Mucinase Activity 385
385
31
Mucinase Activity
Roger M. Stark, Rebecca Wiggins, Elizabeth Walley,
Sally J. Hicks, Gulnaz A. Gill, Stephen D. Carrington,
and Anthony P. Corfield
1. Introduction
Turnover of the mucous “barrier gel” overlying mucosal surfaces is essential for
hydration, mechanical protection, the physical removal of contaminants and toxins,
the generation of sacrificial binding ligands that prevent microbial penetration, and
the provision of a suitable environment to renew other defensive molecules that are
incorporated into mucus. Mucinase activity is crucial to this turnover process in loca-
tions such as the gut and the reproductive tract. Similar activity may also be of rel-
evance at other mucosal surfaces that are not normally colonised by significant
microbial populations, such as the eye and the respiratory tract.
Mucinase activity is owing to a mixture of enzymes that are expected to include
proteinases, peptidases, glycosidases, and sulphatases of prokaryotic or eukaryotic
origin. In addition, the presence and action of other hydrolases, including phosphatases
esterases and lipases should also be considered.
The assay of individual enzyme activity resulting in the release of single compo-
nents from mucin substrates does not give a complete analysis of the total mucin-
degrading potential of the sample under study. The examination of mucinase activity
from any source must be correlated with the nature and origin of the mucin used as
substrate. Mucins are fragmented differently as a result of structural variability
between apomucin peptides and their individual patterns of glycosylation. Equally
significant is the composition of the mucinase activity that they encounter. A careful
examination of the fragments derived from incubations of specific mucins with
mucinase activity from different sources can yield information on the succession of
degradative steps, as well as the enzymes that carry them out.
The development of methods for detecting mucinase activity and resolving it into

its component stages is in its infancy. However, with the increasing knowledge of the
structure and organization of mucin (1,2) it is now becoming possible to match frag-
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
386 Stark et al.
mentation patterns with known sequence information for both peptides and oligosac-
charides. Clearly, this is best assessed using a whole mucin substrate and analyzing
the progressive degradation to small molecular weight fragments. However, the analy-
sis of size-fractionated products for carbohydrate and/or amino acid composition
requires large amounts of mucin substrate, is time-consuming, and is therefore not
suitable for the screening of large numbers of samples. Larger-scale screening for
mucinase activity is possible using radiolabeled mucins prepared from organ or cell
cultures. Mucins can be labeled with suitable precursors; for example, cultures of
colonic mucosa can be labeled with [
35
S]sulfate, [
3
H]glucosamine, and [
3
H]threonine.
The purified mucins are subsequently incubated with a source of mucinase activity,
and the degradation is assessed by gel filtration (3–5).
An alternative to the radioactive methods is the use of biotinylated mucin sub-
strates tagged through either the peptide or carbohydrate moieties of the molecule
(6,7). Such assays have been described for protein substrates and adapted for use with
microtiter plates (8). They require small amounts of pure substrate, have high sensitiv-
ity, are rapid, and can be applied to large numbers of samples. Such assays are suitable
for the detection of “total” mucinase activity. Thus, their main use is for the rapid
screening of sources of mucinase activity that may be enzymatically heterogeneous.

The known structure and properties of mucins, together with the ability to biotinylate
either their protein or carbohydrate domains, has important implications for the inter-
pretation of microtiter plate assays. It is important to be aware that the release of
adherent mucin from a plastic surface may be owing to several factors. First, the deg-
radation of mucin substrates and their release from the plates may be entirely the result
of mucinase action over the whole domain structure of the mucin. Second, it may
result from the cleavage of regions of the mucin molecules which specifically bind to
microtiter plates. A third caveat is the possibility that paradoxical results may be
obtained when digesting mucins labeled through their peptide vs their carbohydrate
residues. In extreme cases, e.g., where adhesion of mucins to the plastic of microtiter
plates is mainly related to hydrophobic binding to peptide sequences, mucins labeled
through their carbohydrate may appear to be fully digested when exposed only to
peptidases. In such cases, however, any released glycopeptides would be protected
from digestion by their carbohydrate chains. Where adhesion is mainly related to ionic
interactions between charged carbohydrates and the microtiter plate, sialidase or sul-
fatase activity alone may release the majority of bound mucin while causing only lim-
ited degradation of the whole molecule. Therefore, the microtitre plate assay may not
appear to be specific for enzyme type with respect to the nature of the mucin biotin
label. In reality, however, binding is probably the result of multiple binding interac-
tions, which tends to reduce the significance of such extremes of binding interaction.
Nevertheless, the composition of mixed enzyme preparations cannot be deduced di-
rectly from the plate assays. To obtain this information, an examination of fragmentation
profiles by size fractionation and electrophoresis would define the pattern and sequence
of degradation of mucin for both methods of biotinylation. Further information can be
obtained by comparing such profiles with those obtained using commercially available
peptidases and glycosides, and by subsequent enzyme inhibition experiments.
Mucinase Activity 387
2. Materials
2.1. Enzyme Sources
The nature of possible samples to be screened for mucinase activity is diverse and

the sources given here serve only as examples.
1. Commercially prepared enzymes: proteases, e.g., trypsin (Sigma, Poole, UK), pronase E
(Boehringer Mannheim, Lewes, UK), pepsin (Calbiochem-Novabiochem, Nottingham,
UK); glycosidases, e.g., α-sialidase, α- and β-galactosidase (Oxford Glycosciences,
Abingdon, UK), α- and β-N-acetylhexosaminidase (Boehringer Mannehim), α-fucosidase
(Sigma), O-glycanase (Calbiochem -Novabiochem).
2. Bacterial culture supernatants and cell suspensions or cell extracts.
3. Animal/human secretions and excretions that contain enzymes can also be used, e.g.,
plasma, urine, tears, fecal extracts, sputum, mucosal washings.
4. Animal and insect cell culture supernatants, cell suspensions, or cell extracts.
2.2. Preparation of Radiolabeled Mucin Substrates
1. Radiolabeled mucin singly or dual labeled with [
3
H]glucosamine or [
3
H]threonine com-
bined with either [
35
S]sulfate or [
14
C]threonine. This mucin must be purified and should
elute as a single high molecular weight peak at the V
o
of a Sepharose CL-2B column. The
preparation of these substrates is detailed in Chapter 19 (see Note 1).
2. Gel filtration buffer: 10 mM Tris-HCl, pH 8.0.
3. Sepharose CL-2B (Pharmacia, Uppsala, Sweden).
2.3. Preparation of Biotinylated Mucin Substrates:
Biotinylation of Mucins
1. Prepare mucin using density gradient centrifugation and gel filtration and characterize for

the presence of noncovalently associated contaminants as described in Chapters 1, 7, and
8(see Note 1).
2. Sephadex G25 (Pharmacia). Sephadex column preparation: hydrate Sephadex G25 in
phosphate-buffered saline (PBS) (see Subheading 2.2.1., item 8) and pack in an all-glass
column approx 1 × 15 cm. Thoroughly equilibrated the packed column in PBS.
3. Protein biotinylation reagent: δ-biotinyl-ε-aminocaproic-N-hydroxysuccinimide ester
(BNHS) (Sigma).
4. Carbohydrate biotinylation reagent: δ-biotinyl-ε-aminocaproic-acid hydrazide (BACH)
(Boehringer Mannheim).
5. Dimethylformamide (DMF) (Sigma).
6. Dimethylsulfoxide (DMSO) (Sigma).
7. Sodium periodate (Sigma).
8. PBS: 0.375 g of sodium dihydrogen phosphate dihydrate, 1.155 g of disodium hydrogen
phosphate, and 8.765 g of sodium chloride in 1000 mL water.
2.4. Mucinase Assay with Biotinylated Mucin
2.4.1. Coating of Plates with Biotinylated Mucin (
see
Notes 4–6)
1. Microtiter plates, 96-well, Nunc-Immuno™ plates, MaxiSorp Surface™ (Nalge Nunc,
Life Technologies, Glasgow, Scotland) (see Note 2).
2. Coating buffers: 0.1 M sodium acetate buffer, pH 5.0, for carbohydrate labelled mucin
and 0.1 M sodium phosphate buffer, pH 7.0, for protein label (see Notes 3 and 4).
388 Stark et al.
2.4.2. Detection of Biotin-Labeled Mucin
1. PBS (see Subheading 2.3.1., item 8).
2. PBST: Add Tween-20 (Sigma) to PBS to give a final concentration of 0.2%.
3. Blocking buffer: 1% bovine serum albumin in PBST. Use enough to fill the well, 200–
300 µL.
4. Streptavidin-horseradish peroxidase (HRP) solution: Streptavidin-HRP conjugate (Vec-
tor, Peterborough, UK) at 1 mg/mL is diluted to 1:1500 in blocking solution (75 µL/well)

5. OPD Solution: 1,2-phenylenediamine dihydrochloride (Dako, High Wycombe, Bucks,
UK). Dissolve four (2 mg) tablets in 12 mL of distilled water and add 5 µL of 30% hydro-
gen peroxide immediately prior to use.
6. Stop solution: 0.5 M sulfuric acid (28 mL of 95–97% acid in 1000 mL of distilled water).
3. Methods
3.1. Biotin Labeling of Mucins in the Protein Moiety
1. Dissolve BNHS in DMF to give a final concentration of 20 mg/mL.
2. Dissolve 1 mg of mucin in 0.9 mL of PBS (larger batches can be prepared at the same
mucin buffer ratio).
3. Add 0.1 mL of BNHS solution and incubate at 4°C overnight or at room temperature for
4 h.
4. Load the biotin/mucin incubation (1 mL) onto a Sephadex G25 column and run in PBS
buffer, collecting 1-mL fractions up to 30 mL total volume. The Sephadex column is
discarded (see Note 2).
5. Test fractions from the column for mucin using the slot blot assay with the periodic acid
Schiff’s stain (see Chapter 4).
6. Test fractions for their biotin labeling by adherence to 96-well microtiter plate assay (see
Subheading 3.4).
7. Pool the labeled fractions (approx 5 × 1 mL) to give a concentration of 20 µg/mL mucin,
and aliquot as 0.2-mL samples. Store at 4°C until used.
3.2. Biotin Labeling of Mucins in the Carbohydrate Moiety
Carbohydrate labeling requires the periodate oxidation of the carbohydrate moi-
eties of the mucin before biotinylation of these oxidized residues. In the case of co-
lonic mucins, the presence of O-acetyl esters will block this oxidation and a
saponification step is needed first.
1. Dissolve 1 mg of mucin in 0.5 mL of 0.1 M sodium hydroxide and incubate for 45 min at
room temperature. Neutralize to approx pH 7.0 with 0.05 mL of 1 M HCl. Check the pH.
2. Adjust to pH 5.5 with 0.1 M acetate buffer. Add sodium periodate so that the final con-
centration is 1 mM.
3. Incubate for 20–60 min at room temperature

4. Apply the oxidised mucin to a Sephadex G25 column as for the biotinylation of protein-
labeled mucin (see Note 6) and collect fractions.
5. Detect mucin containing fractions mucin using the slot-blot assay with the periodic acid
Schiff’s stain (see Chapter 4), and pool these fractions (approx 5 × 1 mL).
6. Add BACH in DMSO to a final concentration of 1 mM.
7. Incubate at room temperature for 2 hours or overnight at 4°C.
8. Separate the biotinylated mucin on a Sephadex G25 column as for protein-labeled mucin.
Mucinase Activity 389
9. Collect fractions and detect mucin using the slot blot assay with the periodic acid Schiff’s
stain and with the 96-well microtiter plate assay (see Subheading 3.4.).
10. Pool the labeled fractions (approx 8 × 1 mL) to give a concentration of 12.5 µg/mL mucin
and aliquot as 0.2-mL samples. Store at 4°C until used.
3.3. Mucinase Assay with Radioactive Substrates (
see
Notes 7 and 8)
1. Mix 20–500 µL of enzyme extract or commercially available enzyme (see Subheading
2.1., item 1) with 5000–50,000 cpm of radiolabeled mucin in incubation buffer in a final
volume of 1 mL, maximum (see Notes 7 and 8).
2. Incubate at 37°C for periods up to 24 h (usually 6, 12, or 24 h, but start with 24 h if the
activity is unknown).
3. Prepare a blank and incubate under the same conditions as step 2 above (see Note 9).
4. After incubation, either load onto Sepharose CL 2B column immediately, or freeze at
–20°C until ready to start gel filtration.
5. Load as a 1 mL sample onto a column 30 × 1cm of Sepharose CL 2B equilibrated in 10 mM
Tris-HCl, pH 8.0, and elute with the same buffer.
6. Collect 30 1-mL fractions
7. Mix the entire fraction (or 0.5 mL where >50,000 cpm counts are present) with scintilla-
tion fluid and measure the radioactivity.
8. Compare profiles of the test with the blank incubations. Identify the region of low
molecular weight product that represents degraded mucin. Figure 1 gives an example

profile.
9. Subtract the blank incubation (background) from the test incubation, and assess the propor-
tion of low molecular weight product formed from the high molecular weight substrate.
3.4. Mucinase Assay with Biotinylated Substrates
3.4.1. Coating Microtiter Plates
1. Dilute the biotinylated mucin in coating buffer (see Notes 6 and 7).
2. Carefully place the chosen volume in the bottom of the microtitre plate well (see Note 5).
Typically about 50 µL is used.
3. Incubate the plates overnight at 4°C for the mucin to adsorb onto the plate.
4. Empty the plates carefully (see Note 5).
5. Wash once with incubation buffer 1 × 50 µL followed by 3 × 200 µl. The buffer used is the
buffer that is to be used in the assay (see Notes 7 and 8).
3.4.2. Digestion of Coated Plates
1. Prepare a suitable dilution of the enzyme sample in incubation buffer (see Notes 7, 8, and 10).
2. Place 60–100 µL of enzyme preparation in each well (i.e., more than the volume of mucin
solution which was used to coat the plate).
3. Incubate at 37°C typically for 1–2 h.
4. Carefully remove the digestion media.
5. Wash four times with 200 µL of PBS.
3.4.3. Detection of Labeled Mucins
1. Block nonspecific binding with 150–300 µL of blocking buffer for 1 h at room tempera-
ture or overnight at 4°C.
2. Empty the plates and wash twice with 200 µL PBS per well.
3. Incubate with streptavidin-HRP solution (75 µL) for 60 min at room temperature.