Laurent Miclo

Angiotensin-I-converting enzyme inhibitory peptides from tryptic hydrolysate of bovine αS2-casein

Angiotensin‐I‐converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine αS2‐casein (αS2‐CN) was extensively investigated. Forty‐three peptide peaks were isolated and tested. Seven casokinins (i.e. CN‐derived ACE inhibitory peptides) were identified and their IC50 values were determined. Four peptides exhibited an IC50 value lower than 20 μM. Peptides αS2‐CN (f174–181) and αS2‐CN (f174–179) had IC50 values of 4 μM. Surprisingly, deletion of the C‐terminal dipeptide of two of these casokinins did not significantly alter their inhibitory activity.

Tryptic hydrolysis of bovine αS2-casein: identification and release kinetics of peptides

Abstract Bovine α S2 -casein was purified by anionic exchange and hydrophobic interaction chromatographies. The purified α S2 -casein was hydrolysed with trypsin; the resulting peptides were identified by amino acids composition and on line reversed-phase high performance liquid chromatography coupled with electrospray mass spectrometry. A study on the kinetics of peptide release enabled discrimination between different protein areas according to their surface-accessibility. Data from the kinetic study were reconciled with secondary structure predictions and a hypothetical model of the structural organisation of purified bovine α S2 -casein in solution was proposed.

Susceptibility of equine κ- and β-caseins to hydrolysis by chymosin

Abstract Equine whole casein was hydrolysed by chymosin and some peptides generated were characterised by microsequencing after reversed-phase high performance liquid chromatography or sodium dodecyl sulphate polyacrylamide gel electrophoresis. β-Casein was readily hydrolysed into amino- and carboxy-terminal fragments after cleavage of the Leu190–Tyr191 bond. These two fragments seemed to be resistant to further chymosin hydrolysis on incubation for up to 24xa0h. Equine κ-casein was purified by affinity chromatography on immobilised wheat germ agglutinin. O-Glycosylated κ-casein was found to represent less than 6.8% of the equine casein components. Equine κ-casein was also hydrolysed and para-κ-casein and glycomacropeptide were generated after cleavage of the Phe97–Ile98 bond.

Rapid determination of the ratios of three aromatic residues in peptides by reversed-phase high-performance liquid chromatography with a high-resolution photodiode-array detector

A method for the simultaneous determination of the ratios of three aromatic residues in peptides by derivative UV spectrophotometry on a spectrophotometer with a resolution of 0.1 nm can be used for the RP-HPLC analysis of peptides because of the recent development of high-resolution photodiode-array detectors (1.2 nm). The difference between the theoretical and experimental ratios of aromatic residues of peptides determined in real time is less than 5%. This method could become a powerful tool for the study of peptides and hydrolysates: A variety of possible applications are discussed.

Conformational studies of a benzodiazepine-like peptide in SDS micelles by circular dichroism,1H NMR and molecular dynamics simulation

The conformation of a benzodiazepine-like decapeptide corresponding to the YLGYLEQLLR fragment of a casein has been examined in a sodium dodecyl sulfate micellar medium using circular dichroism, two-dimensional1H NMR spectroscopy and restrained molecular dynamics simulation. The decapeptide adopts an amphipathic 310-helicoid structure in which the E6...R10 ionic bridge stabilizes the C-terminus.

Action of plasmin on equine β-casein

Abstract Equine β-casein and, in a lesser extent, αs1- and κ-caseins were good substrates for plasmin in solution. The Lys47–Ile48 bond of β-casein was readily hydrolysed leading to the production of γ-like caseins of 23xa0kDa. These γ-like caseins were degraded by plasmin in solution subsequently to their formation. The presence of PP5-like peptides of 20xa0kDa associated to caseins seemed to arise from endogenous plasmin activity in equine milk. Their formation was, however, not obvious in solution, as the amino-terminal region of β-casein seemed to be readily hydrolysed by plasmin. γ-caseins were generated when a sodium caseinate solution was incubated at 37°C and pH 8.0 for 24xa0h or when caseinate was stored in a freeze-dried form at 7°C for 2 years. This might be due to the presence of low amount of endogenous plasmin associated to equine caseinate as evidenced by assays with a specific synthetic substrate.

Analysis of peptides by amino acids composition: contribution of ultraviolet derivative spectrophotometry and retention time prediction

Amino acid composition analysis is sometimes used for the identification of peptides obtained from the hydrolysis of a protein of known sequence. Nevertheless, the interpretation of the analysis can be difficult if only one hydrochloric acid hydrolysis can be performed because of the partial destruction of some amino acids and the lack of cleavage of certain peptidic bonds. In this paper we propose combining the analysis of amino acids by two techniques (derivative UV spectrometry and retention time estimation) associated on-line with the purification step of the peptides (reversed-phase HPLC).A set of 56 peptides from the peptic and chymotryptic hydrolysis of bovine αs1-casein (αs1-CN) were analysed in this manner. The difference between the theoretical and observed retention times was 1.9 min, for aromatic amino acids ratios the difference was 4.0%. The ambiguities coming from repeated residues in the sequence or the presence of Trp residues, destroyed by acidic hydrolysis, were solved and a fragment of the αs1-CN sequence could be attributed to each peptide.