Proteolysis of β-lactoglobulin and β-casein by pepsin in ethanolic media

Dalgalarrondo, Michèle; Dufour, Éric; Chobert, Jean‐Marc; Bertrand-Harb, C.; Haertlé, Tomasz

doi:10.1016/0958-6946(94)p1595-5

Cited by 76 publications

(69 citation statements)

References 21 publications

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“…As expected, both proteins were very resistant to pepsinolysis (data not shown) because ␤-LG is a poor substrate for pepsin [27][28][29][30]. Nevertheless, the duodenal digestion (trypsin and chymotrypsin) of the native and glycated ␤-LG gave rise to a very complex mixture of peptides after 1 h of incubation as was previously observed by reversed-phase LC-UV [13].…”

Section: Identification Of ␤-Lg Peptides Resulting From the In Vitro supporting

confidence: 79%

Mass spectrometric characterization of glycated β-lactoglobulin peptides derived from galacto-oligosaccharides surviving the in vitro gastrointestinal digestion

Moreno

Quintanilla-López

Lebrón-Aguilar

et al. 2008

J. Am. Soc. Mass Spectrom.

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A mass spectrometric study has been carried out to elucidate the structures of glycated peptides obtained after in vitro gastrointestinal digestion of bovine {3-lactoglobulin ({3-LG) glycated with prebiotic galacto-oligosaccharides (GOS). The digests of both native and glycated {3-LG were analyzed by MALDI-MS, LC-ESI-MS, and LC-ESI-MS/MS. MALDI-MS profiles showed marked differences mainly related to the lower intensity of ions corresponding to the digest of glycated {3-LG. Overall, 58 and 23 unglycated peptides covering 97% and 63% of the mature {3-LG sequence could be identified in the digests of native and glycated samples, respectively. The LC-ESI-MS analyses corroborated the MALDI-MS results regarding the unglycated peptides but they also enabled an extensive investigation into the digest of glycated {3-LG. Thus, a total of 19 peptides glycated with GOS from two to seven hexose units could be identified. The tandem mass spectra of glycated peptides were mostly characterized by two neutral losses of 1026/1056, 864/894, 702/732, 540/570, 378/408, and 216/246 u, corresponding to the formation of the furylium ion and its subsequent "CHOH" loss, indicative of the peptide glycation with hepta-, hexa-, penta-, tetra-, tri-, and disaccharides, respectively. Also, other minor ionic species containing the furylium ring linked to different galactose units could be also detected, showing the diversity of the fragmentation pattern of peptides glycated with larger size carbohydrates. Finally, the putative GOS glycation sites could be determined at the NHz-terminal Leu residue and at Lys residues located in positions

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Section: Identification Of ␤-Lg Peptides Resulting From the In Vitro supporting

confidence: 79%

Mass spectrometric characterization of glycated β-lactoglobulin peptides derived from galacto-oligosaccharides surviving the in vitro gastrointestinal digestion

Moreno

Quintanilla-López

Lebrón-Aguilar

et al. 2008

J. Am. Soc. Mass Spectrom.

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“…The analysis of the results of limited proteolysis experiments performed on globular proteins of known structure reveals that helical segments are not the site of proteolytic attack [25]. Therefore, the high helical conformation state acquired by a-lactalbumin dissolved in aqueous buffer containing 40% ethanol [15] might be the origin of this inhibitory effect as it was also reported in the case of a-lactalbumin in the presence of high concentration of TFE [14] or in the case of other globular proteins in high ethanol concentration [15,17,18]. Fig.…”

Section: Proteolysis Of A-lactalbumin As a Function Of Ethanol Concenmentioning

confidence: 76%

Study of ethanol‐induced conformational changes of holo and apo α‐lactalbumin by spectroscopy and limited proteolysis

Wehbi¹,

Pérez²,

Dalgalarrondo³

et al. 2006

Molecular Nutrition Food Res

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This study was performed to contribute to the analysis of alpha-lactalbumin "molten globule" state by using spectral and proteolysis techniques. Samples of holo and apo alpha-lactalbumin in the presence of different concentrations of ethanol were analyzed. Results of fluorescence spectroscopy of both forms showed that as ethanol concentration increased, the tryptophanyl residues became more accessible to the solvent. Near circular dichroism spectra of holo alpha-lactalbumin indicated that its tertiary structure was maintained in 20% ethanol whereas it was altered in 30 and 40% ethanol. For apo alpha-lactalbumin, spectra were similar in all samples studied. Holo alpha-lactalbumin was resistant to trypsinolysis in 0% ethanol, whereas it was easily hydrolyzed in 20 and 30% ethanol. In the case of the apo form and in the absence of ethanol, 70% of the protein was degraded after 1 h. However, in the presence of 20 and 30% ethanol, the overall reaction rate was lowered. Peptides obtained after tryptic hydrolysis were identified by reversed-phase high-performance liquid chromatography coupled to mass spectrometry. Differences in population of produced peptides indicate the changes of folding intermediates present in the studied alpha-lactalbumin solutions. This study demonstrated that proteolytic enzymes are suitable tools to determine protein structure complementing physico-chemical studies.

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“…For peptic hydrolysis, given the resistance of BLG to cleavage by this enzyme, three different concentrations of ethanol [25, 30, and 40% (v/v)] were added to the buffer to generate transient structural changes in the protein (b-sheet e a-helix transition) and provide greater accessibility to cleavage sites [21,22]. BLG (2 mg/ml), solubilized in citric acid/ethanol mixtures (20 mM final concentration in citric acid), pH 2.5, was hydrolyzed by pepsin (aqueous solution: 1 mg/ml) at an E/S ratio of 2/100 (w/w).…”

Section: Preparation Of Hydrolysatesmentioning

confidence: 99%

“…BLG (2 mg/ml), solubilized in citric acid/ethanol mixtures (20 mM final concentration in citric acid), pH 2.5, was hydrolyzed by pepsin (aqueous solution: 1 mg/ml) at an E/S ratio of 2/100 (w/w). The reaction was performed at 20 8C for 40 h and stopped by addition of 1.5 volume of 0.2 M TrisHCl, pH 8.0 [22].…”

Section: Preparation Of Hydrolysatesmentioning

confidence: 99%

“…For enzymatic digests, solvents A and B were 0.11% TFA and 60% acetonitrile/0.09% TFA, respectively. A non-linear gradient (0-35% B in 8 min, 35 -70% B in 40 min, 70 -100% B in 10 min) was applied for elution at 30 8C of P25, P30, and P40 digests [22]. Tryptic digests were eluted at 60 8C according to the following gradient: 0 -22.5% B in 9 min, 22.5 -50% B in 34 min, 50 -60% B in 7 min, and 60 -100% B in 10 min [20].…”

Section: Reversed-phase High-performance Liquid Chromatography (Rp-hplc)mentioning

confidence: 99%

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Foaming characteristics of chemical and enzymatic hydrolysates of bovine β-lactoglobulin

Rahali

Guéguen

2000

Nahrung

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The foaming properties of bovine beta-lactoglobulin (BLG), BNPS-skatole (2-(2'-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine) (BNPS), trypsin (T) and pepsin in 25, 30 and 40% ethanol (P25, P30, P40) hydrolysates were investigated in the 0.2 to 1 mg/ml range. Foaming capacity and foam stability were assessed in terms of drained liquid volume and foam volume. Foam texture was analyzed from video images obtained during foam decay. The foaming capacity of BNPS, P30 and P40 was similar to that of BLG and greater than that of T or P25. All hydrolysates except BNPS were less stable than BLG at all concentrations tested. This result was insured by texture analysis. Principal component analysis confirmed the distribution of the samples into three groups based on their increasing stability: (i): P25, (ii): P30 and P40, and (iii): BLG and BNPS. Tryptic hydrolysate had the poorest foaming properties. The results are considered in relation to the molecular characteristics of the peptides, particularly their size and hydrophobicity.

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Proteolysis of β-lactoglobulin and β-casein by pepsin in ethanolic media

Cited by 76 publications

References 21 publications

Mass spectrometric characterization of glycated β-lactoglobulin peptides derived from galacto-oligosaccharides surviving the in vitro gastrointestinal digestion

Mass spectrometric characterization of glycated β-lactoglobulin peptides derived from galacto-oligosaccharides surviving the in vitro gastrointestinal digestion

Study of ethanol‐induced conformational changes of holo and apo α‐lactalbumin by spectroscopy and limited proteolysis

Foaming characteristics of chemical and enzymatic hydrolysates of bovine β-lactoglobulin

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