Eur J Biochem 269, 1362–1372 (2002) Ó FEBS 2002 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 Iametti1, Patrizia Rasmussen1,2, Hanne Frøkiær2, Pasquale Ferranti3, Francesco Addeo3 and Francesco Bonomi1 Dipartimento di Scienze Molecolari Agroalimentari, University of Milan, Italy; 2Biocentrum, Technical University of Denmark, Lyngby, Denmark; 3Istituto di Scienze dell’Alimentazione, CNR, Avellino, Italy Bovine b-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 °C at neutral pH At these temperatures b-lactoglobulin undergoes significant but reversible structural changes In the conditions 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 Hightemperature 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 The globular protein b-lactoglobulin is found in the whey fraction of the milk of many mammals, but is absent from human milk In spite of numerous physical and 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 concentration, and on the interaction with other components when complex systems such as milk and whey are considered Most of the steps occurring below a given intensity threshold of physical treatment (temperatures below 60–65 °C, or pressures Correspondence to Francesco Bonomi, Dipartimento di Scienze Molecolari Agroalimentari, Via G Celoria 20133 Milano, Italy Fax: + 39 02 58356801, Tel.: + 39 02 58356819, E-mail: francesco.bonomi@unimi.it Abbreviations: BLG, b-lactoglobulin; BAPA, benzoyl-L-argininep-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 28 August 2001, revised December 2001, accepted January 2002) regions of b-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein The immunochemical properties of the products of b-lactoglobulin hydrolysis were assessed by using various b-lactoglobulin-specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases Keywords: bovine b-lactoglobulin; limited proteolysis; partial unfolding; thermal treatment; reduced immunoreactivity below 600 MPa [6,7]) are fully reversible in solutions of the pure protein at neutral pH Transient BLG conformers are formed by either physical treatment in the same conditions, and the properties of these 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 conformational 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 structural 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 wellknown 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 modification 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 High-temperature heat denaturation is most commonly used in the processes for producing extensively hydrolyzed formulae starting from whey proteins, because denaturation by itself leads to the removal of conformational 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 Ó FEBS 2002 Proteolysis of heat-unfolded b-lactoglobulin (Eur J Biochem 269) 1363 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 preparations could stem from the inability of the hydrolytic reaction to address all the sequential epitopes even in the denatured protein In fact, despite the apparent simplicity of the approaches described above, heat denaturation 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 allergenic properties The BLG conformers transiently formed during a physical treatment of subdenaturing intensity may represent ideal substrates for the action of proteases, as ample regions of the hydrophobic protein 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 parts of the protein accessible, at least in principle, to enzymatic hydrolysis In more advanced steps of physical denaturation, collapse of the hydrophobic portion of the structure may occur [6], possibly making the same enzyme attack sites once again as they were inaccessible in the native folded protein In previous studies on limited proteolysis of partially unfolded BLG, we used high-pressure as the physical denaturant, as the intensity threshold of pressure treatment appears less critical than temperature with respect to the aggregation behavior of BLG and of the sensitivity of the aggregation process to protein concentration [10,27] In those studies, several enzymes were tested Trypsin and chymotrypsin gave the best results, both in terms of interpretation of the hydrolysis pattern and of reduced immunoreactivity [28] Trypsin and chymotrypsin were used in the present study, also in view of a possible comparison with the results obtained under pressure In this work we used short time periods (10 min) for the combined proteolytic/thermal treatment of BLG at relatively high enzyme/ BLG ratios (1 : 10 and : 20) and at the highest temperature compatible with retention of enzyme activity and with the reversibility of structural modifications of BLG Limited proteolysis studies on BLG are significant not only to understanding its unfolding mechanism, but may have practical relevance as for decreasing the immunoreactivity of the protein Therefore, we also tested some of the hydrolysis products obtained in this study for their immunoreactivity 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 disulfidelinked dimers, by using HPLC 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-lysylchloromethane treated, type VII) and the synthetic substrates benzoyl-L-arginine-p-nitroanilide (BAPA, trypsin) or N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SUNA, chymotrypsin), also were from Sigma BLG-specific monoclonal antibodies were prepared according to [29] Proteolytic experiments during thermal treatment Thermal treatments at fixed temperature were carried out as reported in [6] with minor modifications Proper aliquots of concentrated solutions of the appropriate proteolytic enzyme were added at °C to mL of BLG (2.5 mgỈmL)1 in 50 mM phosphate buffer, pH 6.8) to a final mass ratio enzyme/BLG of : 20 or of : 10 The protein/protease mixture was then placed in a thermostatted water bath 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 was stopped by lowering the pH of the reaction mixture to by addition of 0.2 mL of 50% (v/v) acetic acid in water All these manipulations were carried out within 1–2 from the end of the thermal treatment Analytical measurements Enzyme activities were determined at 37 °C in 0.1 M Tris/ HCl, pH 8.1, by following the increase in absorbance at 405 nm due to p-nitroanilide released from BAPA or SUNA, as appropriate Although the supplier gives nominal specific activities on synthetic substrates of 10.000 lmolỈmin)1Ỉmg)1 (trypsin, on benzoyl-arginine ethyl ester), and 50 lmolỈmin)1Ỉmg)1 (chymotrypsin, on benzoyl-tyrosine ethyl ester), we measured specific activities in the range of lmolỈmin)1Ỉmg)1 (trypsin, 0.5 mM BAPA) and 50 lmolỈmin)1Ỉmg)1 (chymotrypsin, 0.2 mM SUNA) Residual enzyme activity was measured after heat treatment of each enzyme in the same conditions and concentrations used for proteolysis experiments, in the presence or in the absence of BLG RP-HPLC analysis of the proteolyzed samples was performed directly on aliquots of the acidified material after 10-fold dilution with 0.1% trifluoroacetic acid and centrifugation for at 10 000 g to remove minor amounts of materials made insoluble by the addition of trifluoroacetic acid A Deltapak C18 column (3.9 · 150 mm, Waters), fitted to a Waters 625 HPLC equipped with a Waters 490E dual wavelength detector was used Elution of the hydrolytic products and of the residual intact protein was performed with a linear gradient from 20 to 60% acetonitrile (in 0.1% trifluoroacetic acid) in 30 Flow was 0.8 mLỈmin)1, detection was at 220 nm Residual intact BLG was quantitated by on-line integration, using native BLG as a standard Size-exclusion HPLC separations of the proteolyzed samples was performed directly on aliquots of the acidified material after centrifugation for at 10 000 g to remove insoluble materials 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 mLỈmin)1 Detection was at 220 and 280 nm Electrospray mass spectrometry (ES/MS) analysis was performed using a Platform single-quadrupole mass spectrometer (Micromass), after liophylization of the original materials Peptide samples (10 lL, 50 pmol protein in Ó FEBS 2002 1364 S Iametti et al (Eur J Biochem 269) water) were injected into the ion source at a flow rate of 10 lLỈmin)1; the spectra were scanned from 1400 to 600 at 10 s per scan Mass scale calibration was carried out using the multiple-charged ions of a separate introduction of myoglobin Mass values are reported 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 by analysis of the spectral data using a computer software developed by the instrument manufactureer (Biolynx, Micromass), and confirmed by MS analysis of the samples prior and after a reduction step in 10 mM dithiothreitol for h at 37 °C Immunochemistry ELISA tests were performed as competitive capture ELISA by using BLG specific monoclonal or polyclonal antibodies as capture antibodies All steps were carried out at 30 °C Polyclonal rabbit anti-BLG Ig was diluted 4000–10000 times in carbonate buffer (15 mM Na2CO3; 35 mM NaHCO3, pH 9.6) and coated directly to the wells of microtiter plates, at 0.1 mL per well When using monoclonal antibodies, polyclonal antimouse antibodies (Z109, : 1000, 0.1 mL per well, DAKO) were used for coating followed by incubation of 0.1 mL per well of monoclonal antibody diluted to approximately 250 ngỈmL)1 in KCl/NaCl/Pi buffer containing 0.1% Triton X-100 (KCl/NaCl/Pi/Triton; 1.5 mM KH2PO4; 6.5 mM Na2HPO4; 0.5 M NaCl; 2,7 mM KCl; mLỈL)1 Triton X-100) Plates were washed four times with KCl/ NaCl/Pi/Triton buffer diluted : 10, and the various samples in KCl/NaCl/Pi/Triton were applied to the wells in serial twofold dilutions and incubated for h After four washes, the plates were incubated with a fixed concentration of biotinylated BLG (10–200 ngỈmL)1, depending on the antibody) After incubation and another four washes, plates were incubated with horseradish peroxidase-labeled streptavidin (HRP-streptavidin, DAKO, diluted : 5000 in KCl/NaCl/Pi/Triton), and washed four times Bound HRP activity was measured by using a substrate-containing buffer (0.2 M potassium citrate pH 5.0; mM H2O2; 0.6 mM 3,3¢,5,5¢-tetramethylbenzidine) The reaction was terminated by addition of M H3PO4, 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 the following reasons: (a) neither enzyme is capable of attacking BLG significantly at room temperature [14,15,18]; (b) both enzymes are available at very high purity; (c) both enzymes are highly specific; and (d) they act on complementary sets 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 trypsin and chymotrypsin on transiently temperature-unfolded BLG could be compared with those we obtained on transiently pressure-unfolded BLG [28] The only major drawback in the use of trypsin and chymotrypsin in the experiments reported here was their limited thermostability As shown in Table 1, both enzymes had very little residual activity after at 65 °C, also in the presence of a 20-fold mass excess of the substrate protein Contrarily to what expected for a generic protective effect of added proteins, the residual activity after 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 substrates for binding to the enzymes In our conditions, the assay mixtures for residual protease activity contained from 0.125 to 0.25 mgỈmL)1 BLG (or BLG hydrolysis products), equivalent to 0.075 and 0.15 mM BLG, respectively To test this possibility in a simple way, we performed assays in which native BLG was added 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 mM BAPA when native BLG was added at 0.075 mM and 0.15 mM, respectively Under similar conditions, inhibition figures for chymotrypsin (0.2 mM SUNA as substrate) were and 15%, respectively These figures not fully account for the differences shown in Table 1, that apparently are better explained by assuming an inhibitory effect of the peptides produced by hydrolysis of BLG at high temperature Table Thermal stability of trypsin and chymotrypsin Proteins (0.125 mgỈmL)1 in 50 mM phosphate buffer, pH 6.8) were heated for at the given temperatures in the absence or in the presence of BLG (2.5 mgỈmL)1 in 50 mM phosphate buffer, pH 6.8, corresponding to a : 20 mass ratio enzyme/BLG) Residual enzyme activity after heat treatment was measured spectrophotometrically at 37 °C with 0.02–0.05 mL enzyme in mL of the synthetic substrates BAPA (trypsin), or SUNA (chymotrypsin) Substrates (0.5 mM BAPA and 0.2 mM SUNA) were in 100 mM Tris/HCl, pH 8.1 Activity is given as percentage of that of control enzymes kept at 37 °C in the absence of BLG Residual activity (%) Treatment temp (°C) 55 60 65 BLG present BLG absent Chymotrypsin Trypsin Chymotrypsin Trypsin 43.0 17.0 6.6 77.9 2.3 0.4 28.0 11.0 5.3 9.3 0.5 0.2 Ó FEBS 2002 Proteolysis of heat-unfolded b-lactoglobulin (Eur J Biochem 269) 1365 Fig 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 added at °C to separate aliquots of a BLG solution (1 mL, 2.5 mgỈmL)1 in 50 mM phosphate buffer, pH 6.8) to a final mass ratio enzyme/BLG of : 20 or of : 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 mixtures were placed on 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 the acidified material after 10-fold dilution with 0.1% trifluoroacetic acid in 20% acetonitrile and centrifugation for at 1100 g to remove some trifluoroacetic acid- insoluble material A Deltapak C18 column (3.9 · 150 mm, Waters), fitted to a Waters 625 HPLC equipped with a Waters 490E detector was used Elution was performed by applying a linear gradient from 20 to 60% acetonitrile (v/v) in 0.1% trifluoroacetic acid in 30 Flow was 0.8 mLỈmin)1, detection was at 220 nm 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 protein was degraded by either enzyme in 20 at 37 °C at an enzyme/ BLG mass ratio of : 10 Essentially the same results (i.e less than 10% degradation of the total protein) were obtained under the same conditions with BLG that was previously heated for 10 at 65 °C This confirms earlier reports on the remarkable resistance of BLG to most proteases [13,17], and the reversibility of heat-induced structural modifications in these conditions 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 at temperatures between 55 and 65 °C As summarized in Table 2, the amount of residual BLG decreased with time and with the amount of added Table Residual intact BLG after proteolysis under different conditions The amount of residual intact BLG after proteolysis in the given conditions was determined by integration of the intact BLG peaks from RP-HPLC separations similar to those reported in Fig 1, and is given as percentage of the signal produced by the native protein as a standard Temperature (°C) Enzyme Mass ratio enzyme/BLG Hydrolysis time (min) 55 60 65 Trypsin : 10 10 20 10 20 10 20 10 20 41 17 42 22 16 17 22 17 11 20 10 40 24 18 30 19 17 32 31 30 40 29 41 30 27 52 42 31 49 48 41 : 20 Chymotrypsin : 10 : 20 1366 S Iametti et al (Eur J Biochem 269) enzyme As for the effects of temperature, inactivation of proteases (Table 1) came into play at the highest temperatures and at the lowest ratio enzyme/BLG Besides the decrease in intact protein, the tracings in Fig show that some hydrolysis products were formed during the early phases of hydrolysis, and their concentration 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 it is evident that chymotrypsin gave an extent of proteolysis not very different from that of trypsin on transiently heat-unfolded BLG This is somewhat puzzling, as both enzymes 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 catalytic activity on synthetic substrates at temperatures above 50 °C for either enzyme (not shown) Both enzymes rather underwent a marked decrease in activity above 50 °C, chymotrypsin being much less thermostable than trypsin, as reported in the equilibrium data shown in Table Thus, our observations (and the specific activity data on different synthetic substrates, as reported in Materials and methods) confirm that the 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 the thermal treatment, hydrolysis of BLG was extensive, in spite of significant inactivation of the enzymes This indicates that the transient conformers originating in the course of thermal treatment had exposed novel access sites for either enzyme After thermal treatment in these conditions, there were no major irreversible changes in all structural levels of BLG, at Ó FEBS 2002 least as detectable by spectroscopic and separation techniques [6,8,9,20,27] The increased accessibility 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 even in the absence of spectroscopically detectable irreversible structural modifications 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 sites [7–9,31] Physically induced reversible dimer dissociation at 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 been provided that heat treatment in this temperature range may affect a heat-labile domain of the protein [32], that was hypothesized to be relevant also for the stabilization of associated forms of BLG in solution [20] In this context, it seems significant that chymotrypsin (specific for aromatic residues) gave the same hydrolysis levels obtained with trypsin, in spite of its lower thermal stability This could confirm that buried, compact hydrophobic regions may be transiently unfolded and exposed by the thermal treatment The RP-HPLC tracings in Fig indicate that the major peaks in the hydrolysis products obtained with each protease (added in a : 10 ratio to BLG) after 10 at various temperatures from 37 to 60 °C had similar elution times, suggesting that the same sites of attack were accessible to either protease in this temperature range Some differences among the RP-HPLC tracings 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 of some cleavage sites in the BLG conformer that is predominant at this temperature, or rather to the fact that the rapid Fig RP-HPLC analysis of the products obtained upon enzymatic digestion of BLG for 10 at various temperatures Proper volumes of concentrated solutions of each given enzyme were added at °C to separate aliquots of a BLG solution (1 mL, 2.5 mgỈmL)1 in 50 mM phosphate buffer, pH 6.8) to a final mass ratio enzyme/BLG of : 10 Individual BLG/protease mixtures were placed in a water bath thermostatted at the given temperature for 10 Further sample processing and RP-HPLC separation were performed as detailed in the legend to Fig Ó FEBS 2002 Proteolysis of heat-unfolded b-lactoglobulin (Eur J Biochem 269) 1367 thermal inactivation of both enzymes at this temperature prevented further degradation of some hydrolysis intermediates formed in the earliest 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 reported 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 and 10 kDa) The largest hydrolysis fragments separated by SE-HPLC were named after the enzyme used (T, trypsin; C, chymotrypsin) and after their elution order from a Superdex Peptide column (hence, the P in their names), and correspond to the peaks labeled CP1 and CP2 (or TP1 and TP2) in the chromatograms presented in Fig Analysis of the proteolyzed samples by SDS/PAGE (data not shown) was consistent with the figures reported in Table The extensive proteolysis observed with chymotrypsin resulted in the formation of appreciable amounts of proteolytic products capable of being retained by the gel, according to the SE-HPLC data shown in Fig Confirm- Fig SEC-HPLC analysis of the products obtained upon enzymatic digestion of BLG at 60 °C Size-exclusion chromatography (SEC) was carried out on the acetic acid-treated materials obtained as detailed in the legend to Fig 1, with no further processing A Superdex Peptide column (10/30, Pharmacia Biotech) was used on the same chromatographic system described in the legend to Fig Eluant was 20% acetonitrile in aqueous 0.1% trifluoroacetic acid Flow was 0.5 mLỈmin)1, detection was at 220 nm 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 enzymetreated samples, indicating that proteolysis took place more rapidly than protein aggregation even at 65 °C [27,28] The time-progressive change in the size distribution of hydrolytic products obtained after treatment with either enzyme at different temperatures is reported in Fig Both the SE-HPLC tracings in Fig (obtained with 0.1% trifluoroacetic acid in 20% acetonitrile as eluant) and the time courses in Fig clearly indicate that a limited number of large fragments constituted a set of intermediate hydrolysis products, suggesting a progressive hydrolysis mechanism with either enzyme More specifically, it appears that the concentration of TP1 remained constant during progressive hydrolysis of heated BLG by trypsin, whereas the concentration of the intact protein decreased with an accompanying increase in TP2 The pattern of events observed with chymotrypsin is made somewhat more complicated by the more extensive thermal inactivation of this enzyme However, 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 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 the chromatograms shown in Fig Full symbols, 55 °C; open symbols, 65 °C Excluded peptides (Mr app > 10 000), circles and full lines, TP1 and TP2 (or CP1 and CP2, as appropriate), triangles and dotted lines; low molecular weight material (Mr app < 3000), squares and dashed lines Ó FEBS 2002 1368 S Iametti et al (Eur J Biochem 269) Table Molecular parameters for large-sized fragments obtained from hydrolysis of BLG Fragments were obtained by size-exclusion HPLC of digests carried out as in the legend to Fig after hydrolysis at 55 °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 ion source at a flow rate of 10 lLỈmin)1; the spectra were scanned from 1400 to 600 at 10 s per scan Mass scale calibration 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 the respective mass signal intensity Mass (Da) Fragment Sequence Individual peptides Total fragment CP1 Arg40-Phe82(Cys66-Cys160)His146-Ile162 Val43-Phe82(Cys66-Cys160)Lys150-Ile162 Arg40-Phe82(Cys66-Cys160)Lys150-Ile162 Val43-Phe82(Cys66-Cys160)His146-Ile162 Leu58-Phe82(Cys66-Cys160)Lys150-Ile162 Glu62-Phe82(Cys66-Cys160)Lys150-Ile162 Lys60-Phe82(Cys66-Cys160)Lys150-Ile162 Val41-Lys70(Cys66-Cys160)Leu149-Ile162 Val41-Lys69(Cys66-Cys160)Leu149-Ile162 Leu58-Lys70(Cys66-Cys160)Leu149-Ile162 Trp61-Lys69(Cys66-Cys160)Leu149-Ile162 Trp61-Lys70(Cys66-Cys160)Leu149-Ile162 Leu58-Lys69(Cys66-Cys160)Leu149-Ile162 5015.2 4596.7 5015.2 4596.7 2931.7 2376.0 2690.4 3546.2 3418.0 1619.9 1122.2 1250.1 1491.7 6926.9 6204.6 6623.1 6508.4 4539.6 3983.9 4298.3 5267.3 5239.1 3341.0 2843.3 2971.5 3212.8 CP2 TP1 TP2 temperature, short reaction times and high enzyme concentration) Indeed, no formation of CP2 was detected during chymotryptic hydrolysis of BLG at 65 °C, and significant accumulation of TP2 only occurred at the longest hydrolysis times at this temperature (data not shown) The figures given in Fig for peptides with a Mr app > 10 000 are significantly higher than the amounts of residual native protein detected by RP-HPLC (Table 2) This discrepancy could indicate that formation of proteolytic 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, fragments CP and 2, as well as fragments TP and 2, were further purified by RP-HPLC and SE-HPLC in the same conditions and with the same equipment used for analysis of the whole proteolyzate, and these chromatographically homogeneous peptides were analyzed by ES-MS The results 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, namely the 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 given in Fig 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 spite of the relative abundance of protease-sensitive residues in their sequence (Fig 5), + + + + + + + + + + + + + 1911.7 1607.9 1607.9 1911.7 1607.9 1607.9 1607.9 1721.1 1721.1 1721.1 1721.1 1721.1 1721.1 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 Fig Position of proteolytic fragments CP1/2 and TP1/2 within the primary structure of BLG Residues susceptible to chymotrypsin and trypsin hydrolysis are labeled with ÔcÕ and ÔtÕ superscripts, respectively Cysteines 66 and 160 are underlined Ó FEBS 2002 Proteolysis of heat-unfolded b-lactoglobulin (Eur J Biochem 269) 1369 Fig ELISA assay of unresolved BLG hydrolysates Hydrolysates were obtained after 20 treatment at 55 °C with trypsin (triangles) or chymotrypsin (squares) at an : 10 enzyme/BLG ratio Native BLG, circles A rabbit anti-BLG Ig was used Immunoreactivity of the products of BLG hydrolysis Fig Position of the proteolytic fragments obtained at high temperature within the 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 disulfideforming Cys66 and Cys160 are in yellow ball and stick Structures were generated by using RASMOL [38], and coordinates in file 1B8E deposited in the RCSB Protein Databank [34] on completely different methodological approaches have shown the existence of compact structural regions in BLG, that are not affected by physical treatments [10,32] Figure presents some schematics of the structural relationship of CP1/CP2 and TP1/TP2 with the remainder of the BLG structure It is evident that all 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 bridge to Cys66 [5] As stated in the introduction, one of the goals of this work was to take advantage of the interplay of treatment conditions and enzyme action to produce fragments of sizable mass, but unable to be recognized by specific antibodies by standard immunochemical techniques 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 in conditions of maximum BLG proteolysis (namely, 55 °C, 20 min, : 10 weight ratio of enzyme/BLG) are shown as an example in Fig The decrease in immunoreactivity in the unresolved BLG hydrolysates obtained with either chymotripsin or trypsin at the various temperatures and treatment conditions parallels the decrease in intact BLG (Table 2) This also suggests that nonproteolyzed BLG retained its immunoreactivity 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 CP1/CP2 and TP1/TP2 was found to be immunoreactive against rabbit anti-BLG Ig even at very high fragment concentration (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 the ELISA curves obtained with different antibodies are reported in Fig While most of the monoclonal antibodies did not recognize any of the large proteolytic fragments, as exemplified by monoclonal 5G6 in Fig 8, monoclonals 9G10 and 1E3 recognized CP1 (although at  100-fold the concentration of native BLG), but neither CP2 nor TP1 and TP2 As the only relevant 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 be assessed whether Arg40 is a 1370 S Iametti et al (Eur J Biochem 269) Ó FEBS 2002 Fig Relevance of protease-sensitive residues to the dimeric structure of BLG Fragments CP1 and TP1 are as colored ribbons The orange regions correspond to the shorter TP1 fragments within the CP1 fragments (orange and brown) Residues attacked by proteases are identified by their position in the sequence, and are given as sticks (blue, basic; green, hydrophobic) The disulfide-forming Cys66 and Cys160 are in yellow Structures were generated by using RASMOL [38], and coordinates in file 1BEB deposited in the RCSB Protein Databank [5] Fig ELISA assay of proteolytic fragments of BLG with various monoclonal antibodies Fragments were purified as reported in the text and in Fig 3, and are identified as in Fig Native BLG, squares CP1, full circles; CP2, open circles; TP1, full triangles; TP2, open triangles relevant component of a sequential epitope, or is rather important for proper 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 already available at 37 °C, where little if 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 spite 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 analysis of the data presented 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 of 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 together contribute to make this region virtually inaccessible at neutral pH Both the 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 polypeptide backbone to the action of trypsin The nature of the interactions in this region, as pointed out above, also explains the reversibility of temperature-induced dissociation 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 the bovine BLG dimer represents the primary event for facilitated proteolysis at high temperature The relevance of dimer dissociation to facilitated proteolysis is also evident when considering the 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 from 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 chains, 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 in the structure Ó FEBS 2002 Proteolysis of heat-unfolded b-lactoglobulin (Eur J Biochem 269) 1371 (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 dimer interface The disulfide-connected peptides that originate from early proteolysis steps have some interesting features Perhaps the most striking regards resistance to chymotrypsin of the three strands encompassing residues 40–82, in spite of 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 that characterizes all lipocalins The insensitivity to chymotryptic attack suggests that some degree of folding is retained in this region, although the separation condition we used prevented the possibility of producing direct evidence for retention of structural organization 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 structure [10,32] The region at the dimer interface is the most sensitive to heat or pressure, as demonstrated by spectroscopic and chemical modification studies [6–8,32], and it could be modified without sensible modification in the remainder of the structure [36] On the other hand, treatment in subdenaturing conditions has revealed transient formation of a number of unfolding intermediates that retain an Ôopen barrelÕ conformation [10] In this frame, it should be noted that the protease-resistant region of the barrel that constitutes most of our largest fragments is located at the opposite site of the molecule with respect to the dimerization interface (Fig 9) One of the few hydrophobic residues that are not pointing inwards in the region of native BLG encompassing 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 Further hydrolysis in this region is however, slow enough to allow significant accumulation of the hydrolysis intermediate even in conditions where no intact BLG is left Once the intact protein is removed from the system, there is no residual immunochemical reactivity of the hydrolysis products against monoclonal antibodies or rabbit antisera Only fragment CP1 retains some faint reactivity towards one of the monoclonals used 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 blot experiments [37] These findings may be of practical relevance, in what hydrolysis at temperatures that facilitate access to otherwise buried structural regions of the protein may allow production of hydrolysates with improved qualities with respect to those presently on the market as for their sensory, nutritional and technological properties ACKNOWLEDGEMENTS Work supported by grants 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.) 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transition Eur J Biochem 268, 477–483 Molinari, H., Ragona, L., Varani, L., Musco, G., Consonni, R., Zetta, L & Monaco, H (1996) Partially folded structure of monomeric bovine beta-lactoglobulin FEBS Lett 381, 237–243 Uhrinova, S., Uhrin, D., Denton, H., Smith, M., Sawyer, L & Barlow, P.N (1998) Complete assignment of 1H, 13C and 15N chemical shifts for bovine beta-lactoglobulin: secondary structure and topology of the native state is retained in a partially unfolded form J Biomol NMR 12, 89–107 Bonomi, F., Iametti, S., Restani, P., Gaiaschi, A., Ferranti, P & Addeo, F (2001) Limited proteolysis of transient conformers in heated beta-lactoglobulin yields large, non-immunoreactive peptides Abstract no 233 Communications of the 4th European Symposium of The Protein Society Protein Sci 10, Suppl Sayle, R.A & Milner-White, E.J (1995) RASMOL: Biomolecular graphics for all Trends Biochem Sci 20, 374–376  ... gave the best results, both in terms of interpretation of the hydrolysis pattern and of reduced immunoreactivity [28] Trypsin and chymotrypsin were used in the present study, also in view of a... namely the 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 given in Fig A nonspecific hydrolysis... Enzymatic hydrolysis of whey proteins In? ??uence of heat treatment of alpha-lactalbumin and beta-lactoglobulin on their proteolysis by pepsin and papain Neth Milk Dairy J 47, 15–22 18 Mullally,