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A simple assay for galactokinase using deae-cellulose column chromatography
Clinica Chimica Acta, 74 (1977) l-5 0 Elsevier/North-Holland Biomedical
Press
CCA 7994
A SIMPLE ASSAY FOR GALACTOKINASE COLUMN CHROMATOGRAPHY
Y.S. SHIN-BUEHRING, Kinderklinik
(Received
M. OSANG,
der Universita t Miinchen,
April 14th,
R. ZIEGLER Miinchen
USING DEAE-CELLULOSE
and J. SCHAUB
*
(G. F. R.)
1976)
The radioactive assay for galactokinase activity utilizing paper chromatography was described first by Robinson [l] and later by other authors [Z-4]. Recently, Beutler and Matsumoto [ 51 reported a rapid method using a paper disc instead of the usual paper chromatography. The former method involving paper chromatography is accurate, but time consuming and not very suitable for routine assays. The latter, on the other hand, not only yielded inconsistent duplicates in many cases but also required a large amount of distilled water (500 ml) to remove galactose from the paper disc. Therefore, we developed a chromatographic procedure employing a small DEAE-cellulose column [6] for the separation of galactose l-phosphate from galactose. We found that this separation method is far more convenient than other methods and also fairly accurate in establishing the optimal conditions for the galactokinase assay, Michaelis constants, pH optimum, and the effects of incubation time, protein and Mg’+ concentration on the enzyme activity. According to thin-layer chromatography, [ “C]galactose (60 Ci/mol, Amersham) was contaminated by a small amount of other hexoses and disaccharides. To remove [ “C]glucose which interferes with the assay, the radioactive galactose was treated with hexokinase. The resulting glucose 6-phosphate was then removed by ion exchange column chromatography. The reaction mixture for galactokinase in whole blood contained per 200 ~1 the following: 80 nmol (0.1 PCi) [14C]galactose, 1.6 pmol MgCl*, 0.2% saponin, 40 pmol Tris/HCJ, 1.5 lmol ATP and 0.8 E.tmol NaF. Reaction was initiated by adding 50 ~1 whole blood, stopped by heating the samples to 95°C for 2 min, and the content was then cooled and centrifuged. 100 ~1 of the supematant was applied to the column and chromatographed as described below. The blank was prepared in the same way as above by using heat-inactivated samples. DEAE-cellulose from Serva was washed as described previously [ 61. The OHor phosphate form of DEAE-cellulose can be used for the separation of galac-
* Requests for reprints should be sent to: Dr. Jurgen Lindwurmstrasse 4. D-8000 Munchen 2. G.F.R.
Schaub,
Kinderklinik
der Universitat
Munchen.
2
tose l-phosphate from galactose. Disposable syringes (2 ml). the bottoms of which were plugged with glass wool, were filled with washed DEAE-cellulose up to the O.&ml mark. A simple syringe holder prepared from cardboard was placed over a scintillation vial container and fractions were collected directly into the vials. With this simple construction as many as 80 samples can be chromatographed at the same time and up to 0.5 ml reaction mixture can be satisfactorily chromatographed on this column. The radioactivity was counted in 10 ml scintillation fluid (3.0 g PPO, 0.2 g POPOP, 250 ml Triton X-100 and 1000 ml toluene). Fig. 1 shows the separation of the product, galactose l-phosphate and the substrate, galactose, from the assay mixture. The first three fractions collected with distilled water contained galactose and two fractions collected with 100 mM I-ICI contained galactosc l-phosphate. Galactokinase in erythrocytes is stable for at least 10 days when it is kept as whole blood at 4°C. The enzyme tests were repeated at least twice within 10 days. The relationship between pH of the reaction mixture and enzyme activity is shown in Fig. 2. It was reported that the optimal galactokinase activity was between pH 7.8 and 8.2 [7,8]. Beutler and coworkers, however, routinely used Tris/HCl buffer pH 7.4 for the enzyme assay [4,5]. We studied the pH optimum of the enzyme in erythrocytes from nine various blood samples. As shown in Fig. 2, two kinds of pH curves were obtained: a smooth curve with the maximum around pH 7.7 from two adult, one fetus and one cord blood sample; a curve with a shoulder about pH 7.5 and the maximum around pH 7.9 from two child and two cord blood samples. This observation may not have any significance in terms of characterizing or differentiating the blood, since
R
2
1
3
FRACTION Fig.
1. Separation
column. 100
mM
60 min
Column HCl. (m---j.
of size:
The
the 0.9
reaction
NUMBER
product. cm
Gal-l-l’,
X 1.0
mixture
4
(2
5
ML)
from
galactose
in the
cm.
Fractions:
l-3
were
was
incubated
for
0 min
reaction
collected (o-
mixture with
- - - - -o),
on
distilled for
30 min
the water (A-
DEAE-cellulose and
4-5
- .A) and
with for
3
t ?-
6
0 l-
x 4 I k
I
I
7.0 - 8.0 PH in incubation mixture Fig. 2. pH depydence sample (Hb 13.36gQ);. final mixture.
of galactokinase activity ------A, an adult blood
in two typical blood samples. l r, a cord blood sample (Hb 12.44 g%). The end pH was measured in the
there is no correlation between the shape of the curve and the source of the blood. However, it would be of interest to know whether the formation of the shoulder is due to isoenzymes or several binding sites of the enzyme, or simply the different buffer capacity of the blood. 1 M Tris/HCl buffer, pH 8.0 (the final pH in the incubation mixture was around 7.8) was chosen for the routine assay. The enzyme activity was linear with incubation times of up to 1 h and proportional to haemoglobin concentrations of up to at least 15 g% (Fig. 3). An incubation time of 30 min and undiluted whole blood (Hb 12-15 g%) were selected for the routine assay. The result obtained by varying the Mg2+ concentration in the reaction mix-
3 Fig. 3. The effect zyme velocity.
6 of incubation
s time
(0~
MIN.(*) 12 gHb% Is) ) and the enzyme
concentration
(x-
*-
*X) on the en-
I
1
Mg2+( mM Fig.
4.
The
effect
of
Mg2+
L
20
10 on
the
30
1 enzyme
activity.
Haemoglobin
concentration
in the
sample
was
11.59
g%.
TABLE
I
NORMAL VALUES OF GALACTOKINASE GALACTOSE IN VARIOUS BLOOD SAMPLES
ACTIVITY
The enzyme activities
phosphorylated
THE
Source
of
are presented Expt.
as nmoi
No.
gaiactose Ages
AND
MICHAELIS
per min
per g haemoglobin,
Enzyme
activity
blood Fetus
Cord
Baby
Child
1
18 weeks
*
90.0,
96.8
2
22
*
158.4,
166.0
3
58.7,
4
130.7,
142.3
5
89.4,
82.8
5 months
39.0,
35.5
7
7 months
51.6,
49.0
8
9 months
35.3,
35.4
9
4 years
36.0,
34.1
32.7,
33.0 33.4
5 years
11
7 years
35.6,
12
9 years
33.0,
36.4
32.6,
30.0
13
Patient
* Gestation Letters
years
14
31
years
27.2,
29.5
15
37
years
30.4,
27.0
10
years
0.77,
(F.K.)
4 months
0.65,
0.67
18 (J.O.)
5 months
0.0,
0.0
16.5,
16.8
17
**
24
16 (Sa.0.)
Heterotygote
55.8
6
10
Adult
weeks
**
19
(Sm.0.)
20
(K.K.)
27
8 years years
21
(S1.0.)
27
years
age. in parentheses
indicate
initials
of subjects.
CONSTANTS
7.5, 10.3,
0.55
8.5 10.8
FOR
adult 13 Km=0164mM child 12 Km=0174mM baby 6 Km=0 21 mM
cord 5 Km=032mM
Km=
l/Galactose Fig.
5. Lineweaver-Burk
listed
in Table
plots
I under
Expt.
0 4mM
(mM1
for the Michalis-Menten
constants
of 5 subiects.
The
subjects
are the same
as
NO.
ture is shown in Fig. 4. The velocity of the enzyme reaction is the highest when the ratio of the concentration of ATP to that of Mg” is between 0.6 and 1.3. The normal values of galactokinase activity and KM values for galactose are summarized in Table I. Galactokinase activity assayed by the method described here is somewhat higher that reported in the literature [3,7,9]. It is interesting to note that the affinity of galactose to the enzyme is different in fetal, baby and adult erythrocyte galactokinase, i.e. Kill values decreased with increasing age of the subjects (Fig. 5). The difference in Michaelis constants for galactose in cord and adult erythrocyte galactokinase was observed by Mathai and Beutler [4]; however, they were unable to differentiate cord blood enzyme from adult enzyme by column chromatography. In contrast, Vigneron [lo] found that fetal and adult enzymes show different mobilities when subjected to starch gel electrophoresis. More studies are required to find an explanation for this interesting phenomenon. Acknowledgement This study was supported
by the Deutsche
Forschungsgemeinschaft.
References 1
Robinson,
A.
(1963)
2
Sherman.
J.R.
and
3
Ng,
Donnell,
4
Mathai,
W.G.,
C.K.
5
Beutler,
6
Shin-Buehring.
I
Mayes,
J.S.
8
Blume,
K.
9
Gitzelmann,
10
Vigneron,
and
J. Exp. Adler, G.N.
Guthrie,
and Beutler, R.
(1967)
C. (1971)
R.
M..
Enzyme
J. Lab.
Ziegler.
E. (1971)
J. Biol. 426-432
33.
Clin. Genet.
Chem.
1, 14-23
873-878
J. Lab.
Clin.
Med.,
66.
115-121
224-230
Med.
R. and Schaub.
Biochem.
Res. 12.
238,
(1965)
Enzymologia
(1968)
Pediatr.
Chem.
W.R.
F. (1973)
Osang,
359-370
J. Biol.
E. (1967)
E. and Matsumoto. and
118,
and Bergen.
Beutler.
Y.S.,
Med.
J. (1963)
82,
818-821
J. (1976)
2, 219-230
246,6507-6510
Clin.
Chim.
Acta
70,
371-377
Press
CCA 7994
A SIMPLE ASSAY FOR GALACTOKINASE COLUMN CHROMATOGRAPHY
Y.S. SHIN-BUEHRING, Kinderklinik
(Received
M. OSANG,
der Universita t Miinchen,
April 14th,
R. ZIEGLER Miinchen
USING DEAE-CELLULOSE
and J. SCHAUB
*
(G. F. R.)
1976)
The radioactive assay for galactokinase activity utilizing paper chromatography was described first by Robinson [l] and later by other authors [Z-4]. Recently, Beutler and Matsumoto [ 51 reported a rapid method using a paper disc instead of the usual paper chromatography. The former method involving paper chromatography is accurate, but time consuming and not very suitable for routine assays. The latter, on the other hand, not only yielded inconsistent duplicates in many cases but also required a large amount of distilled water (500 ml) to remove galactose from the paper disc. Therefore, we developed a chromatographic procedure employing a small DEAE-cellulose column [6] for the separation of galactose l-phosphate from galactose. We found that this separation method is far more convenient than other methods and also fairly accurate in establishing the optimal conditions for the galactokinase assay, Michaelis constants, pH optimum, and the effects of incubation time, protein and Mg’+ concentration on the enzyme activity. According to thin-layer chromatography, [ “C]galactose (60 Ci/mol, Amersham) was contaminated by a small amount of other hexoses and disaccharides. To remove [ “C]glucose which interferes with the assay, the radioactive galactose was treated with hexokinase. The resulting glucose 6-phosphate was then removed by ion exchange column chromatography. The reaction mixture for galactokinase in whole blood contained per 200 ~1 the following: 80 nmol (0.1 PCi) [14C]galactose, 1.6 pmol MgCl*, 0.2% saponin, 40 pmol Tris/HCJ, 1.5 lmol ATP and 0.8 E.tmol NaF. Reaction was initiated by adding 50 ~1 whole blood, stopped by heating the samples to 95°C for 2 min, and the content was then cooled and centrifuged. 100 ~1 of the supematant was applied to the column and chromatographed as described below. The blank was prepared in the same way as above by using heat-inactivated samples. DEAE-cellulose from Serva was washed as described previously [ 61. The OHor phosphate form of DEAE-cellulose can be used for the separation of galac-
* Requests for reprints should be sent to: Dr. Jurgen Lindwurmstrasse 4. D-8000 Munchen 2. G.F.R.
Schaub,
Kinderklinik
der Universitat
Munchen.
2
tose l-phosphate from galactose. Disposable syringes (2 ml). the bottoms of which were plugged with glass wool, were filled with washed DEAE-cellulose up to the O.&ml mark. A simple syringe holder prepared from cardboard was placed over a scintillation vial container and fractions were collected directly into the vials. With this simple construction as many as 80 samples can be chromatographed at the same time and up to 0.5 ml reaction mixture can be satisfactorily chromatographed on this column. The radioactivity was counted in 10 ml scintillation fluid (3.0 g PPO, 0.2 g POPOP, 250 ml Triton X-100 and 1000 ml toluene). Fig. 1 shows the separation of the product, galactose l-phosphate and the substrate, galactose, from the assay mixture. The first three fractions collected with distilled water contained galactose and two fractions collected with 100 mM I-ICI contained galactosc l-phosphate. Galactokinase in erythrocytes is stable for at least 10 days when it is kept as whole blood at 4°C. The enzyme tests were repeated at least twice within 10 days. The relationship between pH of the reaction mixture and enzyme activity is shown in Fig. 2. It was reported that the optimal galactokinase activity was between pH 7.8 and 8.2 [7,8]. Beutler and coworkers, however, routinely used Tris/HCl buffer pH 7.4 for the enzyme assay [4,5]. We studied the pH optimum of the enzyme in erythrocytes from nine various blood samples. As shown in Fig. 2, two kinds of pH curves were obtained: a smooth curve with the maximum around pH 7.7 from two adult, one fetus and one cord blood sample; a curve with a shoulder about pH 7.5 and the maximum around pH 7.9 from two child and two cord blood samples. This observation may not have any significance in terms of characterizing or differentiating the blood, since
R
2
1
3
FRACTION Fig.
1. Separation
column. 100
mM
60 min
Column HCl. (m---j.
of size:
The
the 0.9
reaction
NUMBER
product. cm
Gal-l-l’,
X 1.0
mixture
4
(2
5
ML)
from
galactose
in the
cm.
Fractions:
l-3
were
was
incubated
for
0 min
reaction
collected (o-
mixture with
- - - - -o),
on
distilled for
30 min
the water (A-
DEAE-cellulose and
4-5
- .A) and
with for
3
t ?-
6
0 l-
x 4 I k
I
I
7.0 - 8.0 PH in incubation mixture Fig. 2. pH depydence sample (Hb 13.36gQ);. final mixture.
of galactokinase activity ------A, an adult blood
in two typical blood samples. l r, a cord blood sample (Hb 12.44 g%). The end pH was measured in the
there is no correlation between the shape of the curve and the source of the blood. However, it would be of interest to know whether the formation of the shoulder is due to isoenzymes or several binding sites of the enzyme, or simply the different buffer capacity of the blood. 1 M Tris/HCl buffer, pH 8.0 (the final pH in the incubation mixture was around 7.8) was chosen for the routine assay. The enzyme activity was linear with incubation times of up to 1 h and proportional to haemoglobin concentrations of up to at least 15 g% (Fig. 3). An incubation time of 30 min and undiluted whole blood (Hb 12-15 g%) were selected for the routine assay. The result obtained by varying the Mg2+ concentration in the reaction mix-
3 Fig. 3. The effect zyme velocity.
6 of incubation
s time
(0~
MIN.(*) 12 gHb% Is) ) and the enzyme
concentration
(x-
*-
*X) on the en-
I
1
Mg2+( mM Fig.
4.
The
effect
of
Mg2+
L
20
10 on
the
30
1 enzyme
activity.
Haemoglobin
concentration
in the
sample
was
11.59
g%.
TABLE
I
NORMAL VALUES OF GALACTOKINASE GALACTOSE IN VARIOUS BLOOD SAMPLES
ACTIVITY
The enzyme activities
phosphorylated
THE
Source
of
are presented Expt.
as nmoi
No.
gaiactose Ages
AND
MICHAELIS
per min
per g haemoglobin,
Enzyme
activity
blood Fetus
Cord
Baby
Child
1
18 weeks
*
90.0,
96.8
2
22
*
158.4,
166.0
3
58.7,
4
130.7,
142.3
5
89.4,
82.8
5 months
39.0,
35.5
7
7 months
51.6,
49.0
8
9 months
35.3,
35.4
9
4 years
36.0,
34.1
32.7,
33.0 33.4
5 years
11
7 years
35.6,
12
9 years
33.0,
36.4
32.6,
30.0
13
Patient
* Gestation Letters
years
14
31
years
27.2,
29.5
15
37
years
30.4,
27.0
10
years
0.77,
(F.K.)
4 months
0.65,
0.67
18 (J.O.)
5 months
0.0,
0.0
16.5,
16.8
17
**
24
16 (Sa.0.)
Heterotygote
55.8
6
10
Adult
weeks
**
19
(Sm.0.)
20
(K.K.)
27
8 years years
21
(S1.0.)
27
years
age. in parentheses
indicate
initials
of subjects.
CONSTANTS
7.5, 10.3,
0.55
8.5 10.8
FOR
adult 13 Km=0164mM child 12 Km=0174mM baby 6 Km=0 21 mM
cord 5 Km=032mM
Km=
l/Galactose Fig.
5. Lineweaver-Burk
listed
in Table
plots
I under
Expt.
0 4mM
(mM1
for the Michalis-Menten
constants
of 5 subiects.
The
subjects
are the same
as
NO.
ture is shown in Fig. 4. The velocity of the enzyme reaction is the highest when the ratio of the concentration of ATP to that of Mg” is between 0.6 and 1.3. The normal values of galactokinase activity and KM values for galactose are summarized in Table I. Galactokinase activity assayed by the method described here is somewhat higher that reported in the literature [3,7,9]. It is interesting to note that the affinity of galactose to the enzyme is different in fetal, baby and adult erythrocyte galactokinase, i.e. Kill values decreased with increasing age of the subjects (Fig. 5). The difference in Michaelis constants for galactose in cord and adult erythrocyte galactokinase was observed by Mathai and Beutler [4]; however, they were unable to differentiate cord blood enzyme from adult enzyme by column chromatography. In contrast, Vigneron [lo] found that fetal and adult enzymes show different mobilities when subjected to starch gel electrophoresis. More studies are required to find an explanation for this interesting phenomenon. Acknowledgement This study was supported
by the Deutsche
Forschungsgemeinschaft.
References 1
Robinson,
A.
(1963)
2
Sherman.
J.R.
and
3
Ng,
Donnell,
4
Mathai,
W.G.,
C.K.
5
Beutler,
6
Shin-Buehring.
I
Mayes,
J.S.
8
Blume,
K.
9
Gitzelmann,
10
Vigneron,
and
J. Exp. Adler, G.N.
Guthrie,
and Beutler, R.
(1967)
C. (1971)
R.
M..
Enzyme
J. Lab.
Ziegler.
E. (1971)
J. Biol. 426-432
33.
Clin. Genet.
Chem.
1, 14-23
873-878
J. Lab.
Clin.
Med.,
66.
115-121
224-230
Med.
R. and Schaub.
Biochem.
Res. 12.
238,
(1965)
Enzymologia
(1968)
Pediatr.
Chem.
W.R.
F. (1973)
Osang,
359-370
J. Biol.
E. (1967)
E. and Matsumoto. and
118,
and Bergen.
Beutler.
Y.S.,
Med.
J. (1963)
82,
818-821
J. (1976)
2, 219-230
246,6507-6510
Clin.
Chim.
Acta
70,
371-377