J. Inherit. Metab. Dis.

24 (2001) 675^680
# SSIEM and Kluwer Academic Publishers. Printed in the Netherlands.

A uorimetric enzyme assay for the diagnosis

of MPS II (Hunter disease)
YA. V. VOZNYI1 , J. L. M. KEULEMANS2 and O. P. VAN DIGGELEN2 *
1 2
Institute of Organic Chemistry, Moscow, Russia; Department of Clinical Genetics,
Erasmus University, Rotterdam, The Netherlands
* Correspondence: Department of Clinical Genetics, Erasmus University, PO Box
1738, 3000 DR Rotterdam, The Netherlands. E-mail: vandiggelen@kgen.fgg.eur.nl
MS received 17.04.01 Accepted 10.06.01

Summary: 4-Methylumbelliferyl-a-iduronate 2-sulphate was synthesized and

shown to be a specic substrate for the lysosomal iduronate-2-sulphate
sulphatase (IDS). Fibroblasts (n 17), leukocytes (n 3) and plasmas
(n 9) from different MPS II patients showed < 5% of mean normal IDS
activity. The enzymatic liberation of the uorochrome from 4-methyl-
umbelliferyl-a-iduronate 2-sulphate requires the sequential action of IDS and
a-iduronidase. A normal level of a-iduronidase activity was insufcient to
complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl-
a-iduronide formed by IDS. A second incubation step in the presence of
excess puried a-iduronidase is needed to avoid underestimation of the IDS
activity.

Mucopolysaccharidosis type II (MPS II, Hunter disease, McKusick 309900) is

caused by a deciency of lysosomal sulphatase iduronate-2-sulphate sulphatase
activity (IDS; EC 3.1.6.13), causing excessive storage of heparan sulphate and
dermatan sulphate in various tissues (for review see Neufeld and Muenzer 2001).
Clinically, the severe disorder is characterized by coarse facial features, short stature,
skeletal deformities, joint stiffness and mental retardation; a milder form
without mental deterioration and no obvious somatic involvement has also been
described.
IDS activity is routinely assayed using radiolabelled disaccharides derived from
heparin (Bach et al 1973; Hall et al 1978; Hopwood 1979). This IDS assay is cum-
bersome and labour-intensive compared to assays with the 4-methylumbelliferone-
derived substrates used for most other lysosomal enzymes.
We describe a novel, simple uorimetric assay for IDS, using 4-methylum-
belliferyl-a-iduronate 2-sulphate, and show its usefulness in the diagnosis of
MPS II.

675
676 Voznyi et al

MATERIALS AND METHODS

Total leukocytes were isolated from heparinized blood as described previously (van
Diggelen et al 1990) and frozen until used. Skin broblasts were cultured according
to routine procedures in Ham's F10 medium supplemented with 10% fetal bovine
serum and antibiotics. The cells were harvested with trypsin 7 days after the last
subculture and stored at 70C until used. Fibroblasts from patients with MPS
II were obtained from the European Human Cell Bank, Rotterdam, The
Netherlands (W. J. Kleijer).
For the standard iduronate-2-sulphate sulphatase (IDS) assay, homogenates were
prepared by sonication of cell material in water. Reaction mixtures consisted of 10 ml
homogenate (10 mg protein for broblasts or 15 mg for leukocytes) or 10 ml 5diluted
plasma to which was added 20 ml 1.25 mmol/L MU-aIdoA-2S in 0.1 mol/L sodium
acetate buer pH 5.0, containing 10 mmol/L lead acetate and 0.02% sodium azide.
MU-aIdoA-2S is available from Moscerdam Substrates (including puried
a-iduronidase; inquiries to O. P. van Diggelen, Rotterdam). The reaction mixtures
were incubated for 4 h 37C, whereafter 40 ml concentrated McIlvain's buer
pH 4.5 (0.4 mol/L Na-phosphate/0.2 mol/L citrate) and 10 ml (16 mg protein)
of partially puried a-iduronidase from rabbit liver (preparation essentially accord-
ing to Verheijen et al 1982) were added and a second incubation of 24 h at 37C
was carried out. For certain experiments homogenates were dialysed using the
method of Penefsky (1979). Reactions were terminated by the addition of 200 ml
0.5 mol/L Na2 CO3 /NaHCO3 , pH 10.7, and the uorescence of 4-methylum-
belliferone (MU) was measured on a FluoroCount (Packard) Instrument.
Protein was determined as described previously (van Diggelen et al 1990).

RESULTS
The potential of 4-methylumbelliferyl-a-L-iduronate 2-sulphate (MU-aIdoA-2S) as
substate for iduronate-2-sulphate sulphatase (IDS) was tested. IDS activity was very
low in undialysed homogenates and did not increase with increasing amounts of
protein. In dialysed homogenates, commonly used for IDS assays, the activity
increased 3^5-fold and the dependence on protein concentration improved greatly
(data not shown). The dialysis step was not necessary if lead acetate was added
to the reaction mixtures containing crude homogenates. In the presence of lead,
IDS activities increased almost linearly with the amount of protein in cell
homogenates (Figure 1). In plasma, the linearity was up to 2 ml, after which the
increase in activity levelled off greatly (data not shown). Lead acetate had no adverse
effect on the IDS activity in dialysed homogenates (not shown).
Liberation of MU from MU-aIdoA-2S by IDS requires the sequential action of
two enzymes: desulphation by IDS followed by hydrolysis of MU-a-iduronide
by a-iduronidase. To avoid that second step being rate limiting, a second incubation
(24 h) in the presence of additional a-iduronidase (4 nmol/h per reaction mixture)
was carried out routinely. This was the minimal amount of a-iduronidase that in
24 h could quantitatively hydrolyse any amount of MU-a-iduronide between 10

J. Inherit. Metab. Dis. 24 (2001)

Fluorimetric assay for IDS 677

Figure 1 Protein dependence of IDS activity in normal leukocytes (&) and broblasts (~).
Reaction conditions as in standard assay, except for amount of protein

and 2000 pmol. This exogenous enzyme source of a-iduronidase, a puried

glycoprotein fraction from rabbit liver, also contained IDS activity (59 nmol/4
h), however, this activity did not interfere since it could be inhibited completely
by the high concentration of phosphate used in the second incubation. This was
veried in each assay with the substrate blanks, which were also incubated with
exogenous enzymes and were only 30^40 pmol. Omission of the second step resulted
in underestimation of IDS in leukocytes and broblast by 96% and in plasma no
activity could be detected.
In the experiments shown below, IDS was assayed under standard conditions as
described in Materials and Methods (except for the condition that was varied).
The pH optimum of IDS from leukocytes, plasma and broblasts was pH 4.8^5.2
(Figure 2). The apparent Km was estimated to be 0.6 mmol/L for leukocytes
and broblasts (Figure 3). IDS activity increased almost linearly with time up to
4 h (Figure 4).
Using standard assay conditions, the IDS activities were determined in material
from patients and controls. The 37 investigated MPS II patients, originating from
13 European countries, Iran and Turkey, had a profound deciency of IDS activity
in leukocytes, plasma and broblasts, whereas the activity in broblasts from
patients with MPS I was normal (Table 1).

DISCUSSION
We have dened conditions for accurate enzyme analysis for MPS II by IDS deter-
mination using the uorogenic substrate 4-methylumbelliferyl-a-L-iduronate
2-sulphate (MU-aIdoA-2S). Formation of the uorochrome requires the sequential

J. Inherit. Metab. Dis. 24 (2001)

678 Voznyi et al

Figure 2 pH dependence of IDS activity in normal leukocytes (&; left), normal plasma
(*; right), normal broblasts (~; left) and broblasts from an MPS II patient (~; left).
Reaction conditions as in standard assay, except for pH

Figure 3 Michaelis^Menten plot for IDS activity in normal leukocytes (&; left) plasma
(*; right) and broblasts (~; left). Reaction conditions as in standard assay, except for
MU-aIdo-2S concentration

J. Inherit. Metab. Dis. 24 (2001)

Fluorimetric assay for IDS 679

Figure 4 Time course of IDS activity in normal leukocytes (&; left) plasma (*; right) and
broblasts (~; left). Reaction conditions as in standard assay, except for incubation time

Table 1 IDS activity in leukocytes, plasma and broblasts

IDS activitya;b

Leukocytesa Plasmab Fibroblastsa

Normal range 18^57 167-475 26^108

n 10 n 16 n 28
MPS II patients 0^0.7 0^1.1 0^3.5
n 3 n 9 n 17
MPS I patients 31; 53 328; 444 47; 49
n 2 n 2 n 2
a
Activity in nmol/4 h per mg
b
Activity in nmol/4 h per ml

action of both IDS and a-iduronidase and exogenously supplied a-iduronidase is

required to prevent underestimation of IDS activity.
The conventional IDS assay uses radiolabelled disaccharides derived from heparin
(Hopwood 1979), and has been the only alternative until now. Our uorogenic assay
is sensitive and specic and is obviously more convenient than the conventional
assay. The commonly used dialysis of homogenates is not necessary in our IDS assay
because of the presence of lead acetate. It is well known that lysosomal sulphatases
are strongly inhibited by sulphates and phosphates; the eect of lead is probably
due to its capacity to precipitate sulphates and phosphates. On the other hand, heavy

J. Inherit. Metab. Dis. 24 (2001)

680 Voznyi et al

metal ions inhibit many enzymes and this was observed for a-iduronidase but not for
IDS.
Assays with MU substrates requiring more than one enzyme to yield the
uorochrome are not common. This principle was successfully applied for the rst
time for galactose-6-sulphatase, allowing the diagnosis of MPS IVA (van Diggelen
et al 1990). Subsequently we developed new uorogenic substrates for MPS IIIC,
MPS IIID and MPS IIIA. The present IDS assay has now replaced the only remain-
ing radiochemical assay used for the diagnosis of mucopolysaccharidoses.

ACKNOWLEDGEMENTS
We thank the staff of the Bloedbank, Rotterdam for providing indispensable blood
samples from control individuals.

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J. Inherit. Metab. Dis. 24 (2001)