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-

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

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

)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

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

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

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

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

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

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.

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

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

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].

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

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.

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

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