Glycosidase Activity 403
403
33
Glycosidase Activity
Anthony P. Corfield and Neil Myerscough
1. Introduction
The glycosidases and associated hydrolytic enzymes acting on glycoconjugate oli-
gosaccharides form part of the total mucinase activity. This chapter describes some
assay methods for the determination of these enzymes. Our knowledge of the number
of enzymes required for mucin degradation and their regulation in physiological situ-
ations is scanty (1). The degradation of both protein and carbohydrate domains require
specific enzymes which are able to degrade mucin structure. Carbohydrate degrada-
tion may be dependent on prior or concomitant peptide cleavage in mucins. The issues
that need to be addressed include the following:
1. Being able to demonstrate individual substrate specificity in relation to the known mucin
structure (both protein and carbohydrate).
2. The pathogenic or nutritional role of bacterial degradation leading to the loss (degrada-
tion to create receptor binding sites as part of bacterial colonization or infection of the
host) or recycling (utilization of the released products for energy production)
3. The need to consider the mode of growth of bacteria at the mucosal surface, in particular
the role of biofilms (see Chapter 36).
4. The role of “additional” enzymes such as sulfatases, phosphatases, and lipases, which are
important for rarer posttranslational modifications to mucin peptide and oligosaccharide
structure (1).
Many studies with glycosidases have been carried out using synthetic substrates,
such as 4-nitrophenyl- and 4-methyl umbelliferyl-glycosides. These substrates only
give information with regard to the anomeric configuration of the glycoside, but not
with respect to the nature of linkage to the next sugar in an oligosaccharide. Thus, the
results may have limited physiologic relevance. When the degradation of specific
glycoconjugates such as mucins is to be assessed, alternative, novel substrates have to
be prepared. Only in this way can the manner in which mucins are degraded in vivo be

studied. The design of substrates has followed two directions. The first, the use of
intact mucin/glycoprotein or glycopeptide substrates. These assays have relied on the
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
404 Corfield and Myerscough
detection of the individual monosaccharide being released. The sensitivity of these
assays depends on the sensitivity of the monosaccharide product detection. Colorimet-
ric, fluorimetric, radioactive, ultraviolet (UV) and pulsed amperometric detection
define the limits of these assays. Often further separation techniques (gel filtration,
high-performance liquid chromatography [HPLC], ion-exchange, etc.) are necessary
to isolate and quantify the products. As a result, many of the relevant substrates for
studies of degradation of mucin are not available commercially and must be prepared,
and in some cases, the design of suitable glycoconjugate substrates has not proved
possible. The second direction is the chemical synthesis of oligosaccharides with rel-
evant structure and the identification of degradation by chromatographic methods.
This chapter addresses the question of glycosidase activity. Description of the use
of mucin-related substrates together with some of the widely available synthetic sub-
strates gives an approach to the identity of the general range of mucin-degrading gly-
cosidase activities present in enzymatic preparations.
2. Materials
2.1. Glycosidase Substrates
2.1.1. Natural Glycosidase Substrates
Many naturally occurring glycoconjugates can be utilized as physiologic substrates
for glycosidases in the assays described in this chapter. In addition to those listed here,
other purified glycoconjugates can be used in the same way.
1. Fetuin type III (Sigma, Poole, UK).
2. Salivary gland glycoproteins from bovine (2) and ovine (3) sources.
3. α
1

-Acid glycoprotein (Sigma).
4. Antifreeze glycoprotein (4).
5. Porcine seminal gel glycoprotein (5).
6. Asialoglycoproteins: Prepare in all cases by incubation of the sialoglycoprotein at 1 mg/mL
in 0.1 M HCl at 80°C for 60 min. The sialic acid content is measured before and after the
hydrolysis and additional hydrolysis carried out under the same conditions if significant
sialic acid remains (6).
7. Saponification of O-acetyl esters: Carry out saponification on sialic acids in bovine sali-
vary gland mucin at 1 mg/mL in 0.1 M NaOH at room temperature for 45 min, and neu-
tralize the solution to approx pH 7.0 with 1 M HCl.
8. α2-3 and α2-6 Sialyllactose (Sigma).
2.1.2. Synthetic Glycosidase Substrates
Synthetic substrates form the basis of rapid and sensitive colorimetric (4-nitro–
phenyl-glycosides) and fluorimetric (4-methyl umbelliferyl-glycosides) assays. These
substrates are available through several suppliers, including Sigma; Oxford Glyco-
sciences, Abingdon, UK; Dextra, Reading, UK; Boehringer Mannheim, Lewes, UK;
and Chemica Alta, Edmonton, Canada.
1. 4-Nitrophenyl β-galactose: Dissolve in 1/10 volume of methanol and make up to a final
concentration of 4 mM in 100 mM sodium acetate, pH 6.0.
2. 4-Nitrophenyl α-galactose: Dissolve in 1/10 volume of methanol and make up to a final
concentration of 4 mM in 100 mM sodium acetate, pH 6.0.
Glycosidase Activity 405
3. 4-Nitrophenyl β-N-acetylglucosamine: Dissolve in 1/10 volume of methanol and make
up to a final concentration of 4 mM in 100 mM sodium acetate, pH 6.0.
4. 4-Nitrophenyl α-N-acetylgalactosamine: Dissolve in 1/10 volume of methanol and make
up to a final concentration of 4 mM in 100 mM sodium acetate, pH 6.0.
5. 4-Nitrophenyl Galβ1-3GalNAc: Dissolve the substrate directly in 100 mM citrate-phos-
phate, pH 6.0, to give a final concentration of 2 mM.
6. 4-Nitrophenyl α-fucose: Dissolve in 1/10 volume of methanol and make up to a final
concentration of 4 mM in 100 mM sodium acetate, pH 6.0.

7. 4-Methyl umbelliferyl sialic acid: Dissolve the substrate directly in 0.4 M sodium acetate,
pH 4.2, to give a final concentration of 2 mM.
2.1.3. Galactose/
N
-acetyl-D-Galactosamine-Labeled Glycoproteins
Galactose Oxidase and Tritiated Borohydride (
see
Notes 1 and 2)
1. Dissolve 5 mg of the glycoprotein, e.g., antifreeze glycoprotein, asialofetuin, asialo-ovine
submandibular gland mucin, or α
1
-acid glycoprotein, in 500 µL of 50 mM sodium phos-
phate and 5 mM NaCl, pH 7.0.
2. Add 5 U of galactose oxidase (Sigma) to each glycoprotein solution, and incubate for 24 h
at 37°C.
3. Dilute the products five times with 50 mM sodium phosphate and 50 mM NaCl, pH 7.8.
4. Add 12.5 MBq of sodium boro-[
3
H]-hydride (NaB[
3
H]
4
), typically 10 GBq/mmol
(Amersham, UK) to each, and stir the solutions for 60 min at room temperature (Hazard:
radioactive; see Note 1).
5. Add 1.5 mg of solid NaBH
4
(Sigma) to each incubation and stir for a further 30 min
(Hazard: attacks respiratory tract mucous membranes).
6. Add 10-µL aliquots of glacial acetic acid (analytical grade; BDH/Merck, Poole, UK), to

destroy borohydride, until no more bubbles are formed.
7. Add 2 mL of analytical grade methanol, and evaporate to dryness under reduced pressure
below 30°C in a rotary evaporator. Repeat this extraction five times to remove borate.
8. Adjust the pH of the solution to approx 6.5.
9. Desalt the products by repeated runs on a column of Sephadex G25 (30 × 1 cm; Pharmacia,
Milton Keynes, UK) in 0.2 M NaCl with a final run in distilled water.
10. Store the products as 100- to 500-µL aliquots in 0.02% sodium azide (BDH/Merck) at 4°C.
1.4. Sialyl GalNAc [
3
H]-ol from Boar Seminal Gel Mucin (
see
Note 1)
1. Dissolve 25 mg of porcine seminal gel glycoprotein in 4 mL of 0.05 M NaOH.
2. Add 190 mg of solid NaBH
4
(Hazard: attacks respiratory tract mucous membranes).
3. Add 925 MBq of NaB[
3
H]
4
(typically 10 GBq/mmol) (Amersham) in 1 mL of 0.05 M
NaOH, and incubate at 45°C with stirring, for 16 h (see Note 1).
4. Add glacial acetic acid (analytical reagent grade; BDH/Merck) dropwise until no more
bubbles are formed, to destroy excess borohydride (see Note 1).
5. Pass the solution through a column (20 mL) of Dowex 50 H
+
(200–400 mesh) (Bio-Rad,
Hemel Hempstead, UK), and wash with 100 mL of distilled water.
6. Evaporate to dryness under reduced pressure below 30°C using a rotary evaporator. Add
2 mL of methanol and repeat this evaporation five times to remove borate.

7. Dissolve the sample in 5 mL of 0.1 M pyridine acetate, pH 5.0, apply to a column of Bio-
Gel P4 (200–400 mesh, 150 × 2 cm) (Bio-Rad), and elute in the same buffer.
8. Collect 5-mL of fractions and measure radioactivity. Pool the major radioactive peak
(Neu5Acα2-6GalNAc-[
3
H]-ol) (see Note 3).
406 Corfield and Myerscough
9. Evaporate to dryness under reduced pressure below 30°C using a rotary evaporator.
Redissolve in 5 mM pyridine acetate, pH 5.0, and apply to a column (18 × 1 cm) of
Dowex 1 × 2 (–400 mesh, acetate form) (Bio-Rad). Elute with a gradient of 2–350 mM
pyridine acetate pH 5.0 (2 × 250 mL) and collect 5-mL fractions.
10. Pool the radioactive peak and remove the pyridine acetate by rotary evaporation.
11. Convert the oligosaccharide to the sodium salt by titration with Dowex 50 Na
+
(50–100
mesh) (Bio-Rad).
12. Store at 4°C in 2% aqueous ethanol.
2.1.5. Sialyl-[
3
H]-Labeled Sialoglycoproteins
Any sialoglycoprotein can be labeled with tritium after periodate oxidation (see
Note 4). Typically, α
1
-acid glycoprotein and bovine submandibular gland mucin have
been used. The sialic acid content of the glycoprotein must be determined first in order
to make up the correct ratio of sialic acid to periodate in the mild oxidation step. In the
case of some mucin substrates, e.g., bovine submandibular gland mucin, in which
sialic acid O-acetyl esters are expected or suspected, a mild saponification step should
be included before the periodate oxidation (see Subheading 2.1.1., item 7; Note 5).
1. Dissolve 10–50 mg sialoglycoprotein in 100 mM sodium acetate, 150 mM sodium chlo-

ride buffer, pH 5.5, to give a final concentration of 1 mM with respect to sialic acid and
equilibrate at 4°C.
2. Add ice cold 10 mM sodium metaperiodate (Sigma) in water to give a final concentration
of 1 mM periodate (approx 1/10 of the volume of the sialoglycoprotein solution) and stir.
3. Incubate for 10 min at 4°C with stirring.
4. Add glycerol (0.2 mL for each 10 mL of sialoglycoprotein solution) and stir for a further
10 min.
5. Dialyze the solution against three changes of 2.5 L of 0.05 M sodium phosphate, 0.15 M
sodium chloride, pH 7.4, for 24 h at 4°C.
6. Add 500–1000 Mbq of NaB[
3
H]
4
(typically 10 GBq/mmol) (Amersham) in 0.05 M NaOH
adjusted to a final concentration of approx 0.1 M borohydride with unlabeled NaBH
4
in a
volume of 1 mL and stir for 30 min at room temperature.
7. Add 1 mL of 0.1 M NaBH
4
and incubate for a further 30 min.
8. Dialyze the product against two changes of 3 L of 0.1 M sodium acetate, pH 5.5, and then
against two changes of 3 L of distilled water.
9. Concentrate the sialoglycoprotein solution if necessary (see Note 6).
2.2. Buffers and Reagents for Enzyme Assay
1. General 4-nitrophenyl-glycoside assay buffer: 100 mM sodium acetate, pH 6.0.
2. General 4-nitrophenyl-glycoside stop solution: 10% trichloroacetic acid (TCA) in dis-
tilled water.
3. 0.5 M Sodium carbonate.
4. Ovalbumin (Sigma), 80 mg/mL in 0.1 M sodium phosphate, pH 7.0, buffer.

5. 5% phosphotungstic acid (PTA)/15% TCA: 5% (w/v) PTA (BDH/Merck) and 15% (w/v)
TCA (BDH/Merck) in distilled water.
6. O-glycanase incubation buffer: 100 mM citrate-phosphate, pH 6.0. Prepare 100 mM citric
acid and 200 mM disodium phosphate. Take 1 vol of citric acid and titrate to pH 6.0 with
phosphate, and make up to a final volume equivalent to 1:1 of starting volume.
7. Sialidase incubation buffer (colorimetric assay): 100 mM sodium acetate, 20 mM CaCl
2
,
150 mM NaCl, pH 5.5.
Glycosidase Activity 407
8. Reagents for the Warren assay of sialic acids:
a. 0.25 M Periodic acid: 5.7 g of periodic acid (Sigma) in 75 mL phosphoric acid (BDH/
Merck) make up to 100 mL with distilled water. Stock solutions will keep for approx
12 mo at room temp.
b. 0.38 M sodium arsenite in 0.5 M sodium sulfate: 5 g of sodium arsenite (BDH/Merck),
7.1 g of anhydrous sodium sulphate (BDH/Merck) make up to 100 mL with distilled
water. Solution is stable for several months at room temperature.
c. Thiobarbituric acid in 0.5 M sodium sulfate: 0.9 g of thiobarbituric acid (Aldrich,
Gillingham, UK), 7.1 g of anhydrous sodium sulfate (BDH/Merck) made up to 100 mL
with distilled water. Solution is stable for approx 1 wk only (see Note 7).
d. Cyclohexanone analytical reagent (AR) (Aldrich, Gillingham, UK).
e. N-Acetyl neuraminic acid (sialic acid; Sigma): Standard solution for calibration of
the assay is 0.31 mg/mL, use 10 or 20 µL of this solution in a final volume of 100 µL
for the assay (see Subheading 3.7.1.).
9. Sialidase incubation buffer (fluorimetric assay): 400 mM sodium acetate pH 4.2.
10. Sialidase stop buffer (fluorimetric assay): 85 mM glycine/sodium carbonate buffer, pH 10.0.
11. Sialate O-acetyl esterase buffer: 100 mM triethanolamine, pH 7.8.
12. Acetic acid detection assay kit (Boehringer) (see Note 8).
13. Acylneuraminate pyruvate lyase incubation buffer: 200 mM potassium dihydrogen phos-
phate adjusted to pH 7.2 with KOH.

14. Acylneuraminate pyruvate lyase substrate: 10 mM sialic acid (Sigma) in potassium phos-
phate buffer, pH 7.2, containing 0.5–1.0 kBq/mL of [
14
C]-N-acetylneuraminic acid
(Amersham).
15. Acylneuraminate pyruvate lyase from Clostridium perfringens (Sigma).
3. Methods
3.1.
β
-Galactosidase (
see
Notes 9 and 10)
Several different assays are possible for β-galactosidase; synthetic substrates are
widely available for either colorimetric or fluorimetric assay. Radioactive assays with
glycoproteins give data on physiologically significant molecules (see Notes 9–11).
3.1.1. Synthetic Substrate
1. Mix 25 µL of 4 mMp-nitrophenyl β-galactose in 100 mM sodium acetate, pH 6.0 (see
Subheading 2.1.2., item 1), with 25 µL of extract/enzyme.
2. Incubate for 20 min at 37°C.
3. Add 50 µL of 10% trichloracetic acid (see Subheading 2.2., item 2) to stop the reaction
4. Add 1 mL of 0.5 M sodium carbonate.
5. Centrifuge to remove any solid, and read the supernatant at 400 nm.
6. Prepare blank incubations by adding TCA to enzyme extract incubated alone for 20 min,
and then add substrate incubated alone for 20 min. Add 1 mL of 0.5 M sodium carbonate,
centrifuge and read at 400 nm as above (see Note 12).
3.1.2. General Mucin Galactosidase Assay Radioactive Substrates
(
see
Note 13)
Radioactive asialoglycoproteins labeled in their terminal galactosyl residues (see

Subheading 2.1.3.) can be used as substrates in this precipitation assay. Depending on
the nature of the radioactive substrate, this assay can be specific for a particular glyco-
sidic linkage (see Note 14).