Mechanism for the hydrolysis of hyaluronan
oligosaccharides by bovine testicular hyaluronidase
Ikuko Kakizaki*, Nobuyuki Ibori*, Kaoru Kojima, Masanori Yamaguchi and Masahiko Endo
Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, Japan
Introduction
Bovine testicular hyaluronidase (BTH) (hyaluronoglu-
cosaminidase; EC 3.2.1.35) is an endo-b-N-acetyl-d-hex-
osaminidase that hydrolyzes hyaluronan (HA)
at the b1,4-N-acetylglucosaminide bonds [N-acetylglu-
cosamine (GlcNAc)b-(1 fi 4)-glucuronic acid (GlcUA)]
[1–4]. In addition, the enzyme also hydrolyzes chondroi-
tin sulfates at the b-1,4-N-acetylgalactosaminide bonds,
N-acetylgalactosamine (GalNAc)b-(1 fi 4)-GlcUA, but
at a lower efficiency, which is dependent on the structure
of the chondroitin sulfate [1,5]. The wide substrate speci-
ficity of BTH is invaluable for glycotechnological appli-
cations, such as the preparation of glycosaminoglycan
oligosaccharides of varying chain lengths. Exhaustive
digestion with this enzyme yields mainly a mixture of
tetrasaccharides and hexasaccharides with GlcUA at the
nonreducing end [6]. Hyaluronidases simultaneously
display both hydrolytic and transglycosylation activities
[7–10]. Indeed, transglycosylation progresses even under
Keywords
hyaluronan; hydrolysis; oligosaccharide;
testicular hyaluronidase
Correspondence
I. Kakizaki, Department of Glycotechnology,
Center for Advanced Medical Research,
Hirosaki University Graduate School of
Medicine, 5 Zaifu-cho, Hirosaki 036-8562,

The results of the experiments with pyridylaminated oligosaccharides were
entirely consistent with these conclusions, and in addition showed the impor-
tance of the reducing end of the substrate for the enzyme to recognize the
length of the saccharide.
Abbreviations
BTH, bovine testicular hyaluronidase; GlcUA, glucuronic acid; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; HA, hyaluronan;
PA, 2-pyridylamine.
1776 FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS
conditions that are optimal for hydrolysis. The optimal
conditions for the hydrolysis of HA by commercial
BTH are pH 4.0 and the presence of NaCl, whereas for
transglycosylation they are pH 7.0 and the absence of
NaCl [9,11].
The mechanism by which BTH degrades HA is
complex, as the enzyme simultaneously catalyzes deg-
radation and elongation of the substrate by hydrolysis
and transglycosylation, respectively. Because the oligo-
saccharides can act as sequential substrates for both
reactions, it is difficult to identify the reaction products
and establish how they are generated. The mechanism
and kinetics of the hydrolysis by hyaluronidases have
been previously investigated by different methods,
including colorimetric reactions, capillary zone electro-
phoresis, and ion-pair HPLC [6,7,12–15]. In a previous
study, we analyzed the products of hydrolysis of HA
oligosaccharides of known sizes by BTH using ion-
spray MS [6]. In the present study, the mechanism of
hydrolysis of pure synthetic oligosaccharide substrates
labeled at the reducing and ⁄ or nonreducing ends by
BTH was investigated.

5 min and with a small amount of enzyme, multiple
peaks of similar peak area corresponding to 4-mer to
80-mer HA oligosaccharides were generated (Fig. 1B).
When the reaction temperature was increased from
4 °Cto37°C, the peak sizes increased, but the overall
size distribution of reaction products remained
unchanged (Fig. 1C). The distribution of reaction
products and the peak sizes were essentially the same
at 37 °C, 42 °C, and 50 °C (data not shown). Increas-
ing the amount of enzyme or the incubation time
shifted the reaction products to lower-M
r
species,
A
B
C
Fig. 1. Effect of reaction temperature on the hydrolysis of HA by BTH. One milligram of HA (M
r
= 80 000) was incubated with 0.2 mg of
BTH in 100 lL of 0.1
M sodium acetate buffer (pH 4.0) containing 150 mM NaCl at 4 °C (B) or 37 °C (C) for 5 min. One milligram of HA was
incubated with 0.2 mg of heat-inactivated BTH at 100 °C for 10 min as the substrate control (A). The reaction products were analyzed by
HPLC on a polyamine II column (4.6 · 250 mm). The column was eluted with a linear gradient of 100–246 m
M NaH
2
PO
4
at a flow rate of
1.0 mLÆmin
)1

amount of BTH and for a short time (Fig. 2C). How-
ever, none of the peaks was prominent, suggesting the
absence of a specific oligosaccharide product. The
small peak detected at 65 min (Fig. 2B) was apparently
not HA octadecasaccharide, because it was also
detected in the substrate control consisting of boiled
PA–HA eicosasaccharide without enzyme (data not
shown). Increasing the amount of BTH or extending
the reaction time resulted in a shift indicating the pro-
duction of lower-M
r
species. In particular, hexasaccha-
ride and octasaccharide were the predominant
products after 16 h of incubation.
In the reaction catalyzed by hyaluronidase, transgly-
cosylation occurs in equilibrium with the hydrolysis,
even under optimal conditions for hydrolysis. In addi-
tion, the products become sequential substrates for
both reactions. This complicates the interpretation of
our results obtained using PA–HA eicosasaccharide as
the substrate.
Products of hydrolysis by BTH of HA
oligosaccharides
We also used lower-M
r
HA oligosaccharides (tetrasac-
charide to decasaccharide) as substrates for hydrolysis.
A
B
C

Arrows indicate the elution positions of standard PA-HA
oligosaccharides of known chain lengths.
Hydrolytic mechanism of testicular hyaluronidase I. Kakizaki et al.
1778 FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS
These oligosaccharides, prepared by partial digestion
of HA with BTH, have a saturated GlcUA at the non-
reducing end and GlcNAc without any label at the
reducing end. Fifty micrograms of each of the above
oligosaccharides was incubated with 5 lg of BTH
under optimal conditions for hydrolysis at 37 °C for 1,
3 or 24 h, and the reaction mixture was analyzed by
HPLC using a polyamine II column. The results illus-
trated in Fig. 3 show the time sequence changes in the
relative peak area of each HA oligosaccharide. The
ratios of oligosaccharide after 24 h of incubation with
each substrate, as assessed by peak area from the
HPLC traces, are shown in Table 1. The reaction mix-
tures were also analyzed by ion-spray MS. It is clear
that when tetrasaccharide or hexasaccharide was used
as substrate, no degradation product was detected by
HPLC, even after 24 h of incubation (Fig. 3A,B). The
failure to detect degradation products of the hexasac-
charide by HPLC was reproducible, and therefore
appeared to contradict our previous report [6]. How-
ever, the more sensitive MS analysis of the products
revealed trace amounts of molecular ions correspond-
ing to the disaccharide (at m ⁄ z 395.0) and the tetrasac-
charide (at m ⁄ z 775.0) (Fig. 4). This observation is
consistent with the slight decrease in the amount of
hexasaccharide seen in Fig. 3B.

M sodium acetate buffer (pH 4.0) containing 150 mM NaCl at
37 °C for 24 h. The reaction products were analyzed by HPLC on a
polyamine II column (4.6 · 250 mm). The flow rate was set at
1.0 mLÆmin
)1
, and elution was performed over 30 min using a lin-
ear gradient of 50–61.5 m
M NaH
2
PO
4
. Oligosaccharides were
detected by UV absorbance at 215 nm. The percentages shown
are the relative peak areas of the oligosaccharides when the total
peak area of all of the reaction products is defined as 100%.
Substrate HA
oligosaccharide Reaction products (%)
(GlcUA-GlcNAc)
2
(GlcUA-GlcNAc)
2
100
(GlcUA-GlcNAc)
3
(GlcUA-GlcNAc)
3
100
(GlcUA-GlcNAc)
4
(GlcUA-GlcNAc)

I. Kakizaki et al. Hydrolytic mechanism of testicular hyaluronidase
FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS 1779
charide were detected in the reaction mixture. A longer
reaction time decreased the amount of octasaccharide
and increased the amounts of tetrasaccharide and
hexasaccharide (Fig. 3C). A peak (1.8%) correspond-
ing to the decasaccharide, generated by transglycosyla-
tion, was detected after 1 h of incubation but
disappeared after a longer incubation period. This
result was supported by MS analysis (data not shown)
and our previous report.
When the decasaccharide was used as the substrate,
peaks of tetrasaccharide, hexasaccharide, octasaccha-
ride and decasaccharide were detected in the reaction
mixture, and longer incubation periods decreased the
amount of decasaccharide and increased the amounts
of tetrasaccharide, hexasaccharide, and octasaccharide
(Fig. 3D). After 24 h of incubation, hexasaccharide
and tetrasaccharide were detected as the predominant
products. A peak (1.4%) corresponding to dodecasac-
charide, formed from transglycosylation, was detected
after 1 h of incubation, but decreased at 3 h, and was
undetectable at 24 h.
No disaccharide was detected by HPLC with any of
the above oligosaccharides as the substrate.
Products of hydrolysis by BTH of HA
oligosaccharides with unsaturated GlcUA at the
nonreducing end
In order to elucidate the rules that govern the hydrolysis
mediated by BTH, HA oligosaccharides (tetrasaccha-

215 nm. The amount of octasaccharide (starting mate-
rial) decreased with incubation time, indicating that it
was unsaturated, which is consistent with our MS
analysis (data not shown).
When the unsaturated decasaccharide was used as
the substrate, we observed a decrease in the starting
material and an increase in tetrasaccharide and hexa-
saccharide at both wavelengths. A trace amount of
octasaccharide at 215 nm was detected after 1 h and
3 h of incubation, but not after 24 h of incubation
(Fig. 5C,D).
MS analysis confirmed that unsaturated tetrasaccha-
ride and hexasaccharide are not hydrolyzed by BTH,
in agreement with the results from HPLC. MS analysis
also showed that hydrolysis of unsaturated octasaccha-
ride by BTH mainly generated unsaturated and satu-
rated tetrasaccharide, with a trace amount of the
saturated hexasaccharide. This saturated hexasaccha-
ride is presumably a product generated by the transgly-
cosylation reaction using saturated oligosaccharides as
both acceptor and donor. Although MS analysis is not
a quantitative technique, the percentages of unsatu-
rated and saturated tetrasaccharide produced from
unsaturated octasaccharide were assessed by the mag-
nitudes of the fragment ions as 40.8% and 35.8%,
respectively (i.e. approximately equal amounts). The
unsaturated decasaccharide, however, generated only
unsaturated tetrasaccharide and saturated hexasaccha-
ride.
Products of hydrolysis by BTH of HA

showed a series of oligosaccharides whose chain
lengths differed by a disaccharide unit. However, this
observation alone is insufficient proof for this hypothe-
sis, because the action of BTH is complicated by
several factors. First, free disaccharides are not
observed in the HPLC analysis. Second, high-M
r
HA
is quickly degraded to low- M
r
HA oligosaccharides,
which, in turn, can act as substrates for subsequent
reactions. Finally, the reaction of hyaluronidase is an
equilibrium between hydrolysis and transglycosylation.
In the present study, we prepared saturated and
unsaturated oligosaccharides and their pyridylaminated
derivatives with a high degree of purity, to investigate
the hydrolysis reaction mediated by BTH. Our
studies resulted in some novel findings, in addition to
confirming the previously proposed mechanism for the
hydrolysis of HA by BTH.
AB
C
a
b
cd
a
b
cd
D

The HA hexasaccharide was found to be the mini-
mum substrate for BTH [6,7,12,16]. In contrast, a
kinetic study using recombinant hyaluronidases had
suggested that HA octasaccharide was the minimum
substrate [14]. The analytical methods used to analyze
the reaction mixtures in the above studies are different
from each other and from those used in the present
study. In this investigation, using a combination of
HPLC and MS analyses, we confirmed our previous
finding that hexasaccharide is the minimum substrate
for BTH [6]. Oligosaccharides of low M
r
are more
readily ionized than those of high M
r
. Consequently,
MS data do not quantitatively reflect the ratio of
oligosaccharides in the reaction mixture. Nevertheless,
an approximate ratio can be deduced from the relative
intensities of the detected ions. The ratio of oligo-
saccharides in the reaction mixture after 24 h of incu-
bation of saturated hexasaccharide was assessed by MS
to be 91.3% hexasaccharide, 4.5% tetrasaccharide, and
4.2% disaccharide. These results suggest that hexasac-
charide is quite resistant to degradation by BTH but is
converted to disaccharide and tetrasaccharide on pro-
longed incubation. Indeed, in our experiments, hexasac-
charide was often more abundant than tetrasaccharide
in the final reaction mixture of BTH-mediated hydroly-
sis of high-M

is defined as 100%.
Substrate hyaluronen
oligosaccharide Reaction products (%)
(GlcUA-GlcNAc)
2
-PA (GlcUA-GlcNAc)
2
-PA 100
(GlcUA-GlcNAc)
3
-PA (GlcUA-GlcNAc)
3
-PA 100
(GlcUA-GlcNAc)
4
-PA (GlcUA-GlcNAc)
4
-PA 100
(GlcUA-GlcNAc)
5
-PA (GlcUA-GlcNAc)
3
-PA 42.9
(GlcUA-GlcNAc)
4
-PA 13.7
(GlcUA-GlcNAc)
5
-PA 18.3
(GlcUA-GlcNAc)

M sodium acetate buffer (pH 4.0) containing 150 mM NaCl at
37 °C for 24 h. The reaction products were analyzed by HPLC on a polyamine II column (4.6 · 250 mm). The flow rate was set at 1.0 mLÆ-
min
)1
, and the elution was performed over 30 min using a linear gradient of 50–61.5 mM NaH
2
PO
4
. Oligosaccharides were detected by UV
absorbance at both 215 nm and 232 nm. The percentages shown are the relative peak areas of the oligosaccharides when the total peak
area of all reaction products is defined as 100%. D
4
GlcUA-GlcNAc-GlcUA-GlcNAc, D
4
-unsaturated HA tetrasaccharide; D
4
GlcUA-GlcNAc-
(GlcUA-GlcNAc)
2
, D
4
-unsaturated HA hexasaccharide; D
4
GlcUA-GlcNAc-(GlcUA-GlcNAc)
3
, D
4
-unsaturated HA octasaccharide; D
4
GlcUA-Glc-

a
Octasaccharide 0
Decasaccharide 7.40 Decasaccharide 7.40
a
The peak of HA octasaccharide was observed after 1 h and 3 h of incubation, but disappeared after 24 h of incubation (see Fig. 3).
Hydrolytic mechanism of testicular hyaluronidase I. Kakizaki et al.
1782 FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS
HPLC was almost exclusively tetrasaccharide. In MS
analysis, unsaturated and saturated tetrasaccharides
can be distinguished by the mass of their corresponding
deprotonated molecular ions (m ⁄ z 757.0 and m ⁄ z 775.0,
respectively). MS analysis of the products of hydrolysis
of the unsaturated octasaccharide by BTH showed an
approximate 50 : 50 ratio of unsaturated and saturated
tetrasaccharide. This suggests that if the substrate has
an unsaturated bond at the nonreducing end, BTH rec-
ognizes the second rather than the first N-acetylglucos-
aminide bond from the nonreducing end. Therefore,
only unsaturated tetrasaccharides are generated, and
not unsaturated disaccharides. Furthermore, our data
suggest that the unsaturated tetrasaccharide thus gener-
ated can be transferred to the nonreducing ends of
other saturated oligosaccharides. However, the result-
ing unsaturated oligosaccharide does not become the
acceptor for further transglycosylation, because it now
has an unsaturated GlcUA at the nonreducing end.
Our experiments with unsaturated HA oligosaccha-
rides as substrates suggest a general rule for BTH-med-
iated hydrolysis; namely, BTH recognizes the first
N-acetylglucosaminide bond from the nonreducing end

rated tetrasaccharide. This could explain why only
trace amounts of saturated disaccharides are detectable
in the reaction mixture.
The above findings suggest that the initial products
of hydrolysis of HA octasaccharide are a disaccharide
and a hexasaccharide. The disaccharide, retained via
its intermediate form in the active site of BTH, is
immediately transferred to a different disaccharide to
generate a tetrasaccharide by the transglycosylation
activity of BTH. Alternatively, it is immediately trans-
ferred to other oligosaccharides in the reaction mix-
ture, thereby elongating the acceptor oligosaccharides
by a disaccharide unit. Thus, tetrasaccharide and deca-
saccharide are generated (mostly as transglycosylation
products) in addition to hexasaccharide (mostly an
initial hydrolysis product). Any octasaccharides still
found in the reaction mixture are unhydrolyzed
Table 4. Reaction products of pyridylaminated unsaturated HA oligosaccharides after incubation with testicular hyaluronidase for 1 h. Two
micrograms of saturated oligosaccharide was incubated with 5 lg of testicular hyaluronidase in 100 lL of 0.1
M sodium acetate buffer
(pH 4.0) containing 150 m
M NaCl at 37 °C for 1 h. The reaction products were analyzed by HPLC on a polyamine II column (4.6 · 250 mm).
The flow rate was set at 1.0 mLÆmin
)1
, and elution was performed over 30 min, using a linear gradient of 50–61.5 mM NaH
2
PO
4
. Oligosac-
charides were detected by UV absorbance at 215 nm. The percentages shown are the relative peak areas of the oligosaccharides when the

4
GlcUA-GlcNAc-(GlcUA-GlcNAc)
4
-PA 15.6
(GlcUA-GlcNAc)
3
-PA 43.6
D
4
GlcUA-GlcNAc-GlcUA-GlcNAc 40.8
D
4
GlcUA-GlcNAc-(GlcUA-GlcNAc)
5
-PA D
4
GlcUA-GlcNAc-(GlcUA-GlcNAc)
5
-PA 5.5
(GlcUA-GlcNAc)
4
-PA 44.7
(GlcUA-GlcNAc)
5
-PA 23.3
D
4
GlcUA-GlcNAc-(GlcUA-GlcNAc)
2
9.8

charides appropriately modified at the reducing or
nonreducing ends, we have been able to elucidate the
sites in the oligosaccharide substrates that are recog-
nized by the enzyme.
Experimental procedures
Materials
HA (from Streptococcus zooepidemicus, average M
r
of
80 000) was purchased from Kibun Food Chemifa Co., Ltd
(Tokyo, Japan). BTH (type 1-S) was from Sigma-Aldrich
(St Louis, MO, USA), and chondroitin ABC lyase was
from Seikagaku Kogyo Co. (Tokyo, Japan). Other reagents
were of analytical grade and obtained from commercial
sources.
Preparation of HA oligosaccharides
Saturated HA oligosaccharides were prepared by partial
digestion with BTH as follows. One gram of HA was incu-
bated with 200 mg of BTH in 100 mL of 0.1 m sodium
acetate buffer (pH 4.0) containing 150 mm NaCl at 37 °C
for 3 h, and the reaction was stopped by boiling for
10–15 min. The mixture was then clarified by centrifugation
for 10 min at 10 000 g at 4 °C, concentrated to about
20 mL, and desalted on a Sephadex G-25 column equili-
brated with distilled water. Fractions determined to be posi-
tive for uronic acid using the carbazole sulfate method
were pooled and concentrated to 20 mL. The resulting mix-
ture of saturated HA oligosaccharides was fractionated by
perfusion chromatography performed on a Perceptive Bio-
systems Bio Cad Perfusion Chromatography Workstation

strates of BTH. HA oligosaccharides, with an unsaturated
double bond between C4 and C5 of the GlcUA nonreducing
end, were prepared by partial digestion with chondroitin
ABC lyase [18,19]. Two hundred milligrams of HA was
incubated with 1.5 U (0.5 U per 24 h) of chondroitin ABC
lyase in 12 mL of 33.3 mm Tris ⁄ HCl buffer (pH 8.0) con-
taining 33.3 mm sodium acetate at 37 °C for 72 h, and the
reaction was stopped by boiling for 5 min. The reaction mix-
ture was clarified by centrifugation for 10 min at 10 000 g at
4 °C and fractionated by HPLC, using a YMC-Pack poly-
amine II column (10 · 250 mm; YMC Co., Tokyo, Japan).
Fractions were pooled, desalted, concentrated, and subjected
to ion-spray MS to identify the unsaturated oligosaccharides
(disaccharide, tetrasaccharide, hexasaccharide, octasaccha-
ride, decasaccharide, or dodecasaccharide).
Pyridylamination of HA oligosaccharides
Fluorolabeling of the reducing ends of saturated ⁄ unsatu-
rated HA oligosaccharides with PA was performed by a
modification of the method of Hase et al. [20], as described
in our previous report [21]. In order to eliminate contami-
nation of the substrates used with non-PA oligosaccharides,
PA oligosaccharides were purified by HPLC, using a poly-
amine II column. The individual PA oligosaccharides were
collected, and their purity was verified by ion-spray MS;
they were then used as substrates in the hydrolysis reaction
of BTH.
Hydrolysis reaction of BTH
Each HA oligosaccharide (100–200 lg) or PA–HA oligosac-
charide (2–10 lg) was incubated with a suitable amount of
BTH in 0.1 m sodium acetate buffer (pH 4.0), containing

PO
4
. Chondroitin ABC lyase digests were
injected onto the column equilibrated with solution A at a
flow rate of 1.0 mLÆmin
)1
, and then eluted over 60 min
with a linear gradient of solution B from 0% to 94%. The
eluate was monitored for UV absorbance at 232 nm. Con-
ditions for the analysis of BTH-digested HA oligosaccha-
rides were as follows. Two solutions with different
concentrations of NaH
2
PO
4
were prepared for this column
(solutions A and B). The linear gradient varied, depending
on the size range of oligosaccharides to be analyzed (see fig-
ure legends for details). BTH digests containing only satu-
rated HA oligosaccharides were monitored by UV
absorbance at 215 nm. BTH digests containing both satu-
rated and unsaturated HA oligosaccharides were monitored
by UV absorbance using two detectors set at 215 nm and
232 nm; the latter wavelength detected the unsaturated
bonds. PA-HA oligosaccharides were monitored by fluores-
cence detection (excitation at 320 nm; emission at 400 nm)
and by UV absorbance at either 232 nm or 215 nm.
Ion-spray MS
Mass spectra of HA oligosaccharides were obtained on a
PE-Sciex API-100 single-quadrupole mass spectrometer

4 Takagaki K & Kakizaki I (2007) Degradation of
glycosaminoglycans. In Comprehensive Glycoscience:
from Chemistry to Systems Biology (Kamerling JP,
Boons GJ, Lee YC, Suzuki A, Taniguchi N & Voragen
AGJ eds), pp. 171–192. Elsevier Science B. V,
Amsterdam.
5 Meyer K & Rapport MM (1950) The hydrolysis of
chondroitin sulfate by testicular hyaluronidase. Arch
Biochem 27, 287–293.
6 Takagaki K, Nakamura T, Izumi J, Saitoh H, Endo M,
Kojima K, Kato I & Majima M (1994) Characterization
of hydrolysis and transglycosylation by testicular hyal-
uronidase using ion-spray mass spectrometry. Biochem-
istry 33, 6503–6507.
7 Highsmith S, Garvin JH Jr & Chipman DM (1975)
Mechanism of action of bovine testicular hyaluronidase.
Mapping of the active site. J Biol Chem 250, 7473–
7480.
8 Hoffman P, Meyer K & Linker A (1956) Transglycosy-
lation during the mixed digestion of hyaluronic acid
and chondroitin sulfate by testicular hyaluronidase.
J Biol Chem 219, 653–663.
9 Saitoh H, Takagaki K, Majima M, Nakamura T, Mat-
suki A, Kasai M, Narita H & Endo M (1995) Enzymic
reconstruction of glycosaminoglycan oligosaccharide
chains using the transglycosylation reaction of bovine
testicular hyaluronidase. J Biol Chem 270, 3741–3747.
10 Weissmann B (1955) The transglycosylative action of
testicular hyaluronidase. J Biol Chem 216, 783–794.
11 Gorham SD, Olavesen AH & Dodgson KS (1975)

Chem 277, 22438–22446.
19 Chai W, Lawson AM, Gradwell MJ & Kogelberg H
(1998) Structural characterisation of two hexasaccha-
rides and an octasaccharide from chondroitin sul-
phate C containing the unusual sequence (4-sulpho)-
N-acetylgalactosamine-beta1-4-(2-sulpho)-glucuronic
acid-beta1-3-(6-sulpho)-N-acetylgalactosamine. Eur J
Biochem 251, 114–121.
20 Hase S, Ibuki T & Ikenaka T (1984) Reexamination of
the pyridylamination used for fluorescence labeling of
oligosaccharides and its application to glycoproteins.
J Biochem 95, 197–203.
21 Kon A, Takagaki K, Kawasaki H, Nakamura T &
Endo M (1991) Application of 2-aminopyridine fluores-
cence labeling to glycosaminoglycans. J Biochem 110,
132–135.
22 Takagaki K, Kojima K, Majima M, Nakamura T, Kato
I & Endo M (1992) Ion-spray mass spectrometric analy-
sis of glycosaminoglycan oligosaccharides. Glycoconj J
9, 174–179.
Hydrolytic mechanism of testicular hyaluronidase I. Kakizaki et al.
1786 FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS