ENZYMES OF THE HUMAN ERYTHROCYTE

I. PURINE NUCLEOSIDE PHOSPHORYLASE; ISOLATION

PROCEDURE*

BY K. K. TSUBOI AND PERRY B. HUDSON

(From the Departments of Biochemistry and Urology, Fran& Delajidd Hospital,
and the Institute of Cancer Research, College of Phyeiciane and Surgeons,
Columbia University, New York, New York)

(Receivedfor publication, May 31, 1950)

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Numerous reports have appeared concerning the study of a variety of
enzymes present in the erythrocyte. Only a few (e.g. (l-6)), however,
have been prepared and investigated in a significantly purified state. Be-
cause the erythrocyte contains little non-hemoglobin protein, it would
appear to be an excellent source for the preparation of certain enzymes
which it contains at relatively high concentrations. Appropriate pro-
cedures will be described in this and subsequent papers for the purifica-
tion of various enzymes of the human erythrocyte. The present report
will describe methods for isolating, in highly purified form, a purine nu-
cleoside phosphorylase from the human erythrocyte.
The ability of crude hemolysates to degrade adenosine was described
in the early literature by Dische (7). A limited study of the action of
hemolysates on adenosine was included as part of a previous report (8),
at which time it was suggested that adenosine degradation is mediated by
the action of a nucleoside phosphorylase which acts on the deaminated
product (inosine) . Purification of the phosphorylase was undertaken in
order to investigate its specific properties.
Purification of a purine nucleoside phosphorylase from liver tissue has
been described by Kalckar (9), who has contributed much of the available
information on this enzyme (e.g. (10, 11)). Purification of specific pyrimi-
dine nucleoside phosphorylase from bacteria (12) as well as mammalian
tissue (13) and the preparation of both a purine nucleoside phosphorylase
and hydrolase from yeast (14) have also been previously reported.
EXPERIMENTAL

Methods
Preparation of Calcium Phosphate Gel-The preparation and properties
of calcium phosphate gels were extensively investigated. Certain perti-
* Supported by a grant from the United States Atomic Energy Commission,Di-
vision of Biology and Medicine, contract No. AT(30-1)lllQ.
379
880 ENZYMES OF HUMAN ERYTHROCYTE. I

nent results were briefly summarized and included in a previous report

(6). Gels prepared at slightly acid rather than alkaline pH were superior
as adsorbents and were found to be especially suited to techniques which
involve fractional elution of adsorbed proteins. A careful control of the
pH was essential in the preparation of gels of reproducible characteristics.
Gels used in these and subsequent studies were prepared by the following
procedure.
To 200 ml. of 0.5 M NazHPOa solution were added 6.0 ml. of concen-
trated NHdOH solution (28 to 30 per cent NH,), followed immediately
by 1500 ml. of 0.1 M CaC& solution. The pH of this mixture should be

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(PROTEIN

PHOSPHORYLAgE

I
0.5
GEL S”SP?4OOE% PEk-“hi!- R.&C.
FIG. 1. Relative enzyme affinities for tricalcium phosphate gel a~ determined in
the crude hemolysate. The gel and hemolysate were thoroughly mixed and centri-
fuged, and the supernatant solution was assayed for residual enzymes. R.B.C. =
red blood cell.

initially at about 7, with a drop to around 6 within 30 minutes. Following

this period of time, the gel was sedimented by light centrifugation and
repeatedly washed with ion-free water until washings were free of detecta-
ble chloride ion. The washed gel was suspended with water to a fmal 1
liter volume. The gel can be either used immediately or stored for pro-
longed periods in the refrigerator with little measurable change in proper-
ties.
Adsorption of nucleoside phosphorylase, which has an extremely high
affinity for gel prepared in this manner, is illustrated in Fig. 1. The rela-
tive affinit,y of the gel for a variety of other enzymes present in the crude
hemolysate was also determined and the results have been included in
Fig. 1 for purposes of comparison as well as documentation for future refer-
ence in relation to subsequent studies to be reported.
Enzyme Assays---The enzyme was found to degrade guanosine or inosine,
 K. K. TSUBOI AND P. B. HUDSON 881

provided that either phosphate or arsenate was present. Adenosine degra-

dation could be demonstrated only with the crude hemolysate, being abol-
ished wit,h purification and removal of a specific deaminase. As has been
found by Kalckar (lo), working with purified liver enzyme, the equilib-
rium of the reaction in the presence of phosphate ion strongly favors the
formation of the nucleoside. On the other hand, degradation of nucleoside
in the presence of an equimolar concentration of arsenate (arsenolysis)
was found to proceed toward completion. Arsenolysis, rather than phos-
phorolysis of nucleoside, formed the basis for a quantitative method of
enzyme assay, thereby eliminating the necessity of dealing with cumber-
some kinetics. Guanosine rather than inosine was selected as the sub-
The assay procedure as devised was based on the following set of

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strate.
reactions :

(1) Guanosine + arsenate enzyme , guanine + (ribose-l-arsenate)

+HzO
1.
ribose + arsenate
(2) Guanine + phenol reagent 3 blue color (15)

The validity of the method requires that further degradation of guanine

does. not occur. Incubation of guanine under conditions normally em-
ployed for the assay of nucleoside phosphorylase resulted in a slight loss
(as determined with the phenol reagent) with. crude hemolysate and no
measurable loss with material obtained after the first purification step.
The assay procedure employed was as follows: To a final 1.0 ml. reaction
volume were added 0.2 ml. of enzyme (appropriately diluted in 0.001 M
versenate and 0.02 per cent Triton X-100; for previous use see Tsuboi and
Hudson (16)), 0.4 ml. of 0.0125 M guanosine, arsenate at 0.05 M (adjusted
to pH S.O), and 0.1 M acetate buffer at pH 6.0. Guanosine was prepared
every few days in dilute KOH (1.0 ml. of 1.0 N KOH per millimole of gua-
nosine) and standardized by ultraviolet absorption. Guanosine was added
to the reaction vessels just prior to the assays. Incubations were usually
for 30 minutes at 37”, following which time the reactions were stopped by
the addition of 2.0 ml. of 20 per cent sodium carbonate. Guanine was
usually determined directly on these samples by the addition of 2.5 ml. of
water and 0.5 ml. of phenol reagent (17). The necessity for thorough
shaking prior to and during the addition of phenol reagent is essential if
reliable results are to be realized. After the addition of phenol reagent,
the colors were allowed to develop for a 20 minute period at 37” and were
read at 660 mp. Appropriate tissue and substrate blanks were always
carried out with the assay. Because protein reacts with the phenol reagent
(tyrosine), the cruder enzyme preparations were deproteinized with tri-
882 ENZYMES OF HUMAN ERYTHROCYTE. I

chloroacetic acid prior to guanine determination. The over-all reliability

of the method is demonstrated in Fig. 2. A linear relationship between
enzyme concentration as well as reaction time is maintained until some-
what less than 10 per cent of the substrate is degraded, following which
typical first order kinetics is observed.
Determination of Protein-Protein was estimated by micro-Kjeklahl
nitrogen analysis, with use of a vacuum distillation procedure (18), fol-
lowed by nesslerization (19). In addition to the nitrogen analysis, protein
was also estimated by tyrosine measurements by using the phenol re-
agent (17).

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al 02a304 15 3045 60
ENZYME (ML) TIME (MINS.)
.Fro. 2. Guanosine degradation as a function of enzyme concentration, A, and
reaction time, B, with a standard assay procedure with crude (X) and purified (0)
enzyme solutions (for the standard assay procedure, see the text).

RESULTS AND DISCUSSION

Purafiation Procedure
The purification protocol is presented in summarized form in Table I.
Details concerning the purification procedure as carried out on a 509 ml.
batch of washed erythrocytes follow.
Preparation of HemolysaGutdated human blood obtained from the
hospital blood bank served as the source of red cells. The cells were thor-
oughly washed with 1 per cent sodium chloride solution and adhering
leucocytes were removed. The washed cells were hemolyzed by the addi-
tion of 4 volumes of ion-free water.
Enzyme Adsorption on Calcium Phosphate Gel-To the hemolyzed red
cells was added a volume of gel suspension (see under “Methods” for
preparation of gel suspension) equal to the original volume of cells (500
ml. of gel suspension per 500 ml. of original erythrocytes) . After thorough
mixing, the gel containing adsorbed enzyme was removed and washed
by repeated centrifugation and resuspension with ion-free water until the
washings were free from color. Resuspension of gel was facilitated by
 K. K. TSUBOI AND P. B. HUDSON 883

the use of a rubber stirrer driven by a variable speed motor. The rela-
tionship between gel concentration and enzyme adsorption is illustrated
in Fig. 1.
Elution of Enzyme from Gel-The thoroughly washed gel containing
adsorbed enzyme was evenly suspended to 500 ml. volume. To the sus-
pension was added an equal volume of 0.005 M trisodium citrate. After
being mixed, the gel was separated by centrifugation at room temperature.
The resulting eluate was a clear, light pink solution. The use of weaker

TABLE I
Purim Nucleoside Phosphorylase; Purification Protocol
- -

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Purification steps Volume Enzyme Proteint SP&fiC Purifica-
units* activityf tion
L _- .-
ml. m. fold
Hemolysate (1:5) . . . . . . . . . . . . . . 2509 7069 169,909 0.941
1st eluate from (Ca)r(PO,)e.. . . . . . . . . 925 4696 1,699 2.9 70
Following concentration with
(NH,) zS01. . . . . . . . . . . . . . . . . . . . 40 4499 920 4.7 116
Following dialysis.. . .. ... . . .. ... 65 4290 910 4.6 112
6‘ treatment with fullers’
earth............................. 84 4399 629 6.9 168
2nd (Ca)a(POd)z eluate. . . . 75 3299 269 12.3 390
Ammonium sulfate fraction.. . . .. . 4 2100 loo 21.0 510
- - - -
* An enzyme unit refers to that amount liberating 1 pmole of guanine per minute
under the specified assay conditions (see under “Methods”).
t Protein was estimated on the basis of nitrogen determinations for starting ma-
terial and final isolated product; tyrosine or ultraviolet absorption was used for other
fractions.
$ Specific activity = micromoles of guanine liberated per minute per mg. of
protein.

or stronger citrate solutions resulted, respectively, in either too little re-

covery or insufficient purification of the enzyme.
Concentration of Eluate-Concentration of the enzyme from the eluate
was achieved by ammonium sulfate. Solid ammonium sulfate (42 gm. per
100 ml. of eluate) was added to the &rate (previously cooled to nearly 0”)
and the resulting precipitate was removed by centrifugation. The pre-
cipitated protein containing all of the enzyme was dissolved in a minimal
volume of water.
Dialysis-The enzyme solution was transferred to a cellophane mem-
brane (previously washed and soaked in versenate to remove heavy metal
contaminants (16) and dialyzed for 18 hours against frequent changes of
dilute versenate solution (0.0005 M, pH 8.0). The toxicity of unwashed
cellophane, previously demonstrated with another enzyme of the eryth-
884 ENZYMES OF HUMAN ERYTHROCYTEl. I

rocyte (16), was again clearly illustrated with nucleoside phosphorylase.

The sensitivity of the enzyme toward trace metals was attributed to its
sulfhydryl nature (see Paper II of this series).
Treatment with Futkrs Earth-The dialyzed enzyme preparation was
next treated with fullers’ earth (Fisher Scientific Company, Eimer and
Amend Division) which was washed with water prior to use. A sufkient
amount of fullers’ earth was added to adsorb maximally non-enzyme pro-
tein. This usually required about 20 ml. of a 10 per cent suspensionof
fullers’ earth (for 500 ml. of original red blood cells); however, the precise
amounts should be determined with each preparation. The adsorptive
capacity of fullers’ earth for the enzyme increases sharply as the pH is
dropped below 7.

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Readsorption and Elution of Enzyme from Calcium Phosphate Gel-To
the supernatant fluid resulting from treatment with fullers’ earth was
added just sufhcient gel to adsorb approximately 90 per cent of the enzyme
(approximately 50 ml. of gel required). The gel containing adsorbed en-
zyme was washed several times with water and finally eluted by careful
resuspension with 100 ml. of 0.0025 M trisodium citrate.
Ammonium Sulfate Fraction&on---The eluate in 0.1 M tris(hydroxy-
methyl)aminomethane (Tris) buffer, pH 8.0, was treated with solid am-
monium sulfate (42 gm. per 100 ml. of eluate). The resulting sediment
was dissolved in a minimal volume of water and dialyzed against 0.01 M
Tris, pH 8.0. Following dialysis, the enzyme solution was fractionated
with ammonium sulfate. Saturated ammonium sulfate (neutralized) was
added to final 42.5 per cent saturat,ion and the resulting sediment was
removed. To the supernatant solution was added saturated ammonium
sulfate to bring to a final 50 per cent saturation. The resulting sediment
contained most of the remaining enzyme and represented the final product.

Relative Enzyme Purity

Enzyme prepared by procedures described in the previous section was
investigated for purity. These studies were carried out on a preparation
which had been allowed to remain in the refrigerator as a solution (dis-
solved ammonium sulfate precipitate) for approximately a month. During
this int.erval, some inactivation of enzyme and denaturation of protein
occurred. The denatured protein was removed by centrifugation and the
clear supernatant fluid was dialyzed thoroughly against 0.1 M phosphate
buffer at pH 7.5. The dialyzed enzyme solution (pale yellow in color)
was tested for relative purity.
Paper Electrophoresis-The relative homogeneity of the preparation was
investigated by paper electrophoretic analyses in which the Spinco model
R instrument (Durrum type cell) was used. The procedure was as fol-
 K. K. TSUBOI AND P. B. HUDSON 885

lows: 20 ~1. aliquots of the enzyme solution (0.8 per cent protein) were
transferred onto separate paper strips which had been equilibrated pre-
viously with buffer solution (Verona1 buffer at 0.075 ionic strength and
pH 8.5). The electrophoretic run was carried out in a cold room (at
approximately 6”) for a 16 hour period with a constant current of 5 ma.
Protein patterns were obtained after staining with brom phenol blue.
The stained areas were subsequently analyzed for dye concentration with
a recording densitometer (Spinco, Analytrol).
From the electrophoretic patterns obtained, the preparation appeared
to be homogeneous. A slight amount of material was found to be re-
tained at the origin (presumably denatured protein). The remaining

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FIG. 3. Sedimentation pattern. The pattern was obtained after 70 minutes at

59,890 r.p.m. at 13.5” and a bar angle of 30” (see the text for details).
material moved as a single band and covered an average distance of 2 cm.
under the conditions of the experiment. Circumstances did not allow
further investigation of the preparation by conventional electrophoresis.
Limited studies with other enzymes prepared in these laboratories have
resulted in qualitatively similar electrophoretic patterns in which paper
and conventional electrophoretic techniques have been used. Little en-
zyme activity was found to be retained after the electrolytic procedure
with nucleoside phosphorylase on paper (inactivation presumably due to
heavy metal contaminants present in paper).
Ultracentrifugal Studies-The relative purity of the enzyme preparation
was further examined by ultracentrifugation (Spinco model E ultracen-
trifuge). The ultracentrifuge run was carried out as follows: The enzyme
solution was dialyzed thoroughly against 0.1 M phosphate, pH 7.5. The
dialyzed solution contained 0.8 per cent protein. A 0.28 ml. volume of
886 ESZYMES OF HUMAN EHYTHHOCYTE. I

t,he solution was placed into an appropriate cell and the sedimentation
was carried out at 59,890 r.p.m. in t,he analytical rotor T\‘o. A61 at an aver-
age temperature of 13.5”. Photographs were taken at convenient inter-
vals.
The sedimentation pattern obtained following a 70 minute run is repro-
duced in Fig. 3. A faster moving minor contaminant was found to be
present in the preparation. That the slower moving fraction, rather t.han
the minor component, represented the enzyme was ascertained on the basis
of specific activity measurements on samples carefully withdrawn from
the top and bottom halves of the cell following a 70 minute run. A calcu-
lation of the sedimentation constant of the enzyme, approximated to

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standard conditions (i.e. in water at 20” in the customary way (20)), gave
a value of 6.3 Svedberg units (1 S = I X lo-l3 c.g.s.u.).

DISCUSSIOK

Purine nucleoside phosphorylase is apparently present at a relatively

high concentration in the human erythrocyte (presumably present at about
0.5 per cent of the total protein). The enzyme can be obtained in good
yield and in apparently nearly pure form with simple purification proce-
dures from small amounts of starting material. Attempts to obtain a
further isolated product or crystallization of t.he enzyme would have re-
quired larger scale preparations and were not considered to be within the
scope of the present investigations.
As additional evidence of the relatively high purity of the enzyme, cal-
culations showed a turnover of 2100 moles of substrate per minute per
100,000 gm. of protein with our best preparation, the standard assay pro-
cedure being used. For purposes of comparison, the better preparations
of enzyme purified from rat liver were described by Kalckar (10) as forming
2 mg. of hypoxanthine (inosine as substrate) per hour per mg. of protein,
which would be a turnover of approximately 25 moles of substrate per
minute per 100,000 gm. of protein (measurements made at 25” and sub-
saturating substrate).
Investigations relating to stability characteristics as well as specific
kinetic properties of the purified enzyme will be summarized in the follow-
ing report.

The authors wish to t,hank Dr. Kenneth McCarty for carrying out the
ult.racentrifugal analyses and Dr. Elliot Osserman for the electrophoretic
analyses.
SUMMARY

1. Appropriate procedures for the reliable assay of purine nucleoside

phosphorylase with use of a calorimetric procedure were described.
 k. K. TSUBOI AND P. B. HUDSON 887

2. Methods for obtaining purine nucleoside phosphorylase in highly

purified form from the human erythrocyte were presented in detail.
3. Evidence was presented suggesting a high order of enzyme purifica-
tion in the final product, based on ultracentrifugal analyses, electrophoretic
analyses, and the relative turnover capacity of the preparation.

BIBLIOGRAPHY

1. Keilin, D., and Mann, T., Biochem. J., 34, 1163 (1949).
2. Laskowski, M., and Sumner, J. B., Science, 94, 615 (1941).
3. Adams, E., and Smith, E. L., J. Biol. Chem., 198, 671 (1952).
4. Adams, E., Davis, N. C., and Smith, E. L., J. Biol. Chem., 199, 846 (1952).
5. Zittle, C. A., DellaMonica, E. S., and Custer, J. H., Arch. Biochem. and Biophye.,

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43, 43 (1954).
6. Tsuboi, K. K., and Hudson, P. B., Arch. Biochem. and Biophys., 63, 341 (1954).
7. Dische, Z., Naturwissenschaften, 26, 252 (1938).
8. Tsuboi, K. K., and Hudson, P. B., Arch. Biochem. and Biophys., 43, 339 (1953).
9. Kalckar, H. M., J. Biol. Chem., 167, 461 (1947).
IO. Kalckar, H. M., J. Biol. Chem., 167, 477 (1947).
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12. Paege, L. M., and Schlenk, F., Arch. Biochem. and Biophys., 40, 42 (1952).
13. Friedkin, M., and Roberts, D., J. BioZ. Chem., 207, 245 (1954).
14. Heppel, L. A., and Hilmoe, R. J., J. BioZ. Chem., 198, 683 (1952).
15. Hitchings, G. H., J. BioZ. Chem., 139, 843 (1941).
16: Tsuboi, K. K., and Hudson, P. B., Arch. Biochem. and Biophye., 66, 206 (1955).
17. Folin, O., and Ciocalteu, V., J. BioZ. Chem., 73, 627 (1927).
18. Speck, J. F., J. BioZ. Chem., 179, 1387 (1949).
19. Vanselow, A. P., Ind. and Eng. Chem., Anal. Ed., 22, 516 (1940).
20. Svedberg, T., and Pedersen, K. O., The ultracentrifuge, Oxford (lQ49).
 ENZYMES OF THE HUMAN
ERYTHROCYTE: I. PURINE
NUCLEOSIDE PHOSPHORYLASE;
ISOLATION PROCEDURE
K. K. Tsuboi and Perry B. Hudson
J. Biol. Chem. 1957, 224:879-887.

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