Proteinase Activity 393
393
32
Proteinase Activity
David A. Hutton, Adrian Allen, and Jeffrey P. Pearson
1. Introduction
Early studies concerning proteolytic degradation of mucins demonstrated that the
protein core of mucins consisted of two distinct regions, glycosylated regions: pro-
tected from degradation by the densely packed carbohydrate side chains and non-
glycosylated regions susceptible to proteases (1,2). Since the late 1980s, sequencing
of mucin genes has underlined these studies and provided a firm molecular basis for
these concepts (3). Gene-cloning studies have shown that the protein backbone of the
subunits of secreted polymeric mucins can be up to 5000 amino acids in length (approx
20% by weight of the molecule) and consists of two major types of domain that alter-
nate throughout the sequence (3). One type of domain, situated centrally accounts for
about 50% of the protein core and is characterized by tandem repeat (TR) sequences,
rich in threonine, serine, and proline, and the hydroxyl amino acids form the sites of
attachment of the oligosaccharide chains (approx 80% by weight of the molecule).
The other major type of domain, situated at the N- and C-terminals and between regions
of TR sequences, is relatively poor in these three amino acids and relatively rich in
cysteine (3). Some of these cysteine residues can form disulphide bridges with other
mucin monomers (M
r
2–3 × 10
6
) to form large polymeric mucins linked end to end
(M
r
~ 10
7
) and capable of forming gels (4–6). The tandem repeat domains are pro-

tected from proteolysis by the carbohydrate side chains that sterically inhibit protein-
ases from gaining access to the protein core, however, proteinases can hydrolyze the
cysteine-rich regions of accessible nonglycosylated protein, thereby fragmenting the
polymeric mucins (6). The soluble glycopeptides resistant to proteolysis remain of
relatively high Mr (200–700 kD) and recent studies have suggested that individual
mucin gene products may contain different types and lengths of glycosylated domains.
For instance, analysis of high Mr glycopeptides produced by trypsin digestion of the
MUC5B subunit indicated that it contained different types and lengths of glycosylated
domains; one domain of Mr 7.3 × 10
5
, two domains of 5.2 × 10
5
and a third domain of
2 × 10
5
(7). Similarly rat small intestinal Muc2 mucin subunit contains two glycopep-
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
394 Hutton et al.
tides with an estimated mass of 650 and 335 kDa (8). The significance of the differ-
ences in size of these domains is unclear.
Protective essentially insoluble mucus gels adherent to mucosal surfaces are
solubilised by proteinases, e.g., in the lumen of the gastrointestinal tract (6). The
mechanism of solubilisation is cleavage of the susceptible regions of the mucin pro-
tein core which are responsible for the gel-forming properties of mucus gels. This
mucolysis results in degradation of the polymeric mucins (high viscosity, gel-form-
ing) into relatively low Mr glycopeptides (low viscosity, soluble). A dynamic balance
exists between secretion of gel-forming mucins and their degradation by proteinases,
if this relationship is tilted in favor of proteolytic degradation the protective properties

of the mucus gel will be compromized and disease may result. The proportion of poly-
meric mucins present in mucus gels has been shown to be an indicator of gel strength
(9) and evidence exists for enhanced mucolysis by proteinases and/or inferior mucin
polymerisation and consequent impairment of the mucus barrier in peptic ulceration
and inflammatory bowel disease (10,11). Techniques are therefore required for mea-
surement of the integrity of mucin polymeric structure in large numbers of samples
isolated from mucus gels and the mucosa secreting them and methods are needed to
assess the mucolytic potential of host and pathogen secretions.
The digestion of mucin polymers and their reduced subunits by proteases has been
followed by measuring molecular size using analytical ultracentrifugation or light scat-
tering (2,5). These techniques, whilst providing the most empirically quantitative
descriptions of size, rely on highly purified samples, expensive equipment, a high
level of expertise and can require relatively large quantities of sample (e.g., measure-
ment of sedimentation coefficient). Therefore, they are generally unsuitable for the
assessment of the polymeric integrity of large numbers of mucin samples or for the
screening of the mucolytic potential of protease containing samples.
For most laboratories gel-filtration chromatography, commonly on Sepharose CL-
2B or Sephacryl S-500, has provided the most convenient method of assaying mucin
degradation in terms of both integrity of mucins and mucolytic activity (5,10,12,13).
Polymeric mucins are excluded in the void volume of the column, and proteolytically
degraded subunits are partially included (K
av
~ 0.5), eluted mucins being assayed in
solution or after blotting onto nitrocellulose membranes (see Note 1). However, this
technique also requires relatively large quantities of sample and is time consuming in
processing large numbers of samples. The use of radiolabelled mucin samples
improves the sensitivity of the technique, however it remains laborious (14).
Mucin polymers and their reduced subunits do not penetrate polyacrylamide gels
(>3%), however, proteolytically digested subunits will enter gels of >4%. The advent
of readily commercially available precast polyacrylamide gradient gels (4–15%)

requiring small amounts of sample and with rapid running and staining times has vastly
improved the analysis of the in vivo polymeric integrity of mucins isolated from
adherent mucus gels and mucosa and is described below.
Assessment of the mucolytic potential of endogenous secretions or extracts from
invasive pathogens is initially underpinned by measurement of nonspecific proteinase
activity. General protease activity has often been assayed by methods distinguishing
Proteinase Activity 395
hydrolyzed protein from nonhydrolyzed protein by precipitation of the latter, e.g., with
trichloroacetic acid. These methods are reliant on qualitative size and conformational
size changes preventing protein precipitation (15). A sensitive and more accurate
method for estimating proteolytic activity involves measuring trinitrophenylated
derivatives of new N-terminal groups which form on peptide bond hydrolysis (16).
The sensitivity of the assay is improved by blocking existing amino groups on the
protein substrate, e.g., by succinylation. The preparation of succinyl albumin is
described subsequently. The assay can also be used with mucin substrates (12) and the
large size of mucin protein cores means that fewer N-terminals are present on a weight
for weight basis and therefore blocking is unnecessary to achieve low background
absorbances. Large quantities of highly purified mucin are, however, required. The
assay has also been adapted for use after separation of proteinase isoenzymes on aga-
rose gels (16).
Attempts to measure specific mucolytic activity have been dogged by similar prob-
lems to those of general proteinase activity. Cetyl trimethylammoniumbromide
(CTAB) or protamine sulfate precipitation has been used to precipitate only undi-
gested mucins, but again depends upon qualitative assessment of precipitability of
undegraded macromolecules (17,18). Other methods that depend on the appearance of
hydrolysis zones on petri dishes containing mucin/agarose gels often rely on crude
mucin preparations (contaminated with hydrolyzable protein) to provide sufficient ma-
terial or require large amounts of purified mucin (19,20).
Dilute solution viscosity studies measuring mucolysis by following the fall in vis-
cosity of mucin solutions (10,12) have a number of advantages over other methods.

They provide information on the kinetics of digestion and the size (molecular space
occupancy) of the degraded species, and crude mucus samples can be studied as well
as purified mucins. The technique is readily compatible with other techniques, e.g., if
mucolysis is inhibited at particular time points, aliquots of sample can be further stud-
ied by gel filtration or N-terminal analysis (12).
The measurement of mucolysis by quantitating digoxigenin-labeled mucin bound
to and released from microtitre plates requires little mucin substrate and may therefore
be useful for screening large numbers of potentially mucolytic samples (20). Differ-
ences in binding to the plates between undegraded and degraded mucins may, how-
ever, make interpretation of the results difficult (see Note 1).
1.1. Proteinases in the Gastrointestinal Tract
Proteinases from all regions of the gastrointestinal tract degrade mucus gel and
mucins in vivo and in vitro to produce soluble glycopeptide fragments which are fur-
ther degraded by glycosidases in the colon (1).
In the stomach, the secreted mucins in the adherent mucus gel layer are faced with
pepsins (maximum concentration ~0.7 mg/ml in humans). Of the seven pepsin isoen-
zymes in human gastric juice, pepsin 3 is the most abundant with some pepsin 5.
Pepsin 1 is normally a minor component (<5%) but can account for up to 25% of the
proteolytic activity in peptic ulcer patients. Pepsin 1 has increased collagenolytic and
mucolytic activities compared with other pepsins (15). In peptic ulcer patients, there is
396 Hutton et al.
disruption of the mucus layer and an increase in the percentage of nongel forming lower
molecular mass mucin in the adherent gel (21) and this is associated with increased
mucolytic activity of the gastric juice owing to raised levels of pepsin 1 (10).
The human pancreas produces 1–3 g of chymotrypsin, 1–3 g of trypsin and approx
0.5 g of elastase per day, however total fecal levels of pancreatic enzymes are ~ 1 mg/d
(22). This fall in levels is due partly to bacterial degradation and partly to autode-
gradation (23). Human fecal extracts contain proteinases of pancreatic (23) and bacte-
rial (24) origin whereas enzymes associated with inflammation, e.g., white cell elastase
are present only in trace amounts (25). The bacterial microflora can produce copious

amounts of luminal proteinases (24). Fecal proteinases have been demonstrated to
have mucus degrading activity (12,14). Human fecal proteinase activity is raised in
ulcerative colitis patients compared to nonsymptomatic control subjects (26,27) and
this raised luminal proteinase activity probably equates with elevated mucolytic activ-
ity. These observations could explain in part the thinner colonic mucus in ulcerative
colitis (28) by allowing mucin degradation to outweigh mucus secretion.
1.2. Specific Proteolysis of Mucins
Specific sites for proteolytic cleavage are also known to exist in the amino acid
sequences of mucin protein cores. cDNA studies on human and rat MUC2 have shown
that these mucins share a proteolytic cleavage site sequence approx 700 amino acids
from their C-terminus (TGWGD PH(Y/F*)VTFDGLYY) and cleavage of the aspartyl
proline bond generates a C-terminal glycopeptide fragment of approximately 120 kDa
(29). This site is homologous with a site cleaved during the synthesis of rat mammary
sialomucin at an early stage of intracellular transport. The timing of cleavage (during
or after biosynthesis) of intestinal mucins has not been established. A heparin binding
site sequence, SRRARRSPRHLGSG, is also cleaved from the protein core of MUC2
at an early stage of biosynthesis (30).
2. Materials
1. Bovine serum albumin (BSA), Fraction V, (Sigma, Poole, Dorset, UK).
2. Succinic anhydride (Sigma ).
3. Trinitrobenzenesulphonic acid (TNBS; 5% [w/v] aqueous solution) (Sigma).
4. Enzymes: porcine pepsin (pepsin A; EC 3.4.23.1), porcine pancreatic trypsin (E.C
2.4.21.4) and porcine pancreatic elastase (EC 3.4.21.36) (Sigma).
5. Proteinase inhibitor cocktail. 1.0 mM phenylmethylsulfonyl fluoride (PMSF), 50 mM
iodoacetamide, 100 mM aminohexanoic acid, 5 mM benzamidine HCl, 1 mMN-
ethylmaleimide, 1 mg/L
soybean trypsin inhibitor (all from Sigma Chemical Company),
10 mM EDTA (B.D.H. Poole, Dorset, UK) in 0.67 M phosphate buffer, pH 7.5.
6. Sodium dodecyl sulfate (SDS)-polyacrylamide gels for electrophoresis: 4–15% Phast gels
were purchased from Pharmacia, L.K.B. Biotechnology, Uppsala, Sweden.

7. Schiffs reagent, commercial solution (Sigma).
8. Sepharose CL-2B (Pharmacia) use in 1.5 ID × 150 cm glass columns equilibrated with
0.2 M NaCl/0.02%(w/v) NaN
3
(B.D.H., Poole, UK).
9. Pharmacia Phast-gel system (Pharmacia).
10. Contraves low shear 30 viscometer (Contraves A.G. Zurich, Switzerland).
Proteinase Activity 397
3. Methods
3.1. Preparation of Substrate, Blocking of
N
-Terminals
1. Dissolve 10g of BSA in 100ml 0.1M phosphate buffer pH 7.5.
2. Add 1.4 g of succinic anhydride , stir to dissolve and maintain the pH at 7.5 with 2 M
NaOH using a pH stat as the succinic anhydride dissolves (see Note 2).
3. Dialyze the solution exhaustively against distilled water at 4°C, freeze-dry and store the
protein substrate with blocked N-terminals at –20°C (see Note 3).
3.1.1.
N
-Terminal Assay
1. Mix proteinase (see Note 4) or extract containing proteinase activity (200 µL, 0.1–0.5 µg)
with 0.5 mL buffer of choice (see Note 5). Prepare control samples by heating the pro-
teinase preparation at 100°C for 10 min to destroy activity. Alternatively add the sub-
strate immediately prior to step 4 below. (This will give a background value for any free
N-terminals in the proteinase sample). Prepare a reagent blank by replacing enzyme or
extract with buffer.
2. Add substrate (0.5 mL 8mg/mL succinyl albumin) to start the reaction, mix and incubate
samples for 30 min at 37°C in a waterbath.
3. Add 0.5 mL of sodium bicarbonate (to increase the pH of the solution to pH 8.0) and add
0.5 mL 0.05% (w/v) TNBS in water (to trinitrophenylate any free amino groups formed).

Incubate at 50°C for 10 min in a water bath to develop color.
4. Add 0.5 mL 10% (w/v) SDS to prevent protein precipitation. Then add 0.25 mL 1 M HCl
to complete the reaction.
5. Read the absorbance at 340 nm in a spectrophotometer.
6. Calculations: Proteinase activity in millimoles new N-terminals/min/g extract is calcu-
lated using the following equation:
Vt × dilution × A
340
× 10
3
E × t × Vs × g
where Vt = final tube volume (mL); E = molar extinction coefficient of trinitrophenyl
amino acids (1.3 × 10
4
cm
2
/mole); t = time (min); Vs = volume of enzyme; A
340
= absor-
bance at 340 nm; g = wet weight of extract/mL (see Note 6).
The example for g is for proteinase activity in fecal extracts, and g refers to the weight of
material present in 1 mL of a fecal homogenate.
3.1.2. Collection, Biopsies, and Brushings: Purification of Mucin
Samples of human mucin can be obtained from samples obtained at surgery, e.g.,
during routine endoscopy or colonoscopy. Adherent mucus gel is obtained by brush-
ing the mucosa with a cytology brush. Mucosal biopsies provide both intracellular
mucin and adherent mucus gel.
1. Place samples immediately into a cocktail of proteinase inhibitors, i.e., 1.0 mM
phenylmethylsulphonyl fluoride (PMSF), 50 mM iodoacetamide, 100 mM aminohexanoic
acid, 5 mM benzamidine HCl, 1 mMN-ethylmaleimide, 1 mg/L soybean trypsin inhibitor,

10 mM EDTA in 0.67 M phosphate buffer, pH 7.5, at 4°C to minimize proteolytic degra-
dation (see Note 5) and store at –20°C until processed.
2. Solubilize mucins by brief homogenization (30 s, low-speed, hand-held homogenizer)
and centrifuge (8000g, 1 h, 4°C) to remove cell debris.