Open Collections

UBC Faculty Research and Publications

Do whey protein-derived peptides have dual dipeptidyl-peptidase IV and angiotensin I-converting enzyme… Lacroix, Isabelle Marie Estelle; Meng, Guangtao; Cheung, Imelda Wing Yan; Li-Chan, Eunice Dec 11, 2015

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
52383-Lacroix_I_et_al_Do_whey_protein_derived.pdf [ 1.72MB ]
Metadata
JSON: 52383-1.0343557.json
JSON-LD: 52383-1.0343557-ld.json
RDF/XML (Pretty): 52383-1.0343557-rdf.xml
RDF/JSON: 52383-1.0343557-rdf.json
Turtle: 52383-1.0343557-turtle.txt
N-Triples: 52383-1.0343557-rdf-ntriples.txt
Original Record: 52383-1.0343557-source.json
Full Text
52383-1.0343557-fulltext.txt
Citation
52383-1.0343557.ris

Full Text

1  Do whey protein-derived peptides have dual dipeptidyl-peptidase IV and angiotensin I-converting enzyme inhibitory activities?     Isabelle M.E. Lacroix, Guangtao Meng, Imelda W.Y. Cheung and  Eunice C.Y. Li-Chan*  The University of British Columbia Faculty of Land & Food Systems Food Nutrition & Health Program 2205 East Mall, Vancouver, BC, Canada. V6T 1Z4.      * Corresponding author: Eunice C.Y. Li-Chan, The University of British Columbia, Faculty of Land & Food Systems, Food Nutrition & Health Program, 2205 East Mall, Vancouver, BC, Canada, V6T 1Z4. E-mail: Eunice.Li-Chan@ubc.ca, Tel: 1-604-822-6182, Fax: 1-604-822-5143.   2  Do whey protein-derived peptides have dual dipeptidyl-peptidase IV and angiotensin I-converting enzyme inhibitory activities?  Abstract  Inhibition of dipeptidyl-peptidase IV (DPP-IV) and angiotensin I-converting enzyme (ACE) are useful strategies for managing, respectively, diabetes and hypertension, two conditions often occurring together. In this study, debittered and non-debittered whey protein hydrolysates (WPHs) were assessed for their in vitro inhibitory activity against ACE and DPP-IV and characterized for their constituent peptides. All WPHs and several fractions obtained from them had ACE and DPP-IV inhibitory activities, with ACE being generally more strongly inhibited than DPP-IV. Among the identified peptides tested, GYGGVSLPEW derived from -lactalbumin and LKPTPEGDLE from -lactoglobulin were, respectively, the most effective at inhibiting ACE (IC50 = 2 µM) and DPP-IV (IC50 = 42 µM). Although some identified peptides were able to inhibit both enzymes, the majority did not show a dual inhibitory effect. This research provides new insight on the active peptides responsible for the ACE and DPP-IV inhibitory activities of whey protein hydrolysates.   Key words: Angiotensin I-converting enzyme; dipeptidyl-peptidase IV; whey protein hydrolysates; bioactive peptides; dual inhibitors   3  1. Introduction  Diabetes and high blood pressure are among the leading risk factors for atherosclerosis and its complications, including strokes and heart attacks. The two conditions are believed to share common pathways and are frequently observed together in the same patients (Campbell et al., 2011; Cheung & Li, 2012). In the American population, hypertension occurs in 50 to 80% of individuals with type 2 diabetes (Landsberg & Molitch, 2004). Moreover, in a prospective cohort study by Gress, Nieto, Shahar, Wofford, and Brancati (2000), subjects with hypertension have been found to be 2.5 times more likely to develop type 2 diabetes than individuals with normal blood pressure.  Over the last two decades, protein hydrolysates and protein-derived peptides have been studied for their potential to help manage or complement pharmacotherapy in the treatment of chronic diseases (Li-Chan, 2015; Mine, Li-Chan, & Jian, 2010).  Dairy proteins are often considered one of the most important precursors of biologically active peptides, a number of proteins from both the whey and casein fractions of milk having been reported to contain within their sequences peptides with an array of activities (Kamau et al., 2010; Nagpal et al., 2011).   Currently, most of the research in the area of biologically active peptides from milk proteins has focused on peptides with inhibitory activity against the angiotensin I-converting enzyme (ACE) (Ricci, Artacho, & Olalla, 2010), a dipeptidyl 4  carboxypeptidase that promotes sodium retention and vasoconstriction (Brown & Vaughan, 1998). A number of milk protein hydrolysates produced using various enzymatic treatments as well as whey- and casein-derived peptides have been reported in the literature to inhibit ACE activity in vitro and exhibit antihypertensive activity in vivo (FitzGerald, Murray, & Walsh, 2004; Ricci, Artacho, & Olalla, 2010; Wang et al., 2012). ACE-inhibiting protein hydrolysates and protein-derived peptides, however, often present a bitter taste that may limit their application as functional food ingredients (Cheung & Li-Chan, 2010). Recently, our research group reported that the treatment of a bitter whey protein hydrolysate (WPH) with aminopeptidase or carboxypeptidase produced hydrolysates with reduced bitterness, while still exhibiting ACE inhibitory activity and ability to decrease systolic blood pressure in spontaneously hypertensive rats (Cheung, Aluko, Cliff, & Li-Chan, 2015). Although these findings suggested that exopeptidase treatment may be an effective approach to generate ACE-inhibitory hydrolysates with acceptable taste, the specific peptides responsible for the observed inhibition of ACE activity were not identified.    In addition to containing within their sequences peptides with ACE inhibitory activity, dairy proteins, particularly those found in whey, have recently been shown to be precursors of fragments able to inhibit the enzyme dipeptidyl-peptidase IV (DPP-IV) (Konrad et al., 2014; Lacroix & Li-Chan, 2012a; Lacroix & Li-Chan, 2013; Lacroix & Li-Chan, 2014; Nongonierma & FitzGerald, 2013; Silveira, Martínez-Maqueda, Recio, & Hernández-Ledesma, 2013; Uchida, Ohshiba, & Mogami, 2011; 5  Uenishi, Kabuki, Seto, Serizawa, & Nakajima, 2012). The inhibition of DPP-IV, an ubiquitous enzyme involved in the inactivation of the incretin hormones glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1, is considered an effective approach to manage hyperglycemia in type 2 diabetes (Filippatos, Athyros, & Elisaf, 2014). The finding that proteins can be precursors of peptides with DPP-IV inhibitory activity in vitro has triggered a great interest in their potential to complement pharmacotherapy in the management of type 2 diabetes. However, although several protein hydrolysates have been shown to be able to inhibit DPP-IV activity, only a limited number of studies have actually isolated and identified DPP-IV inhibitory peptides from these hydrolysates (Lacroix & Li-Chan, 2014; Silveira, Martínez-Maqueda, Recio, & Hernández-Ledesma, 2013; Uenishi, Kabuki, Seto, Serizawa, & Nakajima, 2012).   Several specific milk protein-derived peptides have been reported to have more than one bioactivity. The -casein-derived hepta-peptide YPFPGPI, for example, has been reported to have opioid, immunomodulating and ACE inhibitory activities (Gobbetti, Vini, & Rizzello, 2004). On the other hand, literature on dairy protein-derived peptides displaying both ACE and DPP-IV inhibitory activities is sparse. To date, only a few short peptides found within the sequence of milk proteins, including the tryptophan-containing peptides WL, WY and LW (Nongonierma & FitzGerald, 2015), have been reported to have a dual inhibitory effect on the two enzymes.  These peptides, however, were not generated empirically by hydrolysis of milk proteins, but were rather chemically synthesized and tested for their effect on ACE 6  and DPP-IV activities. A recent study by Konrad et al. (2014) showed that hydrolysates of whey protein concentrate and -lactoglobulin produced by enzymatic treatment with a serine protease isolated from Asian pumpkin had an inhibitory effect on both ACE and DPP-IV. However, since the authors did not identify the hydrolysates’ constituent peptides, it is unknown whether the peptides responsible for the inhibition of ACE were also the ones causing the inhibition of the DPP-IV enzyme.    The objectives of the present study were to assess whether ACE-inhibiting WPHs debittered by exopeptidase treatment are also able to cause the inhibition of the DPP-IV enzyme in vitro and if so, to determine whether the same whey protein-derived peptides are responsible for the inhibition of both enzymes.   2. Materials and Methods  2.1 Materials  Whey protein isolate (WPI 895, Fonterra, New Zealand; 92.4% protein) donated by Caldic (Delta, BC, Canada) was used to produce the whey protein hydrolysates (WPHs) investigated in this study. The enzymes Thermoase PC10F, Peptidase R, and ProteAX were donated by Amano Enzyme U.S.A, LTD. (Elgin, IL, USA), while Accelerzyme® CGP was donated by DSM Food Specialties B.V (Delft, The 7  Netherlands). The commercially available whey protein hydrolysate HilmarTM 8390, referred to as “H” in this study, was donated by Hilmar Ingredients (Hilmar, CA, USA). Dipeptidyl-peptidase IV (DPP-IV, EC 3.4.14.5, from porcine kidney, ≥10 units per mg protein), angiotensin I-converting  enzyme (ACE, EC 3.4.15.1, from rabbit lung, ≥2.0 units per mg protein), N-hippuryl-His-Leu hydrate (≥98% by HPLC), Ile-Pro-Ile (diprotin A), N-[(S)-3-mercapto-2-methylpropionyl]-L-proline (captopril), teprotide (Glp-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro, ≥95% by TLC) and HPLC peptide standard mixture were purchased from Sigma-Aldrich (Oakville, ON, Canada). Antifreeze protein type 1 was donated by A/F Protein Canada (St Johns, NF, Canada). Gly-Pro-p-nitroanilide (H-Gly-Pro-p-NAHCl) was from Bachem Americas (Torrance, CA, USA). Synthesized peptides (≥ 95% purity) were prepared and purified by GL Biochem (Shanghai) Ltd (Shanghai, China). All other chemicals used were of analytical grade.   2.2 Preparation of whey protein isolate hydrolysates  The hydrolysates (referred to as T, T-AC, T-PR and T-PX) were prepared in duplicate as described in Cheung, Aluko, Cliff, and Li-Chan (2015). Briefly, WPI (3% WPI, w/v, in deionized distilled (dd) H2O, at the unadjusted pH of 6.9) was hydrolyzed with Thermoase PC10F (3 g/100 g WPI) for 180 min at 65C. The mixture was then heated at 90C for 15 min to inactivate the enzyme and centrifuged (10 min at 13,200 x g) using a DuPont Sorvall Centrifuge RC 5B (Mandel Scientific Co. Ltd., 8  Guelph, ON, Canada). The supernatant was collected and freeze-dried (Labconco Corporation, Kansas City, MO, USA). This hydrolysate is referred to as “T”.   The hydrolysate produced after 180 min of hydrolysis with Thermoase PC10F (T) was further treated with one of three exopeptidase enzyme preparations. Briefly, a solution of T (10% hydrolysate, w/v, in ddH2O) was hydrolyzed for 7 h with Accelerzyme® CGP (“AC”; 37C, pH 4), Peptidase R (“PR”; 52C, pH 6) or ProteAX (“PX”; 60C, pH 6) at 4g/100g hydrolysate. The resulting exopeptidase-treated hydrolysates, respectively referred to as “T-AC”, “T-PR” and “T-PX”, were heated at 90C for 15 min to inactivate the enzymes and freeze-dried.  2.3 Isolation of the ACE and DPP-IV inhibitory peptides from the hydrolysates  2.3.1 Size-exclusion chromatography  The hydrolysates H, T, T-AC, T-PR and T-PX were fractionated by size-exclusion chromatography. The samples were dissolved in 20 mM Tris-HCl buffer pH 8.0 to a concentration of 20 mg/mL, 500 µL was loaded into a SuperdexTM Peptide 10/300 GL column (10 x 310 mm, 24 mL, GE Healthcare Uppsala, Sweden) connected to a fast protein liquid chromatography (FPLC) system (ÄKTAPurifier 100/10; GE Healthcare Life Science, Baie d’Urfé, QC, Canada), and eluted with Tris-HCl buffer (20 mM, pH 8.0) at a flow rate of 0.2 mL/min. The eluates were collected, pooled into fractions based on the peaks observed on the elution profiles monitored at 215 9  nm and 280 nm (similar elution profiles were observed at these two wavelengths), freeze-dried and assessed for their ACE and DPP-IV inhibitory activities. The separations were repeated multiple times to obtain enough samples for the subsequent purification and analysis. The antifreeze protein (MW 3240 Da) as well as the peptides DRVYIHPF (MW 1046.2 Da), YGGFM (MW 573.7 Da), YGGFL (MW 555.6 Da), VYV (MW 379.5 Da) and GY (MW 238.2 Da) from the HPLC peptide mixture standard were used to evaluate the molecular weight of the fractions.   2.3.2 Reversed-phase high performance liquid chromatography (RP-HPLC)  The peptide fraction (T-AC-4) obtained from the size-exclusion chromatography of T-AC that displayed the highest ACE and DPP-IV inhibitory activities was further fractionated by RP-HPLC as described in Lacroix and Li-Chan (2014). The collected fractions were assessed for their effect on ACE and DPP-IV activities.   2.4 Identification of peptide sequences by liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS)   The hydrolysates T and T-AC and the purified fractions displaying the highest inhibition of ACE and DPP-IV were analyzed at the Fred Hutchinson Cancer Research Center (Seattle, WA, USA) to identify their constituent peptides by LC–ESI-MS/MS.  10  Samples (0.5-2 µg) were analyzed using a Thermo Scientific Easy-nLC II nano HPLC system coupled to a hybrid Orbitrap Elite ETD mass spectrometer (Thermo Scientific, Waltham, MA, USA) using an instrument configuration as described in Yi, Lee, Aebersold, and Goodlett (2003). In the case of T, T-AC and T-AC-4, the samples were first desalted using C18 ZipTips (Millipore, Billerica, MA, USA). In-line desalting was performed using a reversed-phase trap column (100 μm × 20 mm) packed with Magic C18AQ (5-μm 200Å resin; Michrom Bioresources, Auburn, CA, USA) followed by peptide separations on a Magic C18AQ reversed-phase column (75 μm × 250 mm) directly mounted on the electrospray ion source. Chromatographic separation was realized using a linear gradient of acetonitrile (2-40% in 60 min) and 0.1% formic acid in H2O at a flow rate of 400 nL/min (300 nL/minute for direct inject runs). The heated capillary temperature was set to 300C and a spray voltage of 2500 V was applied to the electrospray tip.  The Orbitrap Elite instrument was operated in the data-dependent mode, switching automatically between MS survey scans in the Orbitrap (automatic gain control (AGC) target value 1,000,000, resolution 240,000, and injection time 250 ms) with MS/MS spectra acquisition in the linear ion-trap (AGC target value of 10,000 and injection time  of100 ms). The 20 most intense ions from the Fourier-transform full scan were selected for fragmentation in the linear ion trap by collision-induced dissociation with a normalized collision energy of 35%. Selected ions were dynamically excluded for 15 s with a list size of 500 and exclusion mass by a mass width of ± 10 ppm. Each sample was run at least twice, one run having +2 and +3 charge states selected for 11  MS/MS (m/z range of 300 to 1800) and the second run having +1, +2 and +3  charge states for MS/MS (m/z range of 300 to 5000).  Collected data were analyzed using Proteome Discoverer 1.4 (Thermo Scientific, San Jose, CA, USA) and the amino acid sequence of each peptide was identified by comparison with peptide sequences from Bos taurus in the UniProt database. Searches were performed with no enzyme; the precursor ion tolerance was set to 10 ppm and the fragment ion tolerance was set to 0.6 Da. Variable modifications included oxidation on methionine and proline (+15.995 Da). The tools Sequest HT and Fixed Value PSM Validator were used for database searching and scoring.  2.5 Determination of ACE inhibitory activity  The effect of the whey protein hydrolysates and synthesized peptides on the activity of ACE was determined using the substrate N-hippuryl-His-Leu (HHL) according to the method described in Cheung and Li-Chan (2010) with some modifications. The hydrolysates were dissolved in 0.05 M Tris-HCl buffer at pH 8.3 containing 0.3 M NaCl (hereinafter referred to as “assay buffer”) while the synthesized peptides were first solubilized in a mixture of acetonitrile and water, then further diluted with the assay buffer to the desired concentrations. Samples (30 µL) were pre-incubated with 30 µL of ACE enzyme (0.083 U/mL in assay buffer) for 15 min at 37C, thereupon 150 µL of HHL (6.5 mM in assay buffer) was added. The enzymatic reaction was carried out at 37C for 60 min after which 250 µL of 1 N HCl was added 12  to inactivate the enzyme, followed by 1 mL of ethyl acetate to extract the hippuryl acid (HA). The mixtures were vortexed for 30 s and centrifuged at 2000 x g for 5 min. After centrifugation, the ethyl acetate layer (0.7 mL) was removed and heated at 120C for 30 min to evaporate the solvent. The HA residues were re-dissolved with 1.3 mL of ddH2O and the absorbance was measured at 228 nm. The positive and negative controls, corresponding to the activity of ACE in the absence of inhibitor and the absence of ACE activity, respectively, were prepared by using assay buffer in lieu of the sample and in lieu of the sample and enzyme solution, respectively. For the synthesized peptides, a mixture of Tris-HCl and acetonitrile was used to prepare the positive controls.  The concentrations of samples required to cause a 50% inhibition of the enzyme activity (IC50) were calculated from the logarithmic regression equations obtained from plotting the percent ACE inhibition against the sample concentrations. The IC50 values of captopril and the angiotensin converting enzyme inhibitory peptide teprotide (Glp-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) were also determined for comparison.           2.6 Determination of DPP-IV inhibitory activity  The effect of the whey protein hydrolysates and synthesized peptides on the activity of DPP-IV was determined using the chromogenic substrate Gly-Pro-p-NA as 13  described by Lacroix and Li-Chan (2014).  The tri-peptide Ile-Pro-Ile (diprotin A) was used as a reference inhibitor.   2.7 Statistical analysis   One-way analysis of variance using the general linear model and pairwise comparison with Tukey’s method were performed using Minitab Statistical Software (Version 17, Minitab Inc., State College, PA, USA). All assays were conducted at least in triplicate and significant difference was established at P < 0.05.  3. Results   3.1 ACE and DPP-IV inhibitory activities of whey protein hydrolysates  The whey protein hydrolysates prepared by treatment of WPI with Thermoase PC10F alone (T) or with Thermoase PC10F followed by one of the exopeptidases, namely Accelerzyme® CPG (T-AC), Peptidase R (T-PR) or ProteAX (T-PX), as well as a commercial whey protein hydrolysate HilmarTM 8390 (H), were tested for their effect on ACE and DPP-IV activities.   Although all five of these hydrolysates were able to inhibit the two enzymes to some extent, they appeared to cause a stronger inhibition of ACE than of DPP-IV (Figure 1). The greatest inhibition of ACE activity was observed with T, whereas the 14  carboxypeptidase treated T-AC was the most effective at inhibiting the DPP-IV enzyme. Treatment of T with ProteAX, an enzyme preparation exhibiting aminopeptidase as well as endoproteinase activity, resulted in the hydrolysate T-PX with the lowest ACE and DPP-IV inhibitory activities. While both the aminopeptidase and carboxypeptidase treatments of T led to a reduction in ACE inhibitory activity, only the aminopeptidase-treated hydrolysates (T-PR and T-PX) showed a significantly reduced ability to inhibit DPP-IV (Figure 1).   3.2 Fractionation of whey protein hydrolysates with ACE and DPP-IV inhibitory activities  3.2.1 Size-exclusion chromatography of H, T, T-AC, T-PR and T-PX  The four whey protein hydrolysates produced in this study (T, T-AC, T-PR and T-PX) and the commercial whey protein hydrolysate (H) were fractionated by size-exclusion chromatography (Figure 2).   Treatments of T with exopeptidase (T-AC-7, T-PR-5 and T-PX-7 in Figures 2C, 2D and 2E, respectively) led to the formation of short peptides and free amino acids, as shown by the presence of fractions eluting at about 30 mL, corresponding to MW < 238.2 Da. Interestingly, the elution profile of the commercial hydrolysate H was also characterized by a prominent peak (H-5) at around 30 mL (Figure 2A).  15  The resulting size exclusion fractions were tested for their effect on both ACE and DPP-IV activities.  With 82% ACE inhibition, T-3 (MW 750-1230 Da) was the most potent size-exclusion fraction obtained from all five hydrolysates. Among the 31 fractions collected, H-3, H-4, T-3, T-AC-4, T-AC-5, T-PR-3, T-PX-2, T-PX-3, T-PX-4 and T-PX-6 were found to be significantly more effective at inhibiting the ACE enzymes than the un-fractionated hydrolysates from which they were obtained. The peptide fractions eluting around 30 mL and having the lowest molecular weight (H-5, T-AC-7, T-PR-5, and T-PX-7) all displayed low or no ACE inhibitory activity.    On the other hand, the fraction T-AC-4 (MW 645-1065 Da), with 47% inhibition, was found to be the most effective at inhibiting DPP-IV activity. While several fractions were able to inhibit the ACE enzyme more strongly than the crude hydrolysates they originated from, only fractions T-AC-4, T-PX-2 and T-PX-4, were more effective at inhibiting DPP-IV than their respective non-fractionated hydrolysates. Similarly to what was observed for the ACE enzyme, peptide fractions eluting around 30 mL had little or no effect on DPP-IV activity (Figure 2).  Displaying the highest DPP-IV inhibitory activity and being among the most effective at inhibiting ACE, T-AC-4 was selected to undergo further purification by RP-HPLC.  3.2.2 Reversed-phase high performance liquid chromatography of T-AC-4  16  As shown in Figure 3, among the seven fractions collected from the RP-HPLC fractionation of T-AC-4, the most non-polar fraction T-AC-4-g, eluting at ~ 27–29 min, caused the strongest inhibition of the DPP-IV enzyme (74% inhibition). The other fractions had some inhibitory activity, but their potency was far lower. Conversely, T-AC-4-g had no effect on the ACE enzyme. Instead, the fractions T-AC-4-b and T-AC-4-f were the most effective ACE inhibitors, causing 77 and 84% inhibition respectively. The enzyme was also inhibited by the other four fractions (T-AC-4-a, T-AC-4-c, T-AC-4-d and T-AC-4-e), but to a lesser extent.   3.3 ACE and DPP-IV inhibitory activities (IC50 values) of fractions isolated from T-AC  The potency of the most active size-exclusion and RP-HPLC fractions isolated from T-AC were determined and compared to the un-fractionated hydrolysates T and T-AC (Table 1).  The fraction T-AC-4 obtained by size exclusion chromatography had significantly lower IC50 values against both ACE and DPP-IV than the un-fractionated hydrolysate T-AC as well as the starting hydrolysate T. Further purification of T-AC-4 by RP-HPLC produced fractions with even lower IC50 values; those displaying the highest ACE inhibitory activity, however, were not the same that caused the strongest inhibition of DPP-IV.   With an IC50 value of 25 µg/mL, the RP-HPLC fraction T-AC-4-f was the most effective at inhibiting the ACE enzyme. The potency of this fraction was roughly two times greater than that of the size exclusion fraction T-AC-4 and four times greater 17  than the hydrolysate T. The more polar RP-HPLC fraction T-AC-4-b also showed high ACE inhibitory activity, with an IC50 value of 36 µg/mL.  In contrast, the hydrolysate T-AC, with an IC50 value of 195 µg/mL, was much less potent than the fractions isolated from it.    On the other hand, the T-AC-4-g fraction caused the strongest inhibition of DPP-IV activity (IC50  = 50 µg/mL). This RP-HPLC fraction was about 3 times more potent against DPP-IV than T-AC-4 and about 5 times more effective at inhibiting the enzyme than the whole hydrolysates T-AC and T.   3.4 Identification by LC–ESI-MS/MS of the peptides present in the hydrolysates T and T-AC and purified fractions T-AC-4, T-AC-4-b, T-AC-4-f and T-AC-4-g   Causing the strongest inhibition of the DPP-IV or ACE enzyme, the RP-HPLC fractions T-AC-4-b, T-AC-4-f and T-AC-4-g were analyzed for their constituent peptides by LC–ESI-MS/MS. The peptides present in the hydrolysates T and T-AC as well as the fraction T-AC-4 from size-exclusion chromatography were also determined and compared to those identified in the potent RP-HPLC fractions.  The sequences of the 378 peptides identified, whose length varied from 4 to 24 amino acid residues, are presented in the Supplementary Table S1. These peptides originated from -lactoglobulin and -lactalbumin, which are the two major protein constituents in the WPI used to produce the hydrolysates investigated in this study. 18  A few peptides derived from minor protein constituents of the WPI, such as lactoferrin and bovine serum albumin, were also identified in the hydrolysates and purified peptide fractions (data not shown).  A total of 201 peptide sequences were identified in T, 162 derived from -lactoglobulin and 39 from -lactalbumin, while 101 peptides derived from -lactoglobulin and 33 from -lactalbumin were identified in the T-AC hydrolysate. Forty-five of these sequences were common to both hydrolysates. The fraction T-AC-4 was found to be composed of 80 peptides derived from -lactoglobulin and 27 from -lactalbumin. Of these, 75 were also identified in T-AC. On the other hand, the three RP-HPLC fractions were found to contain 115 -lactoglobulin- and 48 -lactalbumin-derived peptides. Among these 163 sequences, 63 were also found in T-AC-4. While all peptide sequences identified in T-AC-4 should have also been found in T-AC and all peptides in T-AC-4-b, T-AC-4-f and T-AC-4-g should have been identified in T-AC-4, this was not the case. This discrepancy is likely due to the fact that crude whey protein hydrolysates and their size-exclusion fractions are composed of a complex mixture of peptides, making the identification of each individual peptide sequence by mass spectrometry a very challenging task. It is probable that some peptides, particularly those present in small amount, might not have been found in T, T-AC or T-AC-4 by LC–ESI-MS/MS analysis, but were identified in the more purified RP-HPLC fractions from T-AC-4.           3.5 ACE and DPP-IV inhibitory activities of identified peptides 19   Among the peptides identified in T, T-AC, T-AC-4, T-AC-4-b, T-AC-4-f and/or T-AC-4-g, 15 peptides derived from -lactalbumin and 33 peptides from -lactoglobulin were chemically synthesized and their effect on the activity of the ACE and DPP-IV enzymes was assessed (Figure 4, Table 2). These peptides were selected based on similarities between their amino acid sequences and -lactalbumin- and -lactoglobulin-derived peptides reported to have ACE and/or DPP-IV inhibitory activity, or because of the presence in their sequence of structural features (i.e. particular amino acid residues at certain positions) often observed in ACE and/or DPP-IV inhibitory peptides.      The peptides were found to have various levels of effectiveness on the ACE and DPP-IV enzymes (Figure 4). Among the 48 peptides tested, 20 had no detectable effect on ACE activity, whereas 8 were able to cause at least 50% inhibition of the enzyme at a final assay concentration of 143 µM (Figure 4A). The -lactoglobulin-derived peptides LDIQKVAGTW and LKALPMH, with IC50 values of 21 and 11 µM, respectively, and -lactalbumin derived peptides GYGGVSLPEW and WLAHKAL, with IC50 values of 2 and 29 µM, respectively, were the most effective at inhibiting the ACE enzyme. The potency of these peptides was, however, lower than that of the peptidomimetic ACE inhibitors teprotide and captopril (IC50 = 0.48 and 0.0054 µM, respectively) (Table 2).    20  With the exception of the tetra-peptide RTPE and deca-peptide LDDDLTDDIM, which had no detectable effect on DPP-IV, all other peptides were able to inhibit the enzyme, albeit mostly only to a moderate extent (Figure 4B). Displaying IC50 values of 42 and 57 µM, the -lactoglobulin-derived peptides LKPTPEGDLE and LKPTPEGDLEIL were the most effective at inhibiting DPP-IV. All peptides tested were less potent towards the enzyme than the reference inhibitor diprotin A (IC50 = 4.7 µM) (Table 2).   4. Discussion  While the treatment of WPH with the aminopeptidases Peptidase R and ProteAX or the carboxypeptidase Accelerzyme® CPG generated hydrolysates with reduced ACE inhibitory activity, only the hydrolysates produced by treatment with the aminopeptidases were found to be less effective at inhibiting the DPP-IV enzyme (Figure 1). Peptides with ACE inhibitory activity have been reported to preferentially have hydrophobic/aromatic amino acids at one or both termini, and/or bulky and basic charged residues at the C-terminal (Nongonierma & FitzGerald, 2015). Since all three exopeptidases used were reported to have specificity for hydrophobic amino acids (Cheung, Aluko, Cliff, & Li-Chan, 2015), the removal of these residues from the peptides present in the starting hydrolysate appear to have led to the production of new peptides with reduced inhibitory activity. Unlike ACE which is a carboxypeptidase, DPP-IV releases di-peptides from the N-terminal of its substrates. The finding that only the aminopeptidase-treated 21  T3 showed reduced inhibitory activity suggests that residues at the C-terminal might have little effect on the ability of a peptide to inhibit DPP-IV.   Most fractions isolated from the WPHs tested in this study were able to inhibit the activity of ACE and DPP-IV; however, the extent of the inhibition differed greatly. In general, hydrolysates and fractions with the strongest ACE inhibitory activity were not the most effective at inhibiting the DPP-IV enzyme. Similar findings were recently reported in a study by Konrad et al. (2014) in which the authors determined the ACE and DPP-IV inhibitory activities of RP-HPLC fractions obtained from hydrolysates of a whey protein concentrate and -lactoglobulin. Among their isolated fractions, a number were able to inhibit both enzymes, but their effectiveness against them varied. Since the authors did not identify the constituent peptides in these fractions, it is unknown whether the same peptides were responsible for the inhibition of the DPP-IV enzyme as well as the ACE enzyme.   Although a few of the peptides tested in the present study had inhibitory activity against both ACE and DPP-IV (Figure 4), the susceptibility of the two enzymes to inhibition by the whey protein-derived peptides differed greatly. In fact, the most potent ACE inhibitory peptide GYGGVSLPEW had very weak effect on DPP-IV activity. Similarly, the fragments with the strongest DPP-IV inhibitory activity (LKPTPEGDLEIL and LKPTPEGDLE) had low or no ACE inhibitory activity (Table 2).  All peptides bearing a tryptophan residue at the N- or C-terminal or a histidine residue at the C-terminal showed strong inhibitory activity against ACE. The shorter 22  -lactoglobulin fragment ALPMH (IC50 = 521 µM) (Pihlanto-Leppälä, Koskinen, Piilola, Tupasela, & Korhonen, 2000) and -lactalbumin-derived peptides KGYGGVSLPEW and WLAHK (IC50 = 0.7 and 77 µM, respectively) (Pihlanto-Leppälä, Koskinen, Piilola, Tupasela, & Korhonen, 2000; Tavares et al., 2011) have also been reported to have inhibitory effect against ACE. On the other hand, the most potent DPP-IV inhibitory peptides have a proline residue at their P1’ position, a structural feature found in a number of peptides reported to have inhibitory activity against the DPP-IV enzyme (Lacroix & Li-Chan, 2012b). Even though the amino acid sequence, rather than individual characteristics such as length or net charge, seems to be the predominant factor determining the effect of peptides on the activity of ACE and DPP-IV, the specific sequences required to cause the inhibition of ACE appeared to be different than those needed to inhibit DPP-IV activity.      5. Conclusion  Findings from the present research show that hydrolysates presenting inhibitory activities against both ACE and DPP-IV can be obtained from the enzymatic treatment of whey proteins, including hydrolysates debittered by exopeptidase treatments. By fractionating the hydrolysates and identifying the sequences of peptides in the most active fractions, the possibility of dual inhibitory activity of the constituent peptides in the hydrolysates was tested. Although a few of the identified peptides were able to inhibit both enzymes, most in fact did not display a dual 23  inhibitory activity. These results suggest that the peptides that are responsible for the observed inhibition by the hydrolysate of the ACE enzyme are most likely not the same ones that cause the inhibition of DPP-IV.  The present findings suggest that whey protein hydrolysates could have the potential to help improve both blood pressure and blood glucose regulation by means of their ability to inhibit the ACE and DPP-IV enzymes. While the experimental hydrolysates investigated in this study had been previously shown to have antihypertensive activity in spontaneously hypertensive rats (Cheung, Aluko, Cliff, & Li-Chan, 2015), their in vivo effect on ACE and DPP-IV activities as well as blood glucose regulation are unknown and, therefore, should be further investigated. Since the inhibitory peptides have to first be absorbed through the gastrointestinal wall before reaching the target enzymes, more research is also needed to evaluate the fate of the whey protein-derived peptides during digestion and their cellular permeability.   Acknowledgements  This research was financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC – Council Grant No. 121822-11). The authors would like to thank Lisa Nader Jones and Dr. Philip R. Gafken from the Hutchinson Cancer Research Center for their help with LC–ESI-MS/MS analysis and peptide 24  sequencing as well as Lennie Cheung for preparing the WPI hydrolysates used in this study.   References        Brown, N.J., & Vaughan, D.E. (1998). Angiotensin-converting enzyme inhibitors. Circulation, 97, 1411–1420.  Campbell, N.R.C., Gilbert, R.E., Leiter, L.A., Larochelle, P., Tobe, S., Chockalingam, A., Ward, R., Morris, D., Tsuyuki, R.T., & Harris, S.B. (2011). Hypertension in people with type 2 diabetes. Update on pharmacologic management. Canadian Family Physician, 57, 997–1002.  Cheung, I.W.Y., & Li-Chan, E.C.Y. (2010). Angiotensin-I-converting enzyme inhibitory activity and bitterness of enzymatically-produced hydrolysates of shrimp (Pandalopsis dispar) processing byproducts investigated by Taguchi design. Food Chemistry, 122, 1003–1012.  Cheung, B.M.Y., & Li, C. (2012). Diabetes and hypertension: is there a common metabolic pathway? Current Atherosclerosis Reports, 14, 160–166.  25  Cheung, L.K.Y., Aluko, R.E., Cliff, M.A., & Li-Chan, E.C.Y. (2015). Effect of exopeptidase treatment on antihypertensive activity and taste attributes of enzymatic whey protein hydrolysates. Journal of Functional Foods, 13, 262–275.  Filippatos, T.D., Athyros, V.G., & Elisaf, M.S. (2014). The pharmacokinetic considerations and adverse effects of DPP-4 inhibitors. Expert Opinion on Drug Metabolism & Toxicology, 10, 787–812.  FitzGerald, R.J., Murray, B.A., & Walsh, D.J. (2004). Hypotensive peptides from milk proteins. The Journal of Nutrition, 134, 980S–988S.  Gobbetti, M., Vini, F.M., & Rizzello, C.G. (2004). Angiotensin I-converting-enzyme-inhibitory and antimicrobial bioactive peptides. International Journal of Dairy Technology, 57, 173–188.  Gress, T.W., Nieto, J., Shahar, E., Wofford, M.R., & Brancati, F.L. (2000). Hypertension and antihypertensive therapy as risk factors for type 2 diabetes mellitus. The New England Journal of Medicine, 342, 905–912.  Kamau, S.M., Lu, R.-R., Chen, W., Liu, X.-M., Tian, F.-W., Shen, Y., & Gao, T. (2010). Functional significance of bioactive peptides derived from milk proteins. Food Reviews International, 26, 386–401.  26  Konrad, B., Anna, D., Marek, S., Marta, P., Aleksandra, Z., Józefa, C. (2014). The evaluation of dipeptidyl peptidase (DPP)-IV, -glucosidase and angiotensin converting enzyme (ACE) inhibitory activities of whey proteins hydrolyzed with serine protease isolated from Asian pumpkin (Cucurbita ficifolia). International Journal of Peptide Research and Therapeutics, 20, 483–491.  Lacroix, I.M.E., & Li-Chan, E.C.Y. (2012a). Dipeptidyl peptidase-IV inhibitory activity of dairy protein hydrolysates. International Dairy Journal, 25, 97–102.  Lacroix, I.M.E., & Li-Chan, E.C.Y. (2012b). Evaluation of the potential of dietary proteins as precursors of dipeptidyl peptidase (DPP)-IV inhibitors by an in silio approach. Journal of Functional Foods, 4, 403–422.  Lacroix, I.M.E., & Li-Chan, E.C.Y. (2013). Inhibition of dipeptidyl peptidase (DPP)-IV and -glucosidase activities by pepsin-treated whey proteins. Journal of Agricultural and Food Chemistry, 61, 7500–7506.  Lacroix, I.M.E., & Li-Chan, E.C.Y. (2014). Isolation and characterization of peptides with dipeptidyl peptidase-IV activity from pepsin-treated bovine whey proteins. Peptides, 54, 39–48.  Landsberg, L., & Molitch, M. (2004). Diabetes and hypertension: pathogenesis, prevention and treatment. Clinical and Experimental Hypertension, 26, 621–628. 27   Li-Chan, E.C.Y. (2015). Bioactive peptides and protein hydrolysates: research trends and challenges for application as nutraceuticals and functional food ingredients. Current Opinion in Food Science, 1, 28–37.  Mine, Y., Li-Chan, E., & Jiang, B. (2010). Bioactive proteins and peptides as functional foods and nutraceuticals. Iowa: Wiley-Blackwell IFT Book Series, 420 p.  Nagpal, R., Behare, P., Rana, R., Kumar, A., Kumar, M., Arora, S., Morotta, F., Jain, S., & Yadav, H. (2011). Bioactive peptides derived from milk proteins and their health beneficial potentials: An update. Food & Function, 2, 18–27.  Nongonierma, A.B., & FitzGerald, R.J. (2013). Dipeptidyl peptidase IV inhibitory and antioxidative properties of milk protein-derived dipeptides and hydrolysates. Peptides, 39, 157–163.  Nongonierma, A.B., & FitzGerald, R.J. (2015). Milk proteins as a source of tryptophan-containing bioactive peptides. Food & Function, 6, 2115–2127.  Pihlanto-Leppälä, A., Koskinen, P., Piilola, K., Tupasela, T., & Korhonen, H. (2000). Angiotensin I-converting enzyme inhibitory properties of whey protein digests: concentration and characterization of active peptides. Journal of Dairy Research, 67, 53–64. 28   Ricci, I., Artacho, R., & Olalla, M. (2010). Milk Protein peptides with angiotensin I-converting enzyme inhibitory (ACEI) activity. Critical Reviews in Food Science and Nutrition, 50, 390–402.  Silveira, S.T., Martínez-Maqueda, D., Recio, I., & Hernández-Ledesma, B. (2013). Dipeptidyl peptidase-IV inhibitory peptides generated by tryptic hydrolysis of a whey protein concentrate rich in -lactoglobulin. Food Chemistry, 141, 1072–1077.  Tavares, T., del Mar Contreras, M., Amorim, M., Pintado, M., Recio, I., & Malcata, F.X. (2011). Novel whey-derived peptides with inhibitory effect against angiotensin-converting enzyme: In vitro effect and stability to gastrointestinal enzymes. Peptides, 32, 1013–1019.   Uchida, M., Ohshiba, Y., & Mogami, O. (2011). Novel dipeptidyl peptidase-4–inhibiting peptide derived from -lactoglobulin. Journal of Pharmacological Sciences, 117, 63–66.  Uenishi, H., Kabuki, T., Seto, Y., Serizawa, A., & Nakajima, H. (2012). Isolation and identification of casein-derived dipeptidyl-peptidase 4 (DPP-4)-inhibitory peptide LPQNIPPL from gouda-type cheese and its effect on plasma glucose in rats. International Dairy Journal, 22, 24–30.  29  Wang, X., Wang, L., Cheung, X., Zhou, J., Tang, X., & Mao, X.-Y. (2012). Hypertension-attenuating effect of whey protein hydrolysate on spontaneous hypertensive rats. Food Chemistry, 134, 122–126.  Yi, E.C., Lee, H., Aebersold, R., & Goodlett, D.R. (2003). A microcapillary trap cartridge-microcapillary high-performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation. Rapid Communications in Mass Spectrometry, 17, 2093–2098. Table 1. ACE and DPP-IV inhibitory activities (IC50 values) of whey protein isolate hydrolysates obtained by enzymatic treatment with Thermoase PC10F (T) or with Thermoase PC10F and Accelerzyme® CPG (T-AC), and the most active size-exclusion chromatography (T-AC-4) and RP-HPLC (T-AC-4-b, T-AC-4-f and T-AC-4-g) fractions isolated from T-AC.     Hydrolysate/fraction IC50 (µg/mL)a  ACE DPP-IV T 102 c 250 c T-AC 195 d 268 d T-AC-4 59 b 151 b T-AC-4-b 36 ab ND T-AC-4-f 25 a ND T-AC-4-g ND 50 a  a IC50 values are reported as the mean from at least three determinations and expressed as final assay concentrations. Within the same column, values with different lower case letters are significantly different (P < 0.05). ND, IC50 values were not determined as the samples had no or low inhibitory activity.     Table 2. ACE and DPP-IV inhibitory activities (IC50 values) of synthesized peptides  Origin of peptide Peptide sequence IC50 (µM)a   ACE DPP-IV -Lactoglobulin LDIQKVAGTW 21 ab ND  IQKVAGTW 51 c 329 c  LKPTPEGDLEILb ND 57 a  LKPTPEGDLE ND 42 a  VLDTDY 128 e 471 d  LDTDY 121 de ND  LKALPMH 11 ab 193 b  LSFNPTQ 106 d ND     -Lactalbumin LKGYGGVSLPE ND 486 d  GYGGVSLPEW 2 a ND  WLAHKALb 29 bc 286 c     Diprotin Ac IPIb ND 4.7  Teprotided GlpWPRPQIPP 0.48   ND Captoprild NA 0.0054 ND  a IC50 values are reported as the mean from at least three determinations and expressed as final assay concentrations. Within the same column, values with different lower case letters are significantly different (P < 0.05).  b IC50 values of these peptides against DPP-IV are from Lacroix & Li-Chan (2014). c Diprotin A was used as reference DPP-IV inhibitor. d Teprotide and captopril were used as reference ACE inhibitors. NA, not applicable; ND, IC50 values were not determined as the peptides/peptidomimetics had no or low inhibitory activity. Figure 1. ACE and DPP-IV inhibitory activities of whey protein hydrolysates. H, commercial whey protein hydrolysate HilmarTM 8390; T, whey protein isolate (WPI) hydrolyzed with Thermoase PC10F; T-AC, WPI successively hydrolyzed with Thermoase PC10F and Accelerzyme® CPG; T-PR, WPI isolate successively hydrolyzed with Thermoase PC10F and Peptidase R; T-PX, WPI successively hydrolyzed with Thermoase PC10F and ProteAX. The percent inhibition values of ACE and DPP-IV were determined using 0.143 mg/mL and 0.125 mg/mL of samples, respectively (final assay concentrations). Each bar represents the mean and standard deviation of at least three determinations. Bars with different lower or upper case letters are significantly different (P < 0.05).  Figure 2. Elution profiles obtained by size-exclusion chromatography of H (A), T (B), T-AC (C), T-PR (D) and T-PX (E), and the ACE and DPP-IV inhibitory activities of the unfractionated hydrolysates and resulting fractions. The percent inhibition values of ACE and DPP-IV are reported as the mean from at least three determinations and were obtained using 0.143 mg/mL and 0.125 mg/mL of samples, respectively (final assay concentrations). Within the same row, values with different lower case letters are significantly different (P < 0.05). Molecular weight markers were eluted at the following volumes: antifreeze protein (3240 Da) = 8.90 mL; DRVYIHPF (1046.2 Da) = 15.57 mL; YGGFM (MW 573.7 Da) = 15.94 mL; YGGFL (MW 555.6 Da) = 16.31 mL; VYV (MW 379.5 Da) = 17.36 mL; GY (MW 238.2 Da) = 19.67 mL.  Figure 3. Elution profile obtained by reversed-phase HPLC of T-AC-4, and the ACE and DPP-IV inhibitory activities of the resulting fractions. The percent inhibition values of ACE and DPP-IV are reported as the mean from at least three determinations and were obtained using 0.143 mg/mL and 0.125 mg/mL of samples, respectively (final assay concentrations). Within the same row, values with different lower case letters are significantly different (P < 0.05).  Figure 4. Percent inhibition of ACE (A) and DPP-IV (B) by synthesized peptides. The percent inhibition values of ACE and DPP-IV were determined using 143 µM and 125 µM of peptides, respectively (final assay concentrations). Each bar represents the mean and standard deviation of at least three determinations.        																Figure	1	0 20 40 60 80 H T T-AC T-PR T-PX % Inhibition Hydrolysate ACE DPP-IV a b c c d A A B D C 	  	  	  	  	  	  	  	  	  	  	  	  	  Figure	  2	  !!!!!!!!!!!!!!!!!!!!!!0 0.4 0.8 1.2 1.6 0 5 10 15 20 25 30 35 Absorbance at 280 nm (AU) Elution volume (mL) T-PX-1 T-PX-2 T-PX-3 T-PX-4 T-PX-5 T-PX-6 T-PX-7 !!!!!!!!!!!!!!!!!!0 0.4 0.8 1.2 0 5 10 15 20 25 30 35 Absorbance at 280 nm (AU) Elution volume (mL) T-AC-1 T-AC-2 T-AC-3 T-AC-4 T-AC-5 T-AC-6 T-AC-7 C Enzyme % Inhibition T T-1 T-2 T-3 T-4 T-5 T-6 ACE 59 b -18 c -19 c 82 a 60 b 56 b 65 b DPP-IV 35 ab 10 d 24 c 30 b 21 c 35 a 34 ab !!Enzyme % Inhibition T-AC T-AC-1 T-AC-2 T-AC-3 T-AC-4 T-AC-5 T-AC-6 T-AC-7 ACE 46 c -21 e -14 e 14 d 71 a 63 ab 52 bc 6 d DPP-IV 39 b 5 f 14 e 34 c 47 a 31 c 27 d 1 g !!Enzyme  % Inhibition T-PR T-PR-1 T-PR-2 T-PR-3 T-PR-4 T-PR-5 ACE 37 b -15 d 31 b 76 a 20 c -7 d DPP-IV 29 a  13 b 27 a 18 b  2 c -3 c !!Enzyme  % Inhibition  T-PX T-PX-1 T-PX-2 T-PX-3 T-PX-4 T-PX-5 T-PX-6 T-PX-7 ACE 27 d  18 ef 55 b 42 c 45 c 26 de 65 a 10 f DPP-IV 16 cd 10 e 25 a 12 de 21 b   3 f 17 bc   2 f !!Enzyme % Inhibition H H-1 H-2 H-3 H-4 H-5 ACE 39 c  7 d 43 bc 56 a 48 b 10 d DPP-IV 22 a 16 bc 25 a 10 c  2 d -2 d !!!!!!!!!!!!!!!!!!!!!0 0.4 0.8 0 5 10 15 20 25 30 35 Absorbance at 280 nm (AU) Elution volume (mL) H-1 H-2 H-3 H-4 H-5 A Enzyme % Inhibition T T-1 T-2 T-3 T-4 T-5 T-6 ACE 59 b -18 c -19 c 82 a 60 b 56 b 65 b DPP-IV 35 ab 10 d 24 c 30 b 21 c 35 a 34 ab !!Enzyme % Inhibition T-AC T-AC-1 T-AC-2 T-AC-3 T-AC-4 T-AC-5 T-AC-6 T-AC-7 ACE 46 c -21 e -14 e 14 d 71 a 63 ab 52 bc 6 d DPP-IV 39 b 5 f 14 e 34 c 47 a 31 c 27 d 1 g !!Enzyme  % Inhibition T-PR T-PR-1 T-PR-2 T-PR-3 T-PR-4 T-PR-5 ACE 37 b -15 d 31 b 76 a 20 c -7 d DPP-IV 29 a  13 b 27 a 18 b  2 c -3 c !!Enzyme  % Inhibition  T-PX T-PX-1 T-PX-2 T-PX-3 T-PX-4 T-PX-5 T-PX-6 T-PX-7 ACE 27 d  18 ef 55 b 42 c 45 c 26 de 65 a 10 f DPP-IV 16 cd 10 e 25 a 12 de 21 b   3 f 17 bc   2 f !!Enzyme % Inhibition H H-1 H-2 H-3 H-4 H-5 ACE 39 c  7 d 43 bc 56 a 48 b 10 d DPP-IV 22 a 16 bc 25 a 10 c  2 d -2 d !!!!!!!!!!!!!!!!0 0.4 0.8 1.2 1.6 0 5 10 15 20 25 30 35 Absorbance at 280 nm (AU) Elution volume (mL) T-2 T-3 T-4 T-5 T-6 T-1 B !!!!!!!!!!!!!!!!!!!!!0 0.4 0.8 1.2 1.6 0 5 10 15 20 25 30 35 Absorbance at 280 nm (AU) Elution volume (mL) T-PR-1 T-PR-2 T-PR-3 T-PR-4 T-PR-5 D E Enzyme % Inhibition T T-1 T-2 T-3 T-4 T-5 T-6 ACE 59 b -18 c -19 c 82 a 60 b 56 b 65 b DPP-IV 35 ab 10 d 24 c 30 b 21 c 35 a 34 ab !!Enzyme % Inhibition T-AC T-AC-1 T-AC-2 T-AC-3 T-AC-4 T-AC-5 T-AC-6 T-AC-7 ACE 46 c -21 e -14 e 14 d 71 a 63 ab 52 bc 6 d DPP-IV 39 b 5 f 14 e 34 c 47 a 31 c 27 d 1 g !!Enzyme  % Inhibition T-PR T-PR-1 T-PR-2 T-PR-3 T-PR-4 T-PR-5 ACE 37 b -15 d 31 b 76 a 20 c -7 d DPP-IV 29 a  13 b 27 a 18 b  2 c -3 c !!Enzyme  % Inhibition  T-PX T-PX-1 T-PX-2 T-PX-3 T-PX-4 T-PX-5 T-PX-6 T-PX-7 ACE 27 d  18 ef 55 b 42 c 45 c 26 de 65 a 10 f DPP-IV 16 cd 10 e 25 a 12 de 21 b   3 f 17 bc   2 f !!Enzyme % Inhibition H H-1 H-2 H-3 H-4 H-5 ACE 39 c  7 d 43 bc 56 a 48 b 10 d DPP-IV 22 a 16 bc 25 a 10 c  2 d -2 d !Enzyme % Inhibition T T-1 T-2 T-3 T-4 T-5 T-6 ACE 59 b -18 c -19 c 82 a 60 b 56 b 65 b DPP-IV 34 ab 10 d 24 c 30 b 21 c 35 a 34 ab !!Enzyme % Inhibition T-AC T-AC-1 T-AC-2 T-AC-3 T-AC-4 T-AC-5 T-AC-6 T-AC-7 ACE 46 c -21 e -14 e 14 d 71 a 63 ab 52 bc 6 d DPP-IV 39 b 5 f 14 e 34 c 47 a 31 c 27 d 1 g !!Enzyme  % Inhibition T-PR T-PR-1 T-PR-2 T-PR-3 T-PR-4 T-PR-5 ACE 37 b -15 d 31 b 76 a 20 c -7 d DPP-IV 29 a  13 b 27 a 18 b  2 c -3 c !!Enzyme  % Inhibition  T-PX T-PX-1 T-PX-2 T-PX-3 T-PX-4 T-PX-5 T-PX-6 T-PX-7 ACE 27 d  18 ef 55 b 42 c 45 c 26 de 65 a 10 f DPP-IV 16 cd 10 e 25 a 12 de 21 b   3 f 17 bc   2 f !!Enzyme % Inhibition H H-1 H-2 H-3 H-4 H-5 ACE 39 c  7 d 43 bc 56 a 48 b 10 d DPP-IV 22 a 16 bc 25 a 10 c  2 d -2 d !Enzyme % Inhibition T T-1 T-2 T-3 T-4 T-5 T-6 ACE 59 b -18 c -19 c 82 a 60 b 56 b 65 b DPP-IV 34 ab 10 d 24 c 30 b 21 c 35 a 34 ab !!Enzyme % Inhibition T-AC T-AC-1 T-AC-2 T-AC-3 T-AC-4 T-AC-5 T-AC-6 T-AC-7 ACE 46 c -21 e -14 e 14 d 71 a 63 ab 52 bc 6 d DPP-IV 39 b 5 f 14 e 34 c 47 a 31 c 27 d 1 g !!Enzyme % Inhibition T-PR T-PR-1 T-PR-2 T-PR-3 T-PR-4 T-PR-5 ACE 37 b -15 d 31 b 76 a 20 c -7 d DPP-IV 29 a  13 b 27 a 18 b  2 c -3 c !!Enzyme  % Inhibition  T-PX T-PX-1 T-PX-2 T-PX-3 T-PX-4 T-PX-5 T-PX-6 T-PX-7 ACE 27 d  18 ef 55 b 42 c 45 c 26 de 65 a 10 f DPP-IV 16 cd 10 e 25 a 12 de 21 b   3 f 17 bc   2 f !!Enzyme % Inhibition H H-1 H-2 H-3 H-4 H-5 ACE 39 c  7 d 43 bc 56 a 48 b 10 d DPP-IV 22 a 16 bc 25 a 10 c  2 d -2 d !				Enzyme % Inhibition T-AC-4-a T-AC-4-b T-AC-4-c T-AC-4-d T-AC-4-e T-AC-4-f T-AC-4-g ACE 38 d 77 b 17 e 48 c 13 e 84 a -5 f DPP-IV 23 c 22 c 11 e 16 d 27 b 19 cd 74 a 							-0.2 0.2 0.6 1 1.4 0 5 10 15 20 25 30 Absorbance at 214 nm (AU) Elution time (min) T-AC-4-a T-AC-4-b T-AC-4-c T-AC-4-d T-AC-4-e T-AC-4-f T-AC-4-g 	A B 0 20 40 60 80 100 LIVTQTMKGLDIQKVAGT LIVTQTMKGLDIQ LIVTQTMKGLD LIVTQTMKG LIVTQTMK IVTQTMKGLDIQ IVTQTMKGLD IVTQTMK MKGLDIQKVA LDIQKVAGTW  IQKVAGTW AASDISLLDAQSAPL  AASDI LDAQSAPL LKPTPEGDLEIL LKPTPEGDLE KPTPE IIAEKTKIPAVFKID  IIAEKTKIP  IIAEK VFKIDALNENK  FKIDAL LNENK LVLDTDYKKY  VLDTDY LDTDY LVRTPEV VRTPEVDD RTPE KALKALP LKALPMH LSFNPTQ NPTQ LKDLK LKGYGGVSLPE GYGGVSLPEW TFHTSGYDTQA FHTSGYDTQA AIVQNNDSTE IVQNNDSTEYGLF IVQNNDSTEY IVQNNDSTE LDDDLTDDIM ILDKVGINY ILDKVGIN  ILDK LDKVGINY WLAHKAL % ACE inhibition Peptide 0 20 40 60 80 100 LIVTQTMKGLDIQKVAGT LIVTQTMKGLDIQ LIVTQTMKGLD LIVTQTMKG LIVTQTMK IVTQTMKGLDIQ IVTQTMKGLD IVTQTMK MKGLDIQKVA LDIQKVAGTW  IQKVAGTW AASDISLLDAQSAPL  AASDI LDAQSAPL LKPTPEGDLEIL LKPTPEGDLE KPTPE IIAEKTKIPAVFKID  IIAEKTKIP  IIAEK VFKIDALNENK  FKIDAL LNENK LVLDTDYKKY  VLDTDY LDTDY LVRTPEV VRTPEVDD RTPE KALKALP LKALPMH LSFNPTQ NPTQ LKDLK LKGYGGVSLPE GYGGVSLPEW TFHTSGYDTQA FHTSGYDTQA AIVQNNDSTE IVQNNDSTEYGLF IVQNNDSTEY IVQNNDSTE LDDDLTDDIM ILDKVGINY ILDKVGIN  ILDK LDKVGINY WLAHKAL % DPP-IV inhibition Peptide 	 1	Table	 S1.	β-Lactoglobulin-	 and	α-lactalbumin-derived	 peptide	 sequences	 identified	 in	 the	whey	protein	hydrolysate	T,	the	exopeptidase	treated	hydrolysate	T-AC,	and/or	the	SE-FPLC	and	RP-HPLC	fractions	isolated	from	T-AC.					Protein of origin Peptide sequencea Hydrolysate SE-FPLC fraction RP-HPLC fractions T T-AC β-lactoglobulin LIVTQTMKGLDIQKVAGTWYS 	 	 	 		 LIVTQTMKGLDIQKVAGTW 	 	 	 		 LIVTQTMKGLDIQKVAGT 	 	 	 		 LIVTQTMKGLDIQKVA 	 	 	 		 LIVTQTMKGLDIQK 	 	 	 		 LIVTQTMKGLDIQ 	 	 	 		 LIVTQTMKGLD 	 	 	 		 LIVTQTMKGL 	 	 	 		 LIVTQTMKG 	 	 	 		 LIVTQTMK 	 	 	 		 LIVTQTM 	 	 	 		 LIVTQT 	 	 	 		 IVTQTMKGLDIQKVAGTWYS 	 	 	 		 IVTQTMKGLDIQKVAGTW 	 	 	 		 IVTQTMKGLDIQ 	 	 	 		 IVTQTMKGLD 	 	 	 		 IVTQTMKGL 	 	 	 		 IVTQTMKG 	 	 	 		 IVTQTMK 	 	 	 		 IVTQT 	 	 	 		 TMKGLDIQKVAGTW 	 	 	 		 MKGLDIQKVAGTWYS 	 	 	 		 MKGLDIQKVAGTW 	 	 	 		 MKGLDIQKVAGT 	 	 	 		 MKGLDIQKVAG 	 	 	 		 MKGLDIQKVA 	 	 	 		 MKGLDIQK 	 	 	 		 MKGLDIQ 	 	 	 		 GLDIQKVAGTW 	 	 	 		 GLDIQKVAGT 	 	 	 		 LDIQKVAGTWYSL 	 	 	 		 LDIQKVAGTWYS 	 	 	 		 LDIQKVAGTWY 	 	 	 		 LDIQKVAGTW 	 	 	 		 LDIQKVAGT 	 	 	 		 LDIQKVAG 	 	 	 		 LDIQKVA 	 	 	 		 LDIQK 	 	 	 		 IQKVAGTWYS 	 	 	 		 IQKVAGTW 	 	 	 		 IQKVAGT 	 	 	 		 2	Protein of origin Peptide sequencea Hydrolysate SE-FPLC fraction RP-HPLC fractions T T-AC 	 VAGTWYSLAM 	 	 	 		 VAGTWYSL 	 	 	 		 VAGTWY 	 	 	 		 VAGTW 	 	 	 		 TWYSL 	 	 	 		 LAMAASDISLLDAQSAPLRVY 	 	 	 		 LAMAASDISLLDAQSAPLR 	 	 	 		 LAMAASDISLLDAQSAPL 	 	 	 		 LAMAASDISL 	 	 	 		 LAMAASDIS 	 	 	 		 AMAASDISL 	 	 	 		 MAASDISLLDAQSAPLR 	 	 	 		 MAASDISLLDAQSAPL 	 	 	 		 MAASDISLLDAQSAP 	 	 	 		 MAASDISLL 	 	 	 		 MAASDISL 	 	 	 		 MAASDIS 	 	 	 		 AASDISLLDAQSAPLRVYVEE 	 	 	 		 AASDISLLDAQSAPLRVY 	 	 	 		 AASDISLLDAQSAPLRV 	 	 	 		 AASDISLLDAQSAPLR 	 	 	 		 AASDISLLDAQSAPL 	 	 	 		 AASDISLLDAQSAP 	 	 	 		 AASDI 	 	 	 		 ASDISLLDAQSAPLR 	 	 	 		 SDISLLDAQSAPL 	 	 	 		 DISLLDAQSAPL 	 	 	 		 DISLLDAQSAP 	 	 	 		 ISLLDAQSAPLR 	 	 	 		 ISLLDAQSAPL 	 	 	 		 LLDAQSAPLRVYVEEL 	 	 	 		 LLDAQSAPLRVYVEE 	 	 	 		 LLDAQSAPLRVY 	 	 	 		 LLDAQSAPLRV 	 	 	 		 LLDAQSAPLR 	 	 	 		 LLDAQSAPL 	 	 	 		 LLDAQSAP 	 	 	 		 LDAQSAPLRVYVEELKPTPEGDLE 	 	 	 		 LDAQSAPLRVYVEELKPTPEGDL 	 	 	 		 LDAQSAPLRVYVEEL 	 	 	 		 LDAQSAPLRVYVEE 	 	 	 		 LDAQSAPLRVY 	 	 	 		 LDAQSAPLRV 	 	 	 		 LDAQSAPLR 	 	 	 		 LDAQSAPL 	 	 	 		 LDAQSAP 	 	 	 		 3	Protein of origin Peptide sequencea Hydrolysate SE-FPLC fraction RP-HPLC fractions T T-AC 	 LDAQSA 	 	 	 		 DAQSAPL 	 	 	 		 SAPLRVYVEE 	 	 	 		 LRVYVEEL 	 	 	 		 LRVYVEE 	 	 	 		 VYVEELKPTPEGDLEIL 	 	 	 		 VYVEELKPTPEGDLEI 	 	 	 		 VYVEELKPTPEGDLE 	 	 	 		 VYVEELKPTPEGDL 	 	 	 		 VYVEELKPTPEGD 	 	 	 		 VYVEELKPTPEG 	 	 	 		 VYVEELKPTPE 	 	 	 		 VYVEELK 	 	 	 		 VYVEEL 	 	 	 		 VYVEE 	 	 	 		 VEELKPTPEGDLEIL 	 	 	 		 VEELKPTPEGDLE 	 	 	 		 EELKPTPEGDLEILLQ 	 	 	 		 ELKPTPEGDLEIL 	 	 	 		 ELKPTPE 	 	 	 		 LKPTPEGDLEILLQKWENGE 	 	 	 		 LKPTPEGDLEILLQKW 	 	 	 		 LKPTPEGDLEILLQK 	 	 	 		 LKPTPEGDLEILLQ 	 	 	 		 LKPTPEGDLEILL 	 	 	 		 LKPTPEGDLEIL 	 	 	 		 LKPTPEGDLEI 	 	 	 		 LKPTPEGDLE 	 	 	 		 LKPTPEGDL 	 	 	 		 LKPTPEGD 	 	 	 		 LKPTPEG 	 	 	 		 LKPTPE 	 	 	 		 KPTPEG 	 	 	 		 KPTPEGD 	 	 	 		 KPTPE 	 	 	 		 ILLQKW 	 	 	 		 LQKWENGECAQKK 	 	 	 		 LLQKWENGE 	 	 	 		 LLQKWENG 	 	 	 		 LQKWENGE 	 	 	 		 AQKKIIAEK 	 	 	 		 KIIAEKTKIPA 	 	 	 		 KIIAEK 	 	 	 		 IIAEKTKIPAVFKIDALNENKV 	 	 	 		 IIAEKTKIPAVFKIDALNENK 	 	 	 		 IIAEKTKIPAVFKIDA 	 	 	 		 4	Protein of origin Peptide sequencea Hydrolysate SE-FPLC fraction RP-HPLC fractions T T-AC 	 IIAEKTKIPAVFKID 	 	 	 		 IIAEKTKIPAVFKI 	 	 	 		 IIAEKTKIPAVFK 	 	 	 		 IIAEKTKIPAVF 	 	 	 		 IIAEKTKIPAV 	 	 	 		 IIAEKTKIPA 	 	 	 		 IIAEKTKIP 	 	 	 		 IIAEKT 	 	 	 		 IIAEK 	 	 	 		 IAEKTKIPAVFK 	 	 	 		 IAEKTKIPAV 	 	 	 		 IAEKTKIPA 	 	 	 		 IAEKTKIP 	 	 	 		 AEKTKIPA 	 	 	 		 AEKTKIP 	 	 	 		 EKTKIP 	 	 	 		 KTKIPA 	 	 	 		 TKIPAVFKID 	 	 	 		 IPAVFKIDALNENKV 	 	 	 		 IPAVFKID 	 	 	 		 AVFKIDALNENK 	 	 	 		 VFKIDALNENKVL 	 	 	 		 VFKIDALNENKV 	 	 	 		 VFKIDALNENK 	 	 	 		 VFKIDALNEN 	 	 	 		 VFKIDALNE 	 	 	 		 VFKIDALN 	 	 	 		 VFKIDAL 	 	 	 		 VFKIDA 	 	 	 		 FKIDALNENKV 	 	 	 		 FKIDALNENK 	 	 	 		 FKIDALN 	 	 	 		 FKIDAL 	 	 	 		 IDALNENKVLVLDTDYKKYL 	 	 	 		 IDALNENKVLVLDTDYKKY 	 	 	 		 IDALNENKVLVLDTDYKK 	 	 	 		 IDALNENKVLVLDTDYK 	 	 	 		 IDALNENKVLVLDTDY 	 	 	 		 IDALNENKVLVLDTD 	 	 	 		 IDALNENKVLVLD 	 	 	 		 IDALNENKVLV 	 	 	 		 IDALNENKVL 	 	 	 		 IDALNENKV 	 	 	 		 IDALNENK 	 	 	 		 IDALNEN 	 	 	 		 IDALNE 	 	 	 		 5	Protein of origin Peptide sequencea Hydrolysate SE-FPLC fraction RP-HPLC fractions T T-AC 	 DALNENKVLVLDTDYKKYL 	 	 	 		 DALNENKV 	 	 	 		 DALNENK 	 	 	 		 ALNENKVLVLDTDY 	 	 	 		 ALNENKVLVLDTD 	 	 	 		 ALNENK 	 	 	 		 LNENKVLVLDTDYKKYLL 	 	 	 		 LNENKVLVLDTDYKKYL 	 	 	 		 LNENKVLVLDTDYKKY 	 	 	 		 LNENKVLVLDTDYKK 	 	 	 		 LNENKVLVLDTDYK 	 	 	 		 LNENKVLVLDTDY 	 	 	 		 LNENKVLVLDTD 	 	 	 		 LNENKVLVLD 	 	 	 		 LNENKVL 	 	 	 		 LNENKV 	 	 	 		 LNENK 	 	 	 		 NKVLVLDTDY 	 	 	 		 VLVLDTDYKKYL 	 	 	 		 VLVLDTDYKK 	 	 	 		 VLVLDTDYK 	 	 	 		 VLVLDTDY 	 	 	 		 VLVLDTD 	 	 	 		 LVLDTDYKKYLL 	 	 	 		 LVLDTDYKKYL 	 	 	 		 LVLDTDYKKY 	 	 	 		 LVLDTDYKK 	 	 	 		 LVLDTDYK 	 	 	 		 LVLDTDY 	 	 	 		 LVLDTD 	 	 	 		 VLDTDYKKYL 	 	 	 		 VLDTDYKKY 	 	 	 		 VLDTDYKK 	 	 	 		 VLDTDYK 	 	 	 		 VLDTDY 	 	 	 		 LDTDYKKYL 	 	 	 		 LDTDYKKY 	 	 	 		 LDTDYKK 	 	 	 		 LDTDYK 	 	 	 		 LDTDY 	 	 	 		 DTDYKKY 	 	 	 		 DTDYKK 	 	 	 		 LLFCMENSAEPEQS 	 	 	 		 LFCMENSAEPEQSL 	 	 	 		 LFCMENSAEPEQS 	 	 	 		 FCMENSAEPEQS 	 	 	 		 6	Protein of origin Peptide sequencea Hydrolysate SE-FPLC fraction RP-HPLC fractions T T-AC 	 CMENSAEPEQS 	 	 	 		 MENSAEPEQSL 	 	 	 		 MENSAEPEQS 	 	 	 		 SAEPEQSL 	 	 	 		 LVRTPEVDDEALEKFDKA 	 	 	 		 LVRTPEVDDEALEK 	 	 	 		 LVRTPEVDDEAL 	 	 	 		 LVRTPEVDDEA 	 	 	 		 LVRTPEVDDE 	 	 	 		 LVRTPEVDD 	 	 	 		 LVRTPEVD 	 	 	 		 LVRTPEV 	 	 	 		 LVRTPE 	 	 	 		 VRTPEVDDEALEKFDKA 	 	 	 		 VRTPEVDDEALEKF 	 	 	 		 VRTPEVDDEALEK 	 	 	 		 VRTPEVDDEAL 	 	 	 		 VRTPEVDDEA 	 	 	 		 VRTPEVDDE 	 	 	 		 VRTPEVDD 	 	 	 		 VRTPEVD 	 	 	 		 VRTPE 	 	 	 		 RTPEVDDEALEK 	 	 	 		 RTPE 	 	 	 		 TPEVDDEA 	 	 	 		 VDDEALEK 	 	 	 		 DDEALEK 	 	 	 		 DEALEKFDKA 	 	 	 		 DEALEK 	 	 	 		 LEKFDKALKALP 	 	 	 		 FDKALKALPMHIR 	 	 	 		 FDKALKALPMH 	 	 	 		 FDKALKALPM 	 	 	 		 FDKALKALP 	 	 	 		 KALKALP 	 	 	 		 ALKALPMHIR 	 	 	 		 ALKALP 	 	 	 		 LKALPMHIRLS 	 	 	 		 LKALPMHIR 	 	 	 		 LKALPMHI 	 	 	 		 LKALPMH 	 	 	 		 LKALPM 	 	 	 		 KALPMH 	 	 	 		 LPMHIR 	 	 	 		 IRLSFNPTQLEEQCHI 	 	 	 		 IRLSFNPTQLEEQ 	 	 	 		 7	Protein of origin Peptide sequencea Hydrolysate SE-FPLC fraction RP-HPLC fractions T T-AC 	 IRLSFNPTQ 	 	 	 		 RLSFNPTQ 	 	 	 		 LSFNPTQLEEQCHI 	 	 	 		 LSFNPTQLEEQ 	 	 	 		 LSFNPTQLEE 	 	 	 		 LSFNPTQL 	 	 	 		 LSFNPTQ 	 	 	 		 LSFNPT 	 	 	 		 SFNPTQLEEQCHI 	 	 	 		 SFNPTQLEEQCH 	 	 	 		 SFNPTQLEEQ 	 	 	 		 FNPTQLEEQCHI 	 	 	 		 FNPTQLEEQCH 	 	 	 		 FNPTQLEEQ 	 	 	 		 FNPTQLEE 	 	 	 		 FNPTQ 	 	 	 		 NPTQ 	 	 	 		 PTQLEEQ 	 	 	 		 LEEQCHI 	 	 	 	α-lactalbumin FRELKDLKGYGG 	 	 	 		 LKGYGGVSLPEWVCTT 	 	 	 		 LKDLKGYGGVSLPEW 	 	 	 		 LKDLK 	 	 	 		 DLKGYGG 	 	 	 		 LKGYGGVSLPEWV 	 	 	 		 LKGYGGVSLPEW 	 	 	 		 LKGYGGVSLPE 	 	 	 		 LKGYGGVSLP 	 	 	 		 LKGYGGVSL 	 	 	 		 LKGYGGVS 	 	 	 		 LKGYGG 	 	 	 		 KGYGGVSLPEW 	 	 	 		 KGYGGVSLPE 	 	 	 		 GYGGVSLPEW 	 	 	 		 GYGGVSLPE 	 	 	 		 YGGVSLPEW 	 	 	 		 YGGVSLPE 	 	 	 		 GGVSLPEW 	 	 	 		 GGVSLPE 	 	 	 		 GVSLPEW 	 	 	 		 VSLPEWVCTT 	 	 	 		 TTFHTSGYDTQA 	 	 	 		 TTFHTSGYDTQ 	 	 	 		 TTFHTSGYD 	 	 	 		 TFHTSGYDTQAI 	 	 	 		 TFHTSGYDTQA 	 	 	 		 8	Protein of origin Peptide sequencea Hydrolysate SE-FPLC fraction RP-HPLC fractions T T-AC 	 TFHTSGYDTQ 	 	 	 		 TFHTSGYDT 	 	 	 		 TFHTSGYD 	 	 	 		 TFHTSG 	 	 	 		 FHTSGYDTQAIVQNNDSTEYG 	 	 	 		 FHTSGYDTQAIVQ 	 	 	 		 FHTSGYDTQAI 	 	 	 		 FHTSGYDTQA 	 	 	 		 FHTSGYDTQ 	 	 	 		 FHTSGYDT 	 	 	 		 FHTSGYD 	 	 	 		 FHTSGY 	 	 	 		 HTSGYDTQ 	 	 	 		 TSGYDTQAI 	 	 	 		 TSGYDTQA 	 	 	 		 TSGYDTQ 	 	 	 		 SGYDTQAI 	 	 	 		 AIVQNNDSTEY 	 	 	 		 AIVQNNDSTE 	 	 	 		 IVQNNDSTEYGLFQ 	 	 	 		 IVQNNDSTEYGLF 	 	 	 		 IVQNNDSTEYGL 	 	 	 		 IVQNNDSTEYG 	 	 	 		 IVQNNDSTEY 	 	 	 		 IVQNNDSTE 	 	 	 		 IVQNND 	 	 	 		 LFQINNK 	 	 	 		 LFQINN 	 	 	 		 FQINNKI 	 	 	 		 FQINNK 	 	 	 		 IWCKDDQNPHSSN 	 	 	 		 IWCKDDQNPHSS 	 	 	 		 WCKDDQNPHSSN 	 	 	 		 KDDQNPHSSN 	 	 	 		 DDQNPHSSNICN 	 	 	 		 DDQNPHSSN 	 	 	 		 DQNPHSSNICN 	 	 	 		 DQNPHSSN 	 	 	 		 FLDDDLTDDIM 	 	 	 		 FLDDDLTDD 	 	 	 		 FLDDDLTD 	 	 	 		 FLDDDLT 	 	 	 		 LDDDLTDDIM 	 	 	 		 LDDDLTDD 	 	 	 		 LDDDLTD 	 	 	 		 DLTDDIM 	 	 	 		 9	Protein of origin Peptide sequencea Hydrolysate SE-FPLC fraction RP-HPLC fractions T T-AC 	 LTDDIM 	 	 	 		 ILDKVGINYWL 	 	 	 		 ILDKVGINYW 	 	 	 		 ILDKVGINY 	 	 	 		 ILDKVGIN 	 	 	 		 ILDKVG 	 	 	 		 ILDK 	 	 	 		 LDKVGINYW 	 	 	 		 LDKVGINY 	 	 	 		 LDKVGIN 	 	 	 		 DKVGIN 	 	 	 		 KVGIN 	 	 	 		 INYWL 	 	 	 		 WLAHKAL 	 	 	 		 LDQWLCEKL 	 	 	 	a	Peptides	in	bold	blue	font	were	chemically	synthesized	and	their	effect	on	ACE	and	DPP-IV	activities	was	tested.					

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.52383.1-0343557/manifest

Comment

Related Items