527
Clinica Chimica Acta, @ Elsevier/North-Holland
79 (1977)
527-531
Biomedical
Press
CCA 8514
A SENSITIVE FLUORESCENCE SEPARATE DETERMINATION
HELENE
CHRISTOMANOU
Max-Planck-Institut (G.F.R.)
(Received
December
and K. SANDHOFF
fzir Psychiatric,
13th,
ASSAY FOR THE SIMULTANEOUS OF ARYLSULPHATASES A AND B
Neurochemische
AND
* Abteilung,
Kraepelinstrasse
2, Miinchen
1976)
Summary A sensitive fluorometric assay utilizing 4-methylumbelliferyl sulphate has been developed for the simultaneous determination of arylsulphatases A and B from leucocytes, based on the differential effect of silver ions on the two enzymes. The procedure allows discrimination between normal cases and those with metachromatic leucodystrophy.
Introduction The physiological substrates for arylsulphatases A (AS-A) and B (AS-B) have been identified [1,2] but the activity of these enzymes, which are deficient in metachromatic leucodystrophy (MLD) [ 31 and mucopolysaccharidosis (type VI) [ 4,5], respectively, is usually measured calorimetrically using 2-hydroxy5nitrophenylsulphate (NCS) as substrate [6]. Due to the limitations of this method, however, low concentrations of enzymes cannot be accurately measured. A more sensitive assay employs 4-methylumbelliferyl sulphate (MUS) as substrate [7-111. Delvin et al. [12] demonstrated that this substrate cannot be used for the simultaneous and separate determination of arylsulphatases A and B in cells from normal and pathological tissues. This paper reports that this is feasible in the presence of Ag’ ions which specifically inhibit arylsulphatase A [10,13]. Materials and methods 4-Methylumbelliferyl sulphate sulphate from Sigma, Ampholine
* To
whom
correspondence
should
was obtained from Koch-Light, from LKB, AgNO, from Merck.
be addressed.
nitrocatechol
528
Leucocytes from normal and pathological individuals were used as sources of enzymes. Leucocyte pellets were prepared according to Kampine et al. [14] and homogenized in 2 ml of 0.25% Triton X-100. The homogenates were dialysecl for 12-15 h at 4°C against distilled water. The dialysed samples were isoelectrically focused in a microanalytical column as described previously [15,16]. Fractions were analysed for AS-A and AS-B activities using an automatic enzyme analyser (5030, Eppendorf, Hamburg) as given below. The pooled AS-A and AS-B fractions were used as the enzyme source. If not otherwise stated, the standard incubation mixture for the determination of the total AS-activity contained, in a final volume of 200 ~1, 100 ~1 of 10 mM MUS in 50 mM sodium acetate buffer, pH 5.0, and up to 50 ~1 enzyme solution. For the determination of AS-B activity, AgNO, was added to the above mixture in a final concentration of 0.3 mM unless stated otherwise. Standard incubations were carried out for 15 min at 37”C, the reactions were stopped by addition of 1 ml of 0.2 M glycine/sodium carbonate buffer, pH 10.4. 4-Methylumbelliferone (4-MU) released during the reaction was measured in an Eppendorf fluorimeter at an excitation wavelength of 365 nm and an emission wavelength of 440 nm. 100 fluorescence units correspond to 0.5 PM 4-methylumbelliferone. The determination of AS-A and AS-B activity with the NCS substrate was performed according to Baum et al. [6] modified by Harzer et al. [16] by determination of nitrocatechol (NC) released.
c C1.3-
0I.2-
:o 4
I.1-
h
z c
Fig. 1. Arylsulphatase A and B patterns obtained c’- - - - - -0, total arylsulphatase activity; *A, AgN03. For details see Materials and methods.
after isoelectric arylsulphatase
focusing of leucocyte homogenates. activity in the presence of 0.3 mM
529
AS-8
=
0.001
0.003
1-t
-
.
11 “ ODI
Fig. 2. Activity of focused arylsulphatases details see Materials and methods.
Results
I
*
003
AS-A
I--_
11 0.1.
”
0.2
0.3
A and B as a function
od
of silver nitrate
b
concentration.
For
and discussion
Fig. 1 shows the arylsulphatase pattern of a leucocyte homogenate after isoelectric focusing. The assay of arylsulphatase activity in the presence of silver ions reveals the
IO
-
AS-E
E---O
AS-0
-
AS-A
O--0
AS-A
20
30
+Ag+ions
+ Ag+ions
40
60 Time,
Fig. 3. Arylsulphatase activities see Materials and methods.
in the presence
and absence
mtnutes
of AgN03
as a function
of time. For details
530
A 0.14 -
T 0.6 -
-
AS-A
c---a
AS-
-
AS-B
wthout wth
A + Ag’oons
0.12
0.5 -
Ag+,ons
Ag+,ons
-
0.10 -
0.4 -
2 c
0
0.3 -
3 I
z
4
“E 0.2 . c
1
0.08
-
0.06
-
0.04
-
:::g .n LI :$:::::$s z.;$g $$$N ::::::::::.: ::::::::::;: ::::::x.:
E c 0.1 -
a02
-
y: $y$
\S-A Fig. tions Fig.
4.
Arylsulphatase
activities
are described 5.
under
Arylsulphatase
absence
of
conditions
AgN03.
in the
Materials activity
Enzyme
are described
of
and
presence
absence
of
AgN03
methods.
focused
concentrations
under
and
AS-A. were:
AS-B AS-A:
and 25
their p;
I
as a function
mixture
AS-B:
25
assayed ~1:
AS-A
-8
-
of PH.
in the + AS-B:
Assay
presence 50
condi-
and
pl. Assay
Methods.
two known peaks of AS-B with isoelectric points at pH 8.2 and 9.4. In the absence of silver ions an additional peak of arylsulphatase A activity with an isoelectric point at pH 5.2 is observed. As shown in Fig. 2 only AS-B remains active in the presence of silver nitrate in the range of 0.1 to 0.3 mM whereas AS-A is inhibited by about 96%. This inhibiting effect remains constant during incubation for 90 min (Fig. 3) and is independent of the amount of enzyme used. Variation of the pH in the incubation mixture reveals that AS-A activity
TABLE
I
ARYLSULPHATASE PATIENTS The
activities
WITH
A
AND
B IN
METACHROMATIC
are expressed Age
(years)
as nmoles
LEUCOCYTE
HOMOGENATES
OF
CONTROL
SUBJECTS
LEUCODYSTROPHY 4-methylumbelliferone Arylsulphatase
A
released
per mg protein
Arylsulphatase
Control
50
0.20
0.39
Control
55
0.25
0.82
Control
10
0.24
0.84
Control
13
0.52
0.84
MLD
17
0.011
0.36
MLD
8
0.041
0.89
MLD
3
0.052
0.47
MLD
3
0.00
4.18
MLD
3
0.00
6.02
B
per h.
AND
531
is almost completely abolished in the optimal range between pH 4.5 and 5.6, while AS-B is not affected at all (Fig. 4). By measuring arylsulphatase activity in the presence and absence of silver ions it is possible to determine AS-A and AS-B activities in a mixture of both enzymes (Fig. 5). The same incubation conditions have been used for the assay of AS-S and AS-B in crude leucocyte homogenates. Patients with MLD exhibit very low but definite AS-A activities (Table I). Acknowledgements We express our thanks to Mrs. &p for excellent cooperation and to Dr. Harzer for the providing leucocyte samples from patients with metachromatic leucodystrophy. This work was supported by the Deutsche Forschungsgemeinschaft (grant Sa 257/2). References 1
Mehl.
2
Matalon,
E. and Jatzkewitz,
3
Austin,
J., McAfee.
4
Beratis,
N.-G.,
5
Fluharty,
A.L..
Commun.
64,
R.,
6
Baum.
7
Sherman,
H.,
8
Rinderknecht,
H. (1968)
Arbogast,
B. and
D. and
Turner.
Shearer,
B.M.,
Stevens,
Biochim.
Dorfman. L.
Weiss.
R.L.,
(1974)
(1965)
R. and
Fun&
Biophys.
A.
D..
Acta
Arch. S.
K. and
619427
Biophys.
Neural.
Hirschhorn, Peak,
151.
Biochem.
12,
Res.
Commun.
61.
(1975)
Pediatr.
Kihara.
H.
Res.
(1975)
9, 475-480
Biochem.
Biophys.
Dodgson, W.R.
K.S.
and
and
Spencer.
Stanfield,
H..
E.F.
Geokas,
M.C.,
B. (1959)
(1967)
Clin.
Biochem.
Carmack.
C. and
Chim.
J. 102.
Acta
4. 453-455
905--909
Haverback,
9
Harinath.
B.C.
and
Robins,
E. (1971)
J. Neurochem.
18.
10
Harinath,
B.C.
and
Robins.
E. (1971)
J. Neurochem.
18,
11
Perumal. Delvin,
13
Dubois,
14
Kampine.
A.S. E.E.,
and
J.P.,
15
Harzer,
K.
Harzer,
K.,
Robins,
Pottier.
G. and
16
Chem.
955-961
B.J.
(1970)
Clin.
Chim.
491
12
1450-1457
447-455
A. and
Baumann. Brady,
(1970)
R.O.
Z. Anal.
Stinshoff,
E. (1973)
K.,
J. Neurochem.
Glorieux,
N.
(1973)
and Chem. Mraz,
F.H. Biochem.
Kanfer. 252,
J.N.
(1976)
21,
245-257 459471
Biochem.
Biophys. (1967)
237-244
Res.
Science
J. 157, Commun. 155,
353-356 50.1129-1135
86
170
W. and Jatzkewitz,
H. (1973)
J. Neurochem.
20.
279-287
Acta
29,
481-
Clinica Chimica Acta, @ Elsevier/North-Holland
79 (1977)
527-531
Biomedical
Press
CCA 8514
A SENSITIVE FLUORESCENCE SEPARATE DETERMINATION
HELENE
CHRISTOMANOU
Max-Planck-Institut (G.F.R.)
(Received
December
and K. SANDHOFF
fzir Psychiatric,
13th,
ASSAY FOR THE SIMULTANEOUS OF ARYLSULPHATASES A AND B
Neurochemische
AND
* Abteilung,
Kraepelinstrasse
2, Miinchen
1976)
Summary A sensitive fluorometric assay utilizing 4-methylumbelliferyl sulphate has been developed for the simultaneous determination of arylsulphatases A and B from leucocytes, based on the differential effect of silver ions on the two enzymes. The procedure allows discrimination between normal cases and those with metachromatic leucodystrophy.
Introduction The physiological substrates for arylsulphatases A (AS-A) and B (AS-B) have been identified [1,2] but the activity of these enzymes, which are deficient in metachromatic leucodystrophy (MLD) [ 31 and mucopolysaccharidosis (type VI) [ 4,5], respectively, is usually measured calorimetrically using 2-hydroxy5nitrophenylsulphate (NCS) as substrate [6]. Due to the limitations of this method, however, low concentrations of enzymes cannot be accurately measured. A more sensitive assay employs 4-methylumbelliferyl sulphate (MUS) as substrate [7-111. Delvin et al. [12] demonstrated that this substrate cannot be used for the simultaneous and separate determination of arylsulphatases A and B in cells from normal and pathological tissues. This paper reports that this is feasible in the presence of Ag’ ions which specifically inhibit arylsulphatase A [10,13]. Materials and methods 4-Methylumbelliferyl sulphate sulphate from Sigma, Ampholine
* To
whom
correspondence
should
was obtained from Koch-Light, from LKB, AgNO, from Merck.
be addressed.
nitrocatechol
528
Leucocytes from normal and pathological individuals were used as sources of enzymes. Leucocyte pellets were prepared according to Kampine et al. [14] and homogenized in 2 ml of 0.25% Triton X-100. The homogenates were dialysecl for 12-15 h at 4°C against distilled water. The dialysed samples were isoelectrically focused in a microanalytical column as described previously [15,16]. Fractions were analysed for AS-A and AS-B activities using an automatic enzyme analyser (5030, Eppendorf, Hamburg) as given below. The pooled AS-A and AS-B fractions were used as the enzyme source. If not otherwise stated, the standard incubation mixture for the determination of the total AS-activity contained, in a final volume of 200 ~1, 100 ~1 of 10 mM MUS in 50 mM sodium acetate buffer, pH 5.0, and up to 50 ~1 enzyme solution. For the determination of AS-B activity, AgNO, was added to the above mixture in a final concentration of 0.3 mM unless stated otherwise. Standard incubations were carried out for 15 min at 37”C, the reactions were stopped by addition of 1 ml of 0.2 M glycine/sodium carbonate buffer, pH 10.4. 4-Methylumbelliferone (4-MU) released during the reaction was measured in an Eppendorf fluorimeter at an excitation wavelength of 365 nm and an emission wavelength of 440 nm. 100 fluorescence units correspond to 0.5 PM 4-methylumbelliferone. The determination of AS-A and AS-B activity with the NCS substrate was performed according to Baum et al. [6] modified by Harzer et al. [16] by determination of nitrocatechol (NC) released.
c C1.3-
0I.2-
:o 4
I.1-
h
z c
Fig. 1. Arylsulphatase A and B patterns obtained c’- - - - - -0, total arylsulphatase activity; *A, AgN03. For details see Materials and methods.
after isoelectric arylsulphatase
focusing of leucocyte homogenates. activity in the presence of 0.3 mM
529
AS-8
=
0.001
0.003
1-t
-
.
11 “ ODI
Fig. 2. Activity of focused arylsulphatases details see Materials and methods.
Results
I
*
003
AS-A
I--_
11 0.1.
”
0.2
0.3
A and B as a function
od
of silver nitrate
b
concentration.
For
and discussion
Fig. 1 shows the arylsulphatase pattern of a leucocyte homogenate after isoelectric focusing. The assay of arylsulphatase activity in the presence of silver ions reveals the
IO
-
AS-E
E---O
AS-0
-
AS-A
O--0
AS-A
20
30
+Ag+ions
+ Ag+ions
40
60 Time,
Fig. 3. Arylsulphatase activities see Materials and methods.
in the presence
and absence
mtnutes
of AgN03
as a function
of time. For details
530
A 0.14 -
T 0.6 -
-
AS-A
c---a
AS-
-
AS-B
wthout wth
A + Ag’oons
0.12
0.5 -
Ag+,ons
Ag+,ons
-
0.10 -
0.4 -
2 c
0
0.3 -
3 I
z
4
“E 0.2 . c
1
0.08
-
0.06
-
0.04
-
:::g .n LI :$:::::$s z.;$g $$$N ::::::::::.: ::::::::::;: ::::::x.:
E c 0.1 -
a02
-
y: $y$
\S-A Fig. tions Fig.
4.
Arylsulphatase
activities
are described 5.
under
Arylsulphatase
absence
of
conditions
AgN03.
in the
Materials activity
Enzyme
are described
of
and
presence
absence
of
AgN03
methods.
focused
concentrations
under
and
AS-A. were:
AS-B AS-A:
and 25
their p;
I
as a function
mixture
AS-B:
25
assayed ~1:
AS-A
-8
-
of PH.
in the + AS-B:
Assay
presence 50
condi-
and
pl. Assay
Methods.
two known peaks of AS-B with isoelectric points at pH 8.2 and 9.4. In the absence of silver ions an additional peak of arylsulphatase A activity with an isoelectric point at pH 5.2 is observed. As shown in Fig. 2 only AS-B remains active in the presence of silver nitrate in the range of 0.1 to 0.3 mM whereas AS-A is inhibited by about 96%. This inhibiting effect remains constant during incubation for 90 min (Fig. 3) and is independent of the amount of enzyme used. Variation of the pH in the incubation mixture reveals that AS-A activity
TABLE
I
ARYLSULPHATASE PATIENTS The
activities
WITH
A
AND
B IN
METACHROMATIC
are expressed Age
(years)
as nmoles
LEUCOCYTE
HOMOGENATES
OF
CONTROL
SUBJECTS
LEUCODYSTROPHY 4-methylumbelliferone Arylsulphatase
A
released
per mg protein
Arylsulphatase
Control
50
0.20
0.39
Control
55
0.25
0.82
Control
10
0.24
0.84
Control
13
0.52
0.84
MLD
17
0.011
0.36
MLD
8
0.041
0.89
MLD
3
0.052
0.47
MLD
3
0.00
4.18
MLD
3
0.00
6.02
B
per h.
AND
531
is almost completely abolished in the optimal range between pH 4.5 and 5.6, while AS-B is not affected at all (Fig. 4). By measuring arylsulphatase activity in the presence and absence of silver ions it is possible to determine AS-A and AS-B activities in a mixture of both enzymes (Fig. 5). The same incubation conditions have been used for the assay of AS-S and AS-B in crude leucocyte homogenates. Patients with MLD exhibit very low but definite AS-A activities (Table I). Acknowledgements We express our thanks to Mrs. &p for excellent cooperation and to Dr. Harzer for the providing leucocyte samples from patients with metachromatic leucodystrophy. This work was supported by the Deutsche Forschungsgemeinschaft (grant Sa 257/2). References 1
Mehl.
2
Matalon,
E. and Jatzkewitz,
3
Austin,
J., McAfee.
4
Beratis,
N.-G.,
5
Fluharty,
A.L..
Commun.
64,
R.,
6
Baum.
7
Sherman,
H.,
8
Rinderknecht,
H. (1968)
Arbogast,
B. and
D. and
Turner.
Shearer,
B.M.,
Stevens,
Biochim.
Dorfman. L.
Weiss.
R.L.,
(1974)
(1965)
R. and
Fun&
Biophys.
A.
D..
Acta
Arch. S.
K. and
619427
Biophys.
Neural.
Hirschhorn, Peak,
151.
Biochem.
12,
Res.
Commun.
61.
(1975)
Pediatr.
Kihara.
H.
Res.
(1975)
9, 475-480
Biochem.
Biophys.
Dodgson, W.R.
K.S.
and
and
Spencer.
Stanfield,
H..
E.F.
Geokas,
M.C.,
B. (1959)
(1967)
Clin.
Biochem.
Carmack.
C. and
Chim.
J. 102.
Acta
4. 453-455
905--909
Haverback,
9
Harinath.
B.C.
and
Robins,
E. (1971)
J. Neurochem.
18.
10
Harinath,
B.C.
and
Robins.
E. (1971)
J. Neurochem.
18,
11
Perumal. Delvin,
13
Dubois,
14
Kampine.
A.S. E.E.,
and
J.P.,
15
Harzer,
K.
Harzer,
K.,
Robins,
Pottier.
G. and
16
Chem.
955-961
B.J.
(1970)
Clin.
Chim.
491
12
1450-1457
447-455
A. and
Baumann. Brady,
(1970)
R.O.
Z. Anal.
Stinshoff,
E. (1973)
K.,
J. Neurochem.
Glorieux,
N.
(1973)
and Chem. Mraz,
F.H. Biochem.
Kanfer. 252,
J.N.
(1976)
21,
245-257 459471
Biochem.
Biophys. (1967)
237-244
Res.
Science
J. 157, Commun. 155,
353-356 50.1129-1135
86
170
W. and Jatzkewitz,
H. (1973)
J. Neurochem.
20.
279-287
Acta
29,
481-
