Tài liệu Báo cáo Y học: Proteolysis of bovine b-lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature-modified protein and yields fragments with low immunoreactivity pptx

Proteolysis of bovine b-lactoglobulin during thermal treatment
in subdenaturing conditions highlights some structural features
of the temperature-modified protein and yields fragments
with low immunoreactivity
Stefania Iametti
1
, Patrizia Rasmussen
1,2
, Hanne Frøkiær
2
, Pasquale Ferranti
3
, Francesco Addeo
3
and Francesco Bonomi
1
1
Dipartimento di Scienze Molecolari Agroalimentari, University of Milan, Italy;
2
Biocentrum, Technical University of Denmark,
Lyngby, Denmark;
3
Istituto di Scienze dell’Alimentazione, CNR, Avellino, Italy
Bovine b-lactoglobulin was hydrolyzed with trypsin or
chymotrypsin in th e course of heat treatment a t 55, 60 and
65 °C a t n eutral pH. At these temperatures b-lactoglobulin
undergoes significant but reversible structural changes. In
the c onditions used in the present study, b-lactoglobulin was
virtually insensitive to proteolysis by either enzyme at room
temperature, but underwent extensive proteolysis when
either protease was present during the heat treatment. High-

temperature proteolysis occurs in a progressive manner.
Mass spectrometry analysis of some large-sized breakdown
intermediates formed in the early steps of hydrolysis
indicated that both enzymes effectively hydrolyzed some
regions of b-lactoglobulin that were transiently e xposed
during the physical treatments and that were not accessible
in the native protein. The immunochemical p roperties of the
products of b-lactoglobulin hydrolysis were assessed b y
using various b-lactoglobulin-specific antibodies, a nd most
epitopic sites w ere no lon ger pre sent a fter attack of the
partially unfolded protein by the two proteases.
Keywords: bovine b-lactoglobulin; limited proteolysis;
partial unfolding; thermal treatment; reduced immunoreac-
tivity.
The globular protein b-lactoglobulin is fou nd in the whey
fraction of the milk of many mammals, but is absent from
human milk. In spite of numerous physical a nd biochemical
studies, its function is still not clearly understood [1,2]. The
crystal structure of bovine b-lactoglobulin (BLG) shows a
marked similarity with the plasma retinol binding protein
and the odorant binding protein, that all belong to the
lipocalin superfamily [2–5].
Denaturation of BLG by physical means is a complex
phenomenon, that occurs through a series of intermediate
steps, whose kinetics and equilibrium depend on the
treatment conditions, on the protein c oncentration, and
on the interaction with other components when complex
systems s uch a s milk and whey are considered. Most o f the
steps occurring below a given i ntensity threshold of physical
treatment (temperatures below 60–65 °C, or pressures

below 600 MPa [6,7]) a re fully reversible in s olutions of
the pure protein at neutral pH. Transient BLG conformers
are formed b y e ither physical t reatment in the same
conditions, and the properties of t hese conformers have
been investigated in some detail [7–10].
Limited proteolysis represents a common and powerful
tool for the investigation of protein structure, including
transient c onformational states of proteins generated during
folding or unfolding (reviewed in [11]). This approach has
not been popular for use with BLG in view of its structu ral
toughness, which makes native BLG quite insensitive to
most proteases under nondenaturing conditions [12–16], in
particular at pH values lower than 7.5, where the well-
known Tanford transition of the protein structure occurs.
Most proteolytic studies on unfolded BLG only addressed
the products of severe thermal treatment, i.e. above the
temperature threshold for irreversible structural modifica-
tion of the protein [17,18].
Proteolysis has been used to lower or to eliminate the
antigenicity of milk proteins, including BLG. Indeed, BLG
is among the major causes of intolerance and/or allergenic
response to cow’s milk in humans, that represent a major
challenge to paediatricians, to nutritionists, and to food
technologists. H igh-temperature h eat d enaturation i s m ost
commonly used in the processes for producing extensively
hydrolyzed formulae starting from whey proteins, because
denaturation by itself leads t o the removal of conforma-
tional epitopes [19], and because the thermal precipitation of
heat-denatured BLG allows to minimize the amount of
residual intact protein in the preparation. Similar processes

rely on extensive hydrolysis of the partially modified form of
Correspondence to Francesco Bonomi, Dipartimento di Scienze
Molecolari Agroalimentari, Via G. Celoria 2 20133 Milano, Italy.
Fax: + 39 02 58356801, Tel.: + 39 02 58356819,
E-mail:
Abbreviations: BLG, b-lactoglobulin; BAPA, benzoyl-
L
-arginine-
p-nitroanilide; SUNA, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide;
SE-HPLC, size-exclusion high performance liquid chromatography;
CP1 and CP2 (or TP1 and TP2), the large-sized proteolytic fragments
isolated after chymotryptic (or tryptic) digestion of BLG at
temperatures above 50 °C.
Enzymes: trypsin (EC 3.4.21.4); chymotrypsin (EC 3.4.21.1).
(Received 2 8 August 2001, revised 7 December 2 001, accepted
8 January 2002)
Eur. J. Biochem. 269, 1362–1372 (2002) Ó FEBS 2002
BLG produced at pH > 8.0 [13,20]. However, several
studies have reported a residual antigenic activity in
hydrolysed milk formulae [21–26]. Residual allergenicity in
these p reparations could s tem from t he inability of the
hydrolytic r eaction to address a ll the sequential e pitopes
even in the denatured protein. In fact, despite the a pparent
simplicity of t he approaches described a bove, heat denatur-
ation and aggregation of BLG upon heat treatment may
hide putative sites of attack from the action of proteases,
therefore leaving intact some regions of the protein that may
be relevant to its a llergenic properties.
The BLG conformers transiently formed during a
physical treatment of subdenaturing intensity may represent

ideal substrates for the action o f proteases, as ample regions
of the hydrophobic p rotein core are unfolded, contrarily to
what happens in the very compact native protein or in the
aggregated products of extensive thermal denaturation of
BLG [6,8], thus making even the most inner p arts of the
protein accessible, at least in principle, to enzymatic
hydrolysis. In more advanced steps of physical denatura-
tion, collapse of t he hydrophobic portion of the structure
may o ccur [6], possibly making the same enzyme attack sites
once again as they were inaccessible i n the native folded
protein.
In previous studies on limited proteolysis of p artially
unfolded BLG, w e used high-pressure as the physical
denaturant, a s the intensity threshold o f pressure treatment
appears less critical than temperature with respect to the
aggregation behavior of BLG and o f the sensitivity of the
aggregation process to protein concentra tion [ 10,27]. In
those s tudies, several enzymes were tested. Trypsi n and
chymotrypsin gave the best results, both i n terms of
interpretation of the hydrolysis pattern and o f r educed
immunoreactivity [28]. Trypsin and chymotrypsin were used
in the present study, also in view of a possible c omparison
with the results ob tained under pressure. I n this work w e
used short time periods (10 min) for the combined proteo-
lytic/thermal treatment of BLG at relatively high enzyme/
BLG ratios (1 : 10 and 1 : 20) and at the highest tempera-
ture compatible with rete ntion of e nzyme activity a nd with
the reversibility of structural modifications of BLG. Limited
proteolysis studies on BLG are s ignificant not only t o
understanding its unfolding mechanism, but may have

practical relevance as for decreasing the i mmunoreactivity
of the protein. Therefore, we also tested some of the
hydrolysis products obtained in this study for their immu-
noreactivity towards different sets of various BLG-specific
antibodies.
EXPERIMENTAL PROCEDURES
Proteins
BLG was from Sigma. Each protein batch was tested as
received for its content in multimeric forms or in disulfide-
linked dimers, by using H PLC gel-permeation and SDS/
PAGE under nonreducing conditions. Bovine pancreatic
trypsin (N-a-tosyl-
L
-phenylalanylchloromethane treated,
type XIII) and chymotrypsin (N-a-tosyl -
L
-lysylchloro-
methane t reated, t ype VII) a nd the synthetic substr ates
benzoyl-
L
-arginine-p-nitroanilide (BAPA, trypsin) or
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SUNA, chymo-
trypsin), also were from Sigma.
BLG-specific m onoclonal a ntibodies were prepared
according to [29].
Proteolytic experiments during thermal treatment
Thermal treatments at fixed t emperature were carried out
as reported in [6] w ith minor modifications. Proper aliquots
of concentrated solutions o f the appropriate proteolytic
enzyme were added at 0 °C to 1 mL of BLG (2.5 mgÆmL

)1
in 50 m
M
phosphate buffer, pH 6.8) to a final mass ratio
enzyme/BLG of 1 : 20 or of 1 : 10. The protein/protease
mixture was then placed in a thermostatted water b ath for
the required amount of time. At the end of the heat
treatment the mixtures were placed in an ice/water bath,
and the enzymatic activity w as stopped by lowering the pH
of the r eaction mixture to 3 by addition of 0 .2 mL of 50%
(v/v) acetic acid in water. All these manipulations were
carried out within 1–2 min from the end of the thermal
treatment.
Analytical measurements
Enzyme activities were determined at 37 °Cin0.1
M
Tris/
HCl, pH 8.1, by following the increase in a bsorbance at
405 nm due to p-nitroanilide released f rom BAPA or
SUNA, as appropriate. Although the supplier gives nominal
specific activities on synthetic substrates of 10.000
lmolÆmin
)1
Æmg
)1
(trypsin, on benzoyl-arginine ethyl e ster),
and 50 lmolÆmin
)1
Æmg
)1

(chymotrypsin, on benzoyl-tyro-
sine ethyl ester), we measured specific activities in the range
of 2 lmolÆmin
)1
Æmg
)1
(trypsin, 0.5 m
M
BAPA) and
50 lmolÆmin
)1
Æmg
)1
(chymotrypsin, 0.2 m
M
SUNA).
Residual enzyme activity was measured after h eat t reatment
of each enzyme in the same conditions and c oncentrations
used for p roteolysis experiments, in the presence or in the
absence of BLG.
RP-HPLC analysis o f t he proteolyzed samples was
performed d irectly on aliquots of the acidified material
after 10-fold dilution with 0.1% trifluoroacetic acid and
centrifugation for 5 min at 10 000 g to remove minor
amounts of materials made insoluble by the addition of
trifluoroacetic acid. A Deltapak C
18
column
(3.9 · 150 mm, Waters), fitted to a Waters 625 HPLC
equipped with a Waters 490E dual wavele ngth detector was

used. Elution of the hydrolytic p roducts and o f the residual
intact protein was performed with a linear gradient from 20
to 60% acetonitrile (in 0.1% trifluoroacetic acid) in 3 0 min
Flow was 0.8 mLÆmin
)1
, detection was at 220 nm. Residual
intact BLG was quantitated by on-line integration, using
native BLG as a s tandard.
Size-exclusion HPL C separ ations of the p roteolyzed
samples was performed directly on aliquots o f the acidified
material aft er centrifugation for 5 min at 10 000 g to
remove insoluble m aterials. A Superdex Peptide 10/30
column (Pharmacia) was used, fitted to a Waters 625 HPLC
equipped with a Waters 490E dual wavelength detector. The
eluant was 20% acetonitrile in water containing 0.1%
trifluoroacetic acid, at 0.5 m LÆmin
)1
. Detection was at 220
and 280 nm.
Electrospray mass spectrometry (ES/MS) analysis was
performed using a Platform single-quadrupole mass spec-
trometer (Micromass), after liophylization of t he original
materials. Pept ide samples (10 lL, 50 pmol protein in
Ó FEBS 2002 Proteolysis of heat-unfolded b-lactoglobulin (Eur. J. Biochem. 269) 1363
water) were injected into the ion source at a flow rate of
10 lLÆmin
)1
; the spectra were scanned from 1400 to 600 at
10 s p er scan. Mass scale calibration was carried out using
the multiple-charged ions of a separate in troduction o f

myoglobin. Mass values are r eported as average masses.
Quantitative analysis of individual components was
performed by integrating the signals from the multiple
charged ions of the single species [30]. The peptide identity
was determined b y a nalysis o f t he sp ectral d ata u sing a
computer software developed by the instrument manufac-
tureer (Biolynx, Micromass), and confirmed by MS analysis
of the samples prior and after a reduction step in 10 m
M
dithiothreitol for 2 h at 37 °C.
Immunochemistry
ELISA tests were performed as competitive capture
ELISA by u sing BLG specific mon oclonal or polyclonal
antibodies as capture a ntibodies. A ll steps were c arried
out at 30 °C. Polyclonal rabbit a nti-BLG Ig was diluted
4000–10000 times in carbonate buffer (15 m
M
Na
2
CO
3
;
35 m
M
NaHCO
3
, p H 9.6) and coated directly to the wells
of microtiter plates, a t 0.1 mL per w ell. When using
monoclonal antibodies, polyclonal antimouse antibodies
(Z109, 1 : 1000, 0 .1 mL per w ell, DAKO) were used f or

coating followed by incubation of 0.1 mL per well of
monoclonal antibody diluted to approximately
250 ngÆmL
)1
in KCl/NaCl/P
i
buffer containing 0.1%
Triton X-100 (KCl/NaCl/P
i
/Triton; 1.5 m
M
KH
2
PO
4
;
6.5 m
M
Na
2
HPO
4
;0.5
M
NaCl; 2,7 m
M
KCl; 1 mLÆL
)1
Triton X-100). Plates w ere washed four times with K Cl/
NaCl/P

i
/Triton buffer diluted 1 : 10, and the various
samples in KCl/NaCl/P
i
/Triton were applied to the wells
in serial twofold dilutions and incubated f or 1 h. After
four washes, the plates were incubated with a fixed
concentration of biotinylated BLG (10–200 ngÆmL
)1
,
depending on the antibody). After i ncubation and another
four washes, plates were incubated with horseradish
peroxidase-labeled streptavidin (HRP-streptavidin,
DAKO, diluted 1 : 5000 in KCl/NaCl/P
i
/Triton), and
washed fou r times. Bound HRP activity was measured by
using a sub strate-containing buffer (0.2
M
potassium
citrate pH 5.0; 3 m
M
H
2
O
2
;0.6m
M
3,3¢,5,5¢-tetram-
ethylbenzidine). The reaction was terminated by addition

of 2
M
H
3
PO
4
, 0.1 mL per well. The absorbance at
450 nm was determined on a microtiter plate reader.
RESULTS
Thermal stability of enzymes
Trypsin and chymotrypsin were chosen for this study for t he
following reasons: (a) neither enzyme is capable of attacking
BLG significantly at r oom temperature [14,15,18]; (b) both
enzymes a re available at very high purity; (c) both enzymes
are highly specific; and (d) t hey act on complementary s ets
of amino acids (hydrophobic, chymotrypsin; basic, trypsin).
Other enzymes were tested, but their action was not further
investigated in that they did not comply with all the
requirements listed above, as reported in other studies
[12–14,17]. Furthermore, the results obtained with t rypsin
and chymotrypsin on transiently temperature-unfolded
BLG c ould be c ompared with t hose we obtained on
transiently pressure-unfolded BLG [28].
The only major drawback in the u se of trypsin a nd
chymotrypsin in the experiments reported here w as their
limited thermostability. As shown in Table 1, both enzymes
had ve ry little residual activity a fter 5 min at 65 °C, also in
the presence of a 20-fold m ass excess of the substrate
protein. Contrarily to what expected for a generic protective
effect of added proteins, the r esidual activity a fter heat

treatment in the presence of BLG was lower than in the
absence of BLG.
One explanation is that residual BLG (or BLG hydrolysis
products) in the enzyme assays performed on the heated
BLG/protease mixtures competed with the artificial sub-
strates for binding to the enzymes. In our conditions, the
assay mixtures for residual protease activity contained from
0.125 to 0.25 mgÆmL
)1
BLG (or BLG hydrolysis products),
equivalent to 0.075 and 0.15 m
M
BLG, respectively. To test
this p ossibility i n a simple way, we perfo rmed a ssays in
which native BLG was add ed to a protease assay mixture
containing synthetic substrates at 37 °C and pH 8.1 and the
same amounts of enzyme present in the heated mixtures. We
found 25% and 35% inhibition of trypsin activity on
0.5 m
M
BAPA when native BLG was a dded at 0.075 m
M
and 0.15 m
M
, respectively. Under similar conditions, inhi-
bition figures for chymotrypsin ( 0.2 m
M
SUNA as sub-
strate) were 8 and 15%, respectively. These figures d o not
fully account for the differences shown in Table 1, that

apparently are b etter explained b y assuming an inhibitory
effect of the peptides produced by hydrolysis of BLG at high
temperature.
Table 1 . Thermal stability of trypsin and chymotrypsin. Proteins (0.125 mg ÆmL
)1
in 50 m
M
phosphate buffer, pH 6.8) were heated for 5 min at the
given temperatures in the ab sence or in t he presence of BLG ( 2.5 mgÆmL
)1
in 50 m
M
phosphate buffer, pH 6.8, corresponding to a 1 : 20 mass ra tio
enzyme/BLG). Residual enzyme activity after heat treatment was measured spectrophotometrically at 37 °C with 0.02–0.05 m L enzyme in 1 mL of
the synt hetic substrates BAPA (trypsin), or SUNA (chymotrypsin). Substrates (0.5 m
M
BAPA an d 0.2 m
M
SUNA) were in 100 m
M
Tris/HCl,
pH 8.1. Activity is given as p ercentage of that o f control enzymes kept at 37 °C in the absence of BLG.
Treatment temp.
(°C)
Residual activity (%)
BLG present BLG absent
Chymotrypsin Trypsin Chymotrypsin Trypsin
55 43.0 77.9 28.0 9.3
60 17.0 2.3 11.0 0.5
65 6.6 0.4 5.3 0.2

1364 S. Iametti et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Extent of proteolysis as a function of temperature, time,
and enzyme concentration
BLG appeared to be virtually insensible to hydrolysis by
either trypsin or chymotrypsin in the absence of a thermal
unfolding step, as less than 10% of the native p rotein was
degraded by either enzyme in 20 min at 37 °Catanenzyme/
BLG mass ratio of 1 : 10. Essentially the same results (i.e.
less than 10% degrada tion of the total p rotein) were
obtained under the same conditions with BLG that was
previously heated for 10 min at 65 °C. This confirms earlier
reports on the remarkable resistance of BLG to most
proteases [ 13,17], and the reversibility o f heat-induced
structural modifications in these c onditions.
As shown in Fig. 1, that reports the RP-HPLC profiles
obtained after proteolysis at 60 °C, hydrolysis of BLG
became significant when either protease were allowed to act
on BLG during exposure a t temperatures between 55 and
65 °C. As summarized in Table 2, t he amount of residual
BLG decreased with time and with the amount of added
Fig. 1. RP-HPLC analysis of the products obtained upon enzymatic digestion of BLG at 60 °C. Proper volumes of concentrated solutions of each
given enzyme were a d ded at 0 °C to separate aliquots of a BLG solution (1 mL, 2.5 mgÆmL
)1
in 50 m
M
phosphate buffer, pH 6.8) to a fi nal mass
ratio enzyme/BLG of 1 : 20 or of 1 : 10. Each BLG/protease mixture was then placed in a water bath thermostatted at 60 °C for the given amount
of time . At the end of the heat treatment the mixture s were placed o n ice, and the enzymatic activity was stopped by adding 0 .2 mL of 50% (v/v)
acetic acid in water. RP-HPLC separations on the proteolytic samples were performed directly on aliquots of t he acidified material after 10-fold
dilution with 0.1% trifluoroacetic acid in 20% acetonitrile and centrifugation for 5 min at 1100 g to remove som e trifluo roacetic acid- insoluble

material. A De ltapak C
18
column (3.9 · 150 mm, Waters), fitted to a Waters 625 HPLC equipped with a Waters 490E detector was used. Elution
was p erformed by applying a linear gradient from 2 0 to 60% acetonitrile (v/v) in 0.1% t rifluoroa cetic acid in 3 0 min Flow was 0.8 mLÆmin
)1
,
detection was at 220 nm.
Table 2. Residual intact BLG a fter p roteolysis under different c onditions. The amount o f r esidual intact B LG after proteolysis in the given conditions
was determine d by integration of the intact BLG peaks from R P-HPLC separations similar t o t hose reported in Fig. 1, and is given as percentage of
the signal produced by the native protein as a standard.
Enzyme
Mass ratio
enzyme/BLG
Hydrolysis time
(min)
Temperature (°C)
55 60 65
Trypsin 1 : 10 5 41 20 40
10 17 10 29
20998
1:205424041
10 22 24 30
20 16 18 27
Chymotrypsin 1 : 10 5 17 30 52
10 9 19 42
20 3 17 31
1:205223249
10 17 31 48
20 11 30 41
Ó FEBS 2002 Proteolysis of heat-unfolded b-lactoglobulin (Eur. J. Biochem. 269) 1365

enzyme. As for the effects o f temperature, inactivation of
proteases (Table 1) came into play at the highest temper-
atures and at the lowest ratio enzyme/BLG.
Besides the decrease in intact protein, the t racings in
Fig. 1 s how that some hydrolysis products were formed
during the early phases of hydrolysis, and t heir concentra-
tion remained constant with time. Other hydrolysis products
were only formed in significant amounts during the late
stages of proteolysis, suggesting a possible sequential
mechanism for hydrolysis.
From the data in Table 2 it is e vident that chymotrypsin
gave an extent of proteolysis not very different f rom that of
trypsin o n transiently heat-unfolded BLG. T his i s some-
what puzzling, as both e nzymes were added in the same
weight ratio to BLG, but they had very different specific
activities (at least on synthetic substrates). Appropriate
control experiments did not provide evidence for a
transient increase in c atalytic activity on s ynthetic sub-
strates at temperatures above 50 °C fo r either enzyme (no t
shown). Both enzymes rather underwent a marked
decrease in activity above 50 °C, chymotrypsin being much
less thermostable than trypsin, as reported in the equilib-
rium data shown i n Table 1. Thus, our observations (and
the specific activity data on different synthetic substrates,
as reported i n Materials and methods) confirm that t he
accessibility of cleavage sites on the substrate, rather than
the intrinsic catalytic ability of a given protease, is what
limits the effectiveness of the enzyme action on actual
protein substrates. When proteases were added during t he
thermal treatment, h ydrolysis of BLG was extensive, in

spite of significant inactivation of the enzymes. This
indicates that t he transient c onformers originating in the
course of thermal treatment had exposed novel access sites
for either e nzyme.
After thermal treatment in these conditions, there were no
major irreversible changes in all structural levels of BLG, at
least a s d etectable by spectroscopic a nd separ ation tech-
niques [6,8,9,20,27]. The increased a ccessibility of thermally
treated BLG could indicate that some of the residues
specifically recognized by each protease were exposed to the
enzyme action in the treated protein e ven in the absence of
spectroscopically detectable irreversible structural modifica-
tions. Thermal treatment was shown to promote transient
modifications of the BLG structure at neutral pH, inducing
transient dimer dissociation with concomitant exposure of
previously buried hydrophobic site s [7–9,31]. Physically
induced reversible dimer dissociation a t temperatures below
65 °C [9] results in the exposure of hydrophobic residues
along the ÔIÕ strand of the b fold, and of positively charged
residues on the edge of the large a helix in each monomer [5].
Evidence has b een provided that he at treatment in this
temperature range may affect a heat-labile domain of the
protein [32], that w as hypothesized to be relevant also for
the stabilization of associated forms of BLG in solution [20].
In this context, it seems significant t hat chymotrypsin
(specific for aromatic residues) gave the s ame hydrolysis
levels obtained with t rypsin, in spite of its lower thermal
stability. This could confirm that buried, compact hydro-
phobic regions may be transiently unfolded and exposed by
the thermal treatment.

The RP-HPLC tracings in Fig. 2 i ndicate that the major
peaks in the hydrolysis products obtained with each
protease(addedina1:10ratiotoBLG)after10minat
various temperatures from 37 to 60 °C had similar elution
times, suggesting that the same sites of attack were
accessible to either protease in t his temperature range.
Some differences among the RP-HPLC t racings were
only evident when BLG was hydrolyzed at 65 °C. It is not
clear whether the different proteolysis patterns obtained at
65 °C with either enzyme related to the inaccessibility o f
some cleavage sites in the BLG conforme r that is predom-
inant at this temperature, or rather to the fact that the rapid
Fig. 2. RP-HPLC analysis of the products
obtained upon enzy matic digestion of BLG for
10 min at various temperatures. Proper
volumes of concentrated solutions of each
given enzyme were a dded at 0 °Ctoseparate
aliquots of a BLG solution (1 mL,
2.5 mgÆmL
)1
in 50 m
M
phosphate b uffer,
pH 6.8) to a final mass ratio enzy me/BLG of
1 : 10. Individual BLG/protease m ixtures
were placed in a water b ath thermostatted at
the given temperature for 10 min. Further
sample processing and RP-HPLC separation
were performed as detailed in the legend to
Fig. 1.

1366 S. Iametti et al. (Eur. J. Biochem. 269) Ó FEBS 2002
thermal inactivation of both enzymes at this temperature
prevented further degradation of some hydrolysis interme-
diates formed in the e arliest steps of proteolysis. The
peculiar nature of the hydrolysis products obtained at 65 °C
with either enzyme will be discussed in the following section.
Molecular characterization of the major hydrolysis
products and intermediates
The proteolysis products obtained in the conditions repor-
ted above were separated on the basis of their molecular size
by SE-HPLC. As shown in the different panels of Fig. 3,
that presents data obtained during treatment at 60 °C, the
SE-HPLC patterns obtained at different times show
progressive digestion of the intact protein, and significant
accumulation of hydrolytic fragments of appreciable size
(that is, between 3 a nd 10 kDa). The larges t hydrolysis
fragments separated by SE-HPLC w ere n amed after the
enzyme used (T, t rypsin; C, chymotrypsin) and after their
elution order from a Superdex Peptide column (hence, the P
in their names), and c orrespond to the peaks labeled CP1
and CP2 (or TP1 and TP2) in the chromatograms presented
in Fig. 3.
Analysis of the proteolyzed samples by SDS/PAGE (data
not shown) was consistent with the figures r eported i n
Table 2 . The extensive proteolysis observed with chymo-
trypsin resulted in the formation o f appreciable amounts of
proteolytic products capable of being retained by the gel,
according to the SE-HPLC data shown in F ig. 3. Confirm-
ing previous reports, temperature-induced formation of
covalently linked BLG aggregates in this temperature range

as detected by SDS/PAGE was modest [6]. No formation of
covalently linked aggregates was observed in the enzyme-
treated samples, indicating that proteolysis took place more
rapidly than protein aggregation even at 6 5 °C [27,28].
The time-progressive change in the size d istribution of
hydrolytic products obtained a fter treatment with either
enzyme at different temperatures is reported in Fig. 4. Both
the SE-HPLC tracings in Fig. 3 (obtained with 0 .1%
trifluoroacetic acid in 20% acetonitrile as eluant) and the
time courses in Fig. 4 clearly indicate that a limited number
of large fragments constituted a set of intermediate h ydro-
lysis products, suggesting a progressive hydrolysis mechan-
ism with either enzyme. More specifically, it a ppears that the
concentration of TP1 remained constant du ring progressive
hydrolysis of heated BLG by trypsin, whereas the concen-
tration of the intact protein decreased with a n accompany-
ing increase in TP2. The pattern o f events observed with
chymotrypsin is made somewhat more complicated by the
more extensive thermal inactivation of t his enzyme. How-
ever, also in this case, formation of the larger CP1 fragment
occurred in the early phases of hydrolysis, and this
intermediate was further degraded to the smaller CP2
intermediate (and to even smaller peptides) when enough
enzyme activity was present (that is, at relatively low
Fig. 4. Time course of the formation of fragments having different size
during proteolysis of BLG at 55 and 65 °C. Data are taken from
integration of t he chromatograms shown in F ig. 3. Full symbols,
55 °C; open symbols, 65 °C. Excluded peptid es (M
rapp
> 10 000),

circles and full lines, TP1 and TP2 (or CP1 and CP2, as appropri-
ate), triangles and dotted lines; low molecular weight material
(M
rapp
< 3000), squares and dashed lines.
Fig. 3. SEC-HPLC analysis of the products obtained upon enzymatic
digestion of BL G at 60 °C. S ize-exclusion chromatography (SEC) was
carried out on the acetic acid-treated materials obtained as d etailed i n
the legend t o Fig. 1, with no f urther processing. A Superdex Peptide
column (10/30, Pharmacia Biotech) was used on the same chromato-
graphic system described in the legend to Fig. 1 . Eluant was
20% acetonitrile in aqueous 0.1% t rifluoroacetic a cid. Flow was
0.5 mLÆmin
)1
, detection was at 220 nm.
Ó FEBS 2002 Proteolysis of heat-unfolded b-lactoglobulin (Eur. J. Biochem. 269) 1367
temperature, short reaction times and high e nzyme concen-
tration). Indeed, no formation of CP2 was detected during
chymotryptic hydrolysis of BLG at 65 °C, and significant
accumulation of TP2 only occurred a t the longest hydrolysis
times at this temperature (data not shown).
The figures given in Fig. 4 for peptides with a
M
rapp
> 1 0 000 are significantly higher than the amounts
of residual native protein detected by RP-HPLC (Table 2).
This discrepancy could indicate that formation of proteo-
lytic intermediates having a larger size than TP1/2 and CP1/2
may occur to a significant extent.
To test the hypothesis of progressive hydrolysis, and to

assess the nature of the regions being attacked by proteases
in the thermally unfolded protein, f ragments CP 1 and 2, as
well as fragments TP 1 and 2, were further purified by
RP-HPLC and SE -HPLC in t he same conditions and with
the same equipment used for analysis of the whole
proteolyzate, and these c hromatographically homogeneous
peptides were analyzed by ES-MS.
The r esults obtained by MS are reported in Table 3, and
make it clear that most of the material that contributes to
the microheterogeneity of the isolated fragments originate
from further proteolytic degradation of a limited number of
primary hydrolysis products.
All these fragments share a common feature, n amely t he
presence of the disulfide bridge connecting Cys66 and
Cys160 in the native protein.
The position of these fragments in the primary sequence
of BLG is g iven in Fig. 5. A nonspecific hydrolysis by
trypsin is observed between the two leucine residues 57 and
58. As expected, chymotrypsin also cut in the same position,
indicating that this region of the molecule was accessible to
enzyme action. However, in general terms, the fact that
these fragments were only attacked at their ends under our
conditions (Table 3), in s pite of the relative a bundance
of protease-sensitive residues in their sequence (Fig. 5),
suggests that these fragments could have retained (or could
have assumed) a rather compact conformation even at the
temperatures used in this study. Indeed, recent studies based
Table 3. Molecular parameters for large-sized f ragments obtained from hydrolysis of BLG. Fragments were o btained by size-exclusion H PLC of
digests carried out a s in t he legend to Fig. 3 after hydrolysis at 5 5 °C. ES -MS analysis was performed using a Platform single-quadrupole mass
spectrometer (VG-Biotech), after liophylization of the original materials. Peptide samples (10 lL, 50 pmol protein in water) were injected into the

ionsourceataflowrateof10lLÆmin
)1
; the spectra were scanned from 1400 to 600 at 10 s per scan. Mass scale c alibration was carried out using the
multiple-charged ions of a separate introduction of myoglobin. Actual mass values are reported as average masses. For each fragment, the highest
size precursor is listed first, and the products of its further proteolysis are listed in order of relative abundance in each chromatographic fraction,
derived by comparison of t he respective mass signal intensity.
Fragment Sequence
Mass (Da)
Individual peptides Total fragment
CP1 Arg40-Phe82(Cys66-Cys160)His146-Ile162 5015.2 + 1911.7 6926.9
Val43-Phe82(Cys66-Cys160)Lys150-Ile162 4596.7 + 1607.9 6204.6
Arg40-Phe82(Cys66-Cys160)Lys150-Ile162 5015.2 + 1607.9 6623.1
Val43-Phe82(Cys66-Cys160)His146-Ile162 4596.7 + 1911.7 6508.4
CP2 Leu58-Phe82(Cys66-Cys160)Lys150-Ile162 2931.7 + 1607.9 4539.6
Glu62-Phe82(Cys66-Cys160)Lys150-Ile162 2376.0 + 1607.9 3983.9
Lys60-Phe82(Cys66-Cys160)Lys150-Ile162 2690.4 + 1607.9 4298.3
TP1 Val41-Lys70(Cys66-Cys160)Leu149-Ile162 3546.2 + 1721.1 5267.3
Val41-Lys69(Cys66-Cys160)Leu149-Ile162 3418.0 + 1721.1 5239.1
TP2 Leu58-Lys70(Cys66-Cys160)Leu149-Ile162 1619.9 + 1721.1 3341.0
Trp61-Lys69(Cys66-Cys160)Leu149-Ile162 1122.2 + 1721.1 2843.3
Trp61-Lys70(Cys66-Cys160)Leu149-Ile162 1250.1 + 1721.1 2971.5
Leu58-Lys69(Cys66-Cys160)Leu149-Ile162 1491.7 + 1721.1 3212.8
Fig. 5 . Pos ition o f proteolytic fragme nts CP 1/2 and TP1/2 w ithin the
primary s tructur e o f BLG. Residues su sceptible to chymotrypsin and
trypsin hydrolysis are labeled with ÔcÕ and ÔtÕ superscripts, respectively.
Cysteines 66 and 160 are underlined.
1368 S. Iametti et al. (Eur. J. Biochem. 269) Ó FEBS 2002
on completely different methodological approaches have
shown the existence of compact structural regions in BLG,
that are not affected by physical t reatments [10,32].

Figure 6 presents s ome s chematics of the structural
relationship of CP1/CP2 and TP1/TP2 with the remainder
of the BLG structure. It is evident that a ll these fragments
include a generous portion of the b-barrel structure (strands
B, C and part of strand D), along with part of the distant I
strand in the C-terminus region that includes Cys160 and
connects to the leftovers of strand D via a disulfide b ridge to
Cys66 [5].
Immunoreactivity of the products of BLG hydrolysis
As stated in the introduction, one of the goals of this work
was to t ake advantage of the interplay of treatment
conditions and enzyme action t o produce fragments of
sizable mass, but unable t o be recognized by specific
antibodies by standard immunochemical t echniques.
ELISA was used to assess residual immunochemical
reactivity in the unresolved digests and in the TP and CP
fragments discussed in the previous section.
The results obtained with a rabbit anti-BLG Ig on
unresolved hydrolyzates obtained i n con ditions of maxi-
mum BLG proteolysis (namely, 5 5 °C, 20 min, 1 : 10
weight ratio of enzyme/BLG) are shown as an example in
Fig. 7. The decrease in immunoreactivity in the unresolved
BLG hydrolysates obtained with either c hymotripsin or
trypsin at the various temperatures and treatment condi-
tions parallels the decrease in intact BLG (Table 2). This
also suggests that nonproteolyzed BLG retained its immuno-
reactivity in spite of the thermal treatment, confirming that
the structural changes in the temperature range investigated
here were fully reversible.
None of the isolated proteolytic fragments C P1/CP2 and

TP1/TP2 was found to be immunoreactive against rabbit
anti-BLG Ig even at very high fragment c oncentration (not
shown). When epitope-specific monoclonal antibodies were
used to test the same material, the immunoreactivity of the
purified fragments was found to depend on the monoclonal
antibody used for the assay. Some of t he ELISA curves
obtained with different antibodies are reported in F ig. 8.
While most of the monoclonal antibodies did not
recognize any of the large proteolytic fragments, as exem-
plified by monoclonal 5G6 in Fig. 8, monoclonals 9G10
and 1 E3 recogn ized CP1 (although a t  100-fold the
concentration of n ative BLG), but neither CP2 nor TP1
and T P2. A s the only r elevant difference among the
fragments is the presence of Arg40 in CP1 (Fig. 5, Table 3),
it could be possible that this residue determined the
recognizability of CP1 by these particular antibodies.
However, it remains to b e assessed whether Arg40 is a
Fig. 6. Position of the proteolytic fragments obtained at high tempera-
ture within t he structure of the BLG monomer. In both schemes, the
appropriate proteolytic fragments are as colored ribbons: red and
purple, CP1 (TP1); purple, CP2 (TP2). Residues attacked by proteases
are given as sticks (blue, basic; green, hydrophobic). The disulfide-
forming Cys66 and Cys160 are in yellow ball and stick. Structures were
generated by using
RASMOL
[38], and coordinates in file 1B8E depo sited
in the RCSB Protein Databank [34].
Fig. 7. ELISA assay of unresolved BLG hydrolysates. Hydrolysates
were obtained after 20 m in treatm ent at 55 °C with trypsin (triangles)
or chymotrypsin (squares) at an 1 : 10 enzyme/BLG ratio. Native

BLG, circles. A rabbit anti-BLG Ig was used.
Ó FEBS 2002 Proteolysis of heat-unfolded b-lactoglobulin (Eur. J. Biochem. 269) 1369
relevant component of a sequential epitope, or is rather
important for p roper structuring of a conformational
epitope.
DISCUSSION
Increasing temperature greatly facilitated proteolytic attack
of BLG by both trypsin and chymotrypsin. However, no
ÔnovelÕ hydrolysis sites were made available at temperatures
in the 50–60 °C range with respect to those a lready available
at 37 °C, where little i f any hydrolysis occurred at the times
and enzyme concentrations used here. Rather, an increased
accessibility of attack sites in the ÔswollenÕ form of the BLG
monomer that represented the most abundant species in the
temperature range exploited here [6,8] should account for
increased proteolysis, in s pite of thermal inactivation of
both trypsin and chymotrypsin at the highest temperatures
used here.
More details on the structural modifications induced by
temperature may be derived from careful ana lysis of the
data p resented in this paper. A first point concerns tryptic
attack on Arg148. This residues is located in the I strand
that, as shown in Fig. 9, is the closest contact point between
monomers in the BLG dimer. There are a number o f
interprotein hydrogen bonds in this region, in addition to
hydrophobic interactions (such as those between Ile147 and
Leu149 and the corresponding residues on the facing
antiparallel strand) that all t ogether contribute to make
this region virtually inaccessible a t neutral pH. Both t he
network of H-bonds and the hydrophobic interactions are

disrupted when the temperature is raised, so that the BLG
dimer may dissociate [8,9], therefore exposing the polypep-
tide backbone to the action of trypsin. The nature of the
interactions in this region, as pointed out above, also
explains the reversibility of tempe rature-induced dissoci-
ation of the BLG dimer. Although this will have to be tested
with true monomeric BLGs (such as the ones found in
mare’s or sow’s milk), it is likely that dissociation of t he
bovine BLG dimer represents the primary event for
facilitated proteolysis at high t emperature. The relevance
of dimer dissociation to facilitated proteolysis is also e vident
when considering t he high hydrolysis yields obtained at
pH > 8.0 (i.e. above the Tanford transition at pH 7.5 [13]),
although the structural features of the BLG monomer
obtained at high pH [ 20,33,34] appear different f rom those
of monomers obtained at low pH [35] or under pressure [10].
Arg148 is not the only buried arginine in the structure of
BLG. The whole side chain of Arg40 (the other main point
of trypsin action) in the native structure of the protein is
deeply buried inside a hydrophobic pocket that comprises
several side c hains, and provides an envelope for the
guanidinium function (Fig. 6). A number of spectroscopic
studies have shown a reversible exposure of hydrophobic
regions of BLG in the temperature range considered in this
study [6,8,32]. This may be instrumental in facilitating
tryptic attack on Arg40, and the action of chymotrypsin on
the adjacent Leu39. Given their position i n the structure
Fig. 8. ELISA assay o f proteolytic fragments of BLG with various
monoclonal antibodies. Fragmentswerepurifiedasreportedinthetext
and in Fig. 3, and are identified as in Fig. 5. Native BLG, squares.

CP1, full circles; CP2, open circles; TP1, full triangles; TP2, open tri-
angles.
Fig. 9. Relevance of protease-sensitive residues to t he dimeric structure
of BLG. Fragments CP1 and TP1 are as colored ribbons. The o range
regions correspond to the shorter TP1 fragments within the CP1 frag-
ments ( orange and brown). Residues attacked by proteases are iden -
tified b y their position in the s equenc e, and are give n as sticks (blue,
basic; gree n, h y drophobic). The disulfide-form ing Cys66 and C ys160
are i n yellow. Structures were generated by using
RASMOL
[38], and
coordinates in file 1BEB deposited in the RCSB Protein Databank [5].
1370 S. Iametti et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Fig. 9), both residues appear to be much more accessible to
proteases after proteolytic removal of the long exposed helix
and of strand I at the d imer interface.
The disulfide-connected peptides that originate f rom
early proteolysis steps have some interesting features.
Perhaps the most striking regards resistance t o chymotryp-
sin o f the three strands encompassing residues 40–82, in
spite o f the relative abundance of residues sensitive to this
protease. Most of these hydrophobic residues are pointing
inwards in the native protein structure, in which they line the
hydrophobic cavity t hat characterizes all lipocalins. The
insensitivity to chymotryptic attack suggests that some
degree of folding is retained i n this r egion, although the
separation condition we used prevented the possibility of
producing d irect evidence for r etention of structural organ-
ization in CP1.
Our observation on the resistance of this region to

physical denaturants is consistent with recent independent
observations on the different stability of secondary structure
elements with respect to physical denaturation in peculiar
regions of the BLG s tructure [10,32]. The r egion at the
dimer interface is the most sensitive to heat or pressure, a s
demonstrated by spectroscopic a nd chemical modification
studies [6–8,32], and it could be modified without se nsible
modification in the re mainder of the structure [36]. On the
other hand, treatment in subdenaturing conditions has
revealed transient formation of a n umber o f unfolding
intermediates that retain a n Ôopen barrelÕ conformation [10].
In this frame, it should be noted that the protease-resistant
region of the barrel that constitutes most o f our largest
fragments is located at the opposite site of the molecule with
respect to the dimerization in terface (Fig. 9).
One o f t he few hydrophobic r esidues that are not
pointing inwards in the region of native BLG encompas-
sing residues 39–70 is Leu57, which is attacked by both
proteases, but only after release of the primary proteolysis
products, CP1 and TP1, from the remainder of the
protein structure. F urther hydrolysis in this region is
however, slow enough to allow s ignificant accumulation
of the hydrolysis intermediate even in c onditions where no
intact BLG is left.
Once the intact protein is removed f rom the system,
there is n o residual i mmunochemical reactivity of t he
hydrolysis products against monoclonal antibodies or
rabbit antisera. Only fragment CP1 retains some faint
reactivity towards one of the monoclonals u sed in this
study. We also obtained preliminary evidence that none of

the large-sized proteolysis products of BLG discussed
above was recognized by sera from allergic pediatric
patients in Western b lot experiments [37]. These findings
may be of p ractical re levance, in what hydrolysis at
temperatures that fac ilitate access to o therwise buried
structural regions of the protein may allow production of
hydrolysates with improved qualities with respect to those
presently on t he m arket as for their sensory, nutr itional
and technological properties.
ACKNOWLEDGEMENTS
Work supported by g rants from the Ministry of University and
Scientific Research (MURST-FIRST, Rome, Italy, to S . I .) and from
the Ministry of Policies for Agriculture and Forestry (MiPAF, Rome,
Italy, to F. B.). This is publication no. 1 of the Project ÔTOLLELATÕ.
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