86
89
75
49
19

429
450

- ._

QX CoA
QY adenine

-

0
0
0
0
0.009
0.011
0
0
0
0.16
0.66
0.70
0.76
0.70
0.58
0.28

* Calculated from optical density at 260 mp on the basis of a molecular extinction

t Calculated on a value of 316 units of CoA per PM (1).
$ Filtrate collected during the adsorption and washing.

coefficient of 15.9 X lo6 sq. cm. per mole.

salt concentration of the CoA solution.  Therefore, when the farthest edge
of the front has progressed approximately 1 cm., the upper 1 cm. layer of
resin is gently stirred up with the overlying solution.  Following this treat-
ment the progress of the front appears uniform.  After the CoA solution
has been applied, the column is washed with two 100 ml. portions of water
and is eluted with a solution containing 25.8 ml. of formic acid (88 per
cent) per liter (0.6 M) and 18.9 gm. of ammonium formate per liter (0.3 M).
The rate of flow (attained with a hydrostatic pressure of 2 to 5 feet) is
about 450 ml. per hour.  500 ml. fractions are collected and assayed for
GOA and for adenine (optical density at 260 mu).

E. R. STADTMAN AND A. KORNBERG 51

Results showing the development of a typical chromatogram are given
in Table 11.  Elution of the CoA begins when 20 to 23 resin bed volumes
(eight or nine fractions) of eluate have been collected.  The CoA is com-
pletely eluted when an additional 10 to 12 resin bed volumes (Fractions 9
to 15) are collected.  In the experiment described, the CoA-adenine ratio
of Fractions 9 to 15 varied from 0.16 to 0.76.  In other experiments ratios
as high as 1.0 have been obtained.  The over-all recovery of CoA from the
chromatograms is generally 80 to 95 per cent; however, in some instances
recoveries as low as 70 per cent have been observed.

Concentration of Eluate-Since the CoA from the Dowex 1. column is dis-
tributed in a large volume (2500 to 3000 ml.) of strong formate buffer, it is
desirable to concentrate it and remove the excess salt before attempting
to recover the CoA by acetone precipitation.  This is accomplished by
readsorbing the CoA on a small charcoal column and eluting with ammoni-
acal acetone.  To obtain GOA of highest purity, the eluates from the ion
exchange chromatogram in which the CoA-adenine ratio is 0.6 or greater
are pooled; the pH is adjusted to 2.0 with 6 N HCl (about 100 ml. per 2000
ml. of eluate) and the solution is passed through a small charcoal column
(2.5 cm. in diameter X 12 em.) at a rate of 1.5 liters per hour.  The optical
density of the effluent at 260 mp should not exceed 0.020.  The column is
washed with 300 ml. of water and with 300 ml. of 40 per cent acetone.
The CoA is finally eluted with 40 per cent acetone containing 1 ml. of
concentrated ammonium hydroxide per liter.  The flow rate is adjusted to
about 3.0 ml. per minute.  50 ml. fractions are collected.  The alkaline
fractions are neutralized with 4 N nitric acid.  The CoA is generally all
eluted when the pH of the effluent is alkaline.

The CoA in these fractions is sufficiently concentrated to permit detec-
tion by the nitroprusside test.  Drops of the various fractions are placed
on a strip of No. 3 Whatman filter paper and dried, and the paper is dipped
first into Reagent I and then into Reagent I1 of Toennies and Kolb (7).
The appearance of a red spot, which is greatly intensified by shaking the
paper strip in ethyl ether, indicates the presence of CoA.  The fractions
containing CoA (usually Fractions 3 to 6) are pooled (final volume, 150 to
200 ml.).  The pooled sample is acidified to pH 1.7 with 4 N nitric acid
and the CoA is precipitated by the addition of 7 volumes of cold acetone.
The precipitate is allowed to settle overnight, the supernatant solution is
decanted, and thc CoA is transferred to a centrifuge tube.  The precipitate
is washed two times with acetone (room temperature) , once with ether,
and dried in a vacuum desiccator over P,Os.

From 15 gm. of CoA concentrate (10 units per mg.) 300 to 400 mg. of
purified material are obtained containing 200 to 270 units of CoA per mg.
The over-all yield is generally 50 to 55 per cent.  Occasionally, yields as

52 PURIFICATION OF COENZYME A

3.32
3.07
3.13
3.00

low as 40 per cent and as high as 70 per cent have been obtained.  In
addition to this CoA of relatively high purity, CoA preparations containing
150 to 200 units per mg. can be obtained as a by-product by working up
the eluates from the Dowex 1 column having a CoA-adenine ratio of less
than 0.6.

Composition-The compositions of some purified preparations are given
in Table 111.  For comparison, analytical data on a sample of Pabst CoA
are also presented.  The contents of phosphorus, adenine, and enzymatic-
ally active CoA are 65 to 67 per cent of theory.  The rather good stoi-
chiometry among these three components precludes the presence of other
adenine nucleotides as major contaminants.

1.06
1.10
1.02
1.00

     TABLE I11
Analysis of CoA Preparations

Sample No.

VIIa. .............
XIII. .............
Pabst (Lot 401-27).
Pure CoAS.. ......

Total P*

p~ per mg.
2.56
2.55
2.66
3.9

_____-

Adeninet

p~ per mg.
0.82
0.91
0.87
1.3

CoA$

per mg.
0.77
0.83
0.85
1.3

-

* Determined by the method of Fiske and Subbarow (8).
t Calculated from optical density at 260 mp on the basis of an assumed molecular

f Determined by arsenolysis of acetyl P with transacetylase.

extinction coefficient of 15.9 X lo6 sq. cm. per mole.

Theoretical values (1).

It should be pointed out that CoA samples of lesser purity prepared from
the fractions just preceding the peak fractions from the Dowex 1 column
(Le. the material with a CoA-adenine ratio of less than 0.6) do contain
significant amounts of ATP.
Since no reductive steps are employed in the above chromatographic
procedure, the CoA isolated should be present mainly as the mixed disulfide
derivative of other sulfhydryl compounds (1, 9, 10).  Paper chromatog-
raphy of the isolated material in an 80 per cent phenol-20 per cent water
solvent shows that the CoA moves to a spot identical with a ninhydrin-
reactive material (Rp = 0.35). Following reduction of the CoA with
hydrogen sulfide it moves with an RF of 0.51, while the amino compound
has an RF of 0.46, which is identical with that found for glutathione.
These observations suggest that the CoA isolated may be present largely
as the mixed disulfide derivative of glutathione.

Chromatography on Cation Resin-A further purification of the CoA may
be achieved by reduction with hydrogen sulfide followed by chromatog-

E. R. STADTMAN AND A. KORNBERG 53

raphy on Dowex 50 (Hf) to remove the amino compounds as previously
described (2).  In a single experiment, 100 mg. of CoA (263 units per mg.)
were neutralized with KOH (400 pes.), and hydrogen sulfide was passed
through the solution (25 ml.) for 3.5 hours.  The solution was adjusted to
pH 2.5 and gassed with helium to remove the hydrogen sulfide.  The
acidified solution (3.0 ml.) was passed through a Dowex 50 (H+) column
(3 cm. X 1 cm. square) and the CoA in the percolate (6.0 ml.) was pre-
cipitated by the addition of 70 ml. of acetone.  45 mg. of precipitate were
recovered containing 328 units of CoA per mg., corresponding to a purity
of 78 per cent.  Since all of the adenine-absorbing material initially present
was recovered in the percolate from the Dowex 50 column, the low recovery
of CoA (55 per cent) is due either to incomplete acetone precipitation of the
reduced CoA or to decomposition of the CoA on the acid resin.

Comments

The ion exchange chromatography of CoA concentrates has also been
used on a scale one-third, one-half, and 2 times that described.  In these
instances the size of the resin column mas varied proportionally by varying
the cross-sectional area of the column while maintaining the height of the
resin bed constant. The rate of elution and volumes of the fractions
collected were varied correspondingly.  The elution pattern, yields of CoA,
and purity of the product obtained were similar, except that a somewhat
poorer over-all yield (40 per cent) was obtained in a single experiment at
2 times the scale described.

Usually 14 to 16 hours are required to elute the CoA from the Dowex 1
column.  In the absence of an automatic fraction collector which permits
uninterrupted elution overnight, the elution can be discontinued and fin-
ished on the following day.

In addition to the charcoal-treated , yeast acetone powder concentrates
a number of other CoA concentrates have been tested as sources of CoA
for ion exchange Chromatography.  Of those tested, an Armour CoA con-
centrate of hog liver (13 units per mg.) and a Sigma triphosphopyridine
nucleotide concentrate (10 per cent triphosphopyridine nucleotide, 6.5 units
of CoA per mg.) gave products of lower purity (about 150 units per mg.)
and lower yields (30 to 35 per cent), while chromatography of a CoA
concentrate (6.0 units per mg.) obtained from Streptomyces fradiae gave
poor results.  On the other hand, a crude Pabst CoA concentrate from
yeast containing only 1.3 units of CoA per mg. gave results similar to those
obtained with the more purified starting material prepared by us.  How-
ever, the capacity of the resin column in terms of CoA was only about one-
seventh as great as when the more purified material was used.  A pre-
liminary charcoal purification of the Pabst CoA concentrate gave a CoA

54 PURIFICATION OF COENZYME A

preparation (10 units per mg.) which for resin chromatography is equally
as good as the material prepared by us from crude yeast extracts.  Thus,
charcoal treatment of other commercially available crude GOA concentrates
may provide suitable starting materials for the ion exchange chromatog-
raphy.

SUMMARY

A method has been developed for the purification of coenzyme A by
charcoal and anion exchange chromatography.  When the procedure was
applied to hot water extracts of dried yeast, preparations containing 200
to 270 units of coenzyme A per mg. (50 to 65 per cent pure) were obtained
in yields of 50 to 55 per cent.

BIBLIOGRAPHY

1. Gregory, J. D., Novelli, G. D., and Lipmann, F., J. Am. Chem. SOC., 74, 854
  (1952).

2. Beinert, H., Von Korff, R. W., Green, D. E., Buyske, D. A., Handschumacher,
  R. E., Higgins, H., and Strong, F. M., J. Am. Chem. SOC., 74, 854 (1952).
3. Stadtman, E. R., Novelli, G. D., and Lipmann, F., J. Bid. Chem., 191, 365 (1951).

4. Stadtman, E. R., J. Bid. Chem., 196, 527 (1952).
5. Lipmann, F., and Tuttle, L. C., J. Bid. Chem., 168, 505 (1945).
6. Stadtman, E. R., J. Bid. Chem., 196, 535 (1952).
7. Toennies, G., and Kolb, J. J., And. Chem., 23, 823 (1951).
8. Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 66, 375 (1925).
9. DeVries, W. H., Govier, W. M., Evans, J. S., Gregory, J. D., Novelli, G. D..

Soodak, M., and Lipmann, F., J. Am. Chem. SOC., 72, 4838 (1950).

10. Brown, G. M., and Snell, E. E., J. Bid. Chem., 198, 375 (1952).