Alain Driou

Characterization of α -casozepine, a tryptic peptide from bovine α s1-casein with benzodiazepine-like activity

Caseins are a known source of biologically active peptides. In this study, we have shown evidence of a novel anxiolytic activity in a tryptic hydrolysate of bovine αs1‐casein. Injection of 3 mg/kg of this hydrolysate significantly reduced the epileptic symptoms caused by pentylenetetrazole in rats. Anxiety reduction was also observed when the hydrolysate was tested in the elevated plus‐maze and in the conditioned defensive burying rat models. Peptides isolated from the hydrolysate were examined for their affinity for the γ‐amino‐butyric acid (GABA) type A receptor. Only one peptide, named α‐casozepine, corresponding to the 91–100 fragment from bovine αs1‐casein, expressed affinity for GABAA receptor. In vitro, the peptide had 10,000 less affinity for the benzodiazepine site of the GABAA than did diazepam. However, in the conditioned defensive burying paradigm it was 10‐fold more efficient than diazepam. The difference observed between the in vitro and in vivo activity of α‐casozepine could not been explained by an action via the peripheral‐type benzodiazepine receptor; α‐casozepine had no affinity for this receptor. The α‐casozepine amino acid sequence could be related to the carboxy‐terminal sequence of the polypeptide diazepam binding inhibitor, an endogenous ligand of the central GABAA and peripheral‐type benzodiazepine receptors.

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.

A solid-phase synthesis of three aza-, iminoaza- and reduced aza-peptides from the same precursor

Using the Boc-strategy, a step-by-step synthesis on the PAM solid support of three aza-, iminoaza- and reduced aza-peptide homologues is described. From the same hydrazinocarbonyl peptide-PAM precursor, the coupling of either a Boc-amino acid or a Boc-amino aldehyde gives rise to an aza-peptide or an iminoaza-peptide, containing the Cα-CO-NH-Nα-CO-NH-Cα or Cα-CH=N-Nα-CO-NH-Cα surrogate, of the peptide motif, respetively. In situ reduction of the latter by NaBH3CN leads to a reduced aza-peptide containing the Cα-CH2-NH-Nα-CO-NH-Cα moiety. The key step synthesis of the hydrazinocarbonyl peptide-PAM precursor is carried out by coupling on the growing peptide chain theN-Boc-azaamino acid chloride obtained by the action of triphosgene on the, correspondingN-Boc-hydrazine. These modifications have been introduced in position 1–2 of the YLGYLEQLLR benzodiazepine-like decapeptide.

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.