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αS1-Casein was isolated from Haflinger mare's milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare's αS1-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrospray ionization mass spectrometry. The results revealed the existence of a full-length protein and 7 variants resulting from posttranscriptional modifications; that is, exon skipping involving exon 7, exon 14, or both and use of a cryptic splice site encoding a glutamine residue. The determination of the different phosphorylation degrees of the native isoforms of αS1-casein was finally achieved by electrospray ionization mass spectrometry analysis after fractionation of the isoforms by ion-exchange chromatography. Thus, 36 different variants of equine αS1-casein were identified with several phosphate groups ranging from 2 to 6 or 8 depending on whether exon 7 was skipped. [ABSTRACT FROM AUTHOR]

Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine β-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. β-Casein prepared from Haflinger mare's milk by hydrophobic interaction chromatography was fractionated by ion-exchange chromatography according to the degree of phosphorylation. Isoforms were identified by mass spectrometry; they corresponded to the full-length protein having 3 to 7 phosphate groups and to the splicing variant involving exon 5 and containing 4 to 7 phosphate groups. Investigations of nonenzymatic deamidation showed that β-casein did not deamidate spontaneously in stored milk and during the different steps of chromatography, but deamidation could occur when 2-dimensional electrophoresis was performed, increasing the β-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10°C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of β-casein was achieved by mixing each phosphorylation isoform in its native state with the whole β-casein fraction. [ABSTRACT FROM AUTHOR]

Abstract: Individual goat milk samples, taken from animals of the Indigenous Greek breed and from the international breeds Saanen and Alpine, were studied by RP-HPLC regarding the qualitative and quantitative characteristics of their proteins. Thirty-two samples from the Indigenous Greek breed and 17 from the international breeds were characterised further by RP-HPLC/ESI-MS. The mean total protein content of the milk samples from the Greek breed was higher 38.8g/l, compared to 31.9g/l of those from international breeds, due to the great difference between their mean αs1-Cn contents (6.90 and 3.02g/l, respectively). In the milk samples of the Greek breed, the strong αs1-Cn variants B3, B4 and As/B1 predominated, whereas in the milk samples from international breeds the medium variant E and the defectives F and null predominated. Variant A of αs2-Cn followed by variant C and the κ-Cn D (former B) were the most abundant in both groups. αs2-Cn F and the rare κ-Cn variant C/B were observed in the milk samples from the Greek breed. The β-caseins A and C were present in both groups of samples. Finally, the level of phosphorylation of the different genotypes is showed. [Copyright &y& Elsevier]

A great number of milk-derived peptides have been shown to exhibit angiotensin converting enzyme (ACE) inhibitory properties and thus potential utility in the regulation of blood pressure. The present work aimed to investigate the effects of 2 milk trypsin hydrolysates from αS1- and αS2-casein (CH1 and CH2, respectively) on ACE activity evaluated in human umbilical vein endothelial cells (HUVEC) in vitro, rat aortic tissues ex vivo, and renovascular hypertensive rat in vivo. Incubation of HUVEC and rat aortic tissues with CH1 or CH2 induced a concentration-dependent inhibition of hydrolysis of the ACE substrate hippuryl-histidyl-leucine (HHL), the hydrolysates being much less potent than perindopril (an ACE inhibitor). However, in contrast to perindopril, CH1 and CH2 failed to modify angiotensin I-induced aortic ring vasoconstriction. The HPLC profiles of rat plasma after intragastric administration were variable among individuals but none of the observed peaks corresponded to peptides comprising CH1 or CH2 or to fragments of these peptides. During 4 wk of cardiovascular monitoring, in hydrolysate-fed renovascular hypertensive rats, systolic blood pressure weakly decreased compared with the control group. However, the CH1-fed hypertensive rats exhibited a decrease of heart rate during the nocturnal period of activity. To conclude, our results show that CH1 and CH2 inhibited ACE activity in HUVEC and rat aortic tissue but failed to antagonize the aortic-constricting effects of the natural agonist angiotensin I. Moreover, we demonstrated that CH1, to a greater extent than CH2, can slightly affect cardiovascular parameters although the ingested bioactive peptides could not be detected in the blood. [ABSTRACT FROM AUTHOR]

Abstract: Milk-clotting activity found in ammonium sulfate-precipitated protein extracts from Albizia lebbeck and Helianthus annuus seeds was studied. Specific clotting activity of albizia seed extract was 15 times higher than that of sunflower seed extract. Zymogram analysis revealed several proteolytic bands in albizia seed extract and one diffuse proteolytic band for sunflower seed extract. Whole bovine casein was incubated with the plant seed extracts or chymosin and some breakdown products were characterized by reversed-phase high-performance liquid chromatography and electrophoresis. Similar to chymosin, the two seed extracts exhibited proteolytic activity toward κ-casein, α s-casein and β-casein, with the highest activity observed for the albizia seed extract. Mass spectrometry analysis showed that the sunflower extract hydrolyzed κ-casein at the Phe105–Met106 bond, as does chymosin. The albizia extract also displayed activity on κ-casein, but the Lys116–Thr117 bond was its preferred target. [Copyright &y& Elsevier]

Abstract: Thermolysin was used to hydrolyze bovine α-lactalbumin at 25 and 70°C under non-reducing conditions. The breakdown products were identified by mass spectrometry. At 25°C, the low proportion of α-lactalbumin in an unfolded state in equilibrium with the native state underwent limited hydrolysis leading to the production of peptides, that were no longer degraded (final peptides). At 70°C, the protein was in a molten globule-like state according to circular dichroism and complete cleavage of the protein was achieved. At 70°C, the protein was first quickly cleaved, then unfolded, leading to the release of intermediate peptides, from which final peptides were eventually produced. The amino-terminal 1–58 and carboxy-terminal 95–123 regions were readily cleaved, whereas the central region including the calcium-binding domain was more resistant. Some peptides were produced at 70°C, but not at 25°C. The choice of accurate experimental conditions may be of importance for the preparation of functional peptides. [Copyright &y& Elsevier]

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 23 kDa. These γ-like caseins were degraded by plasmin in solution subsequently to their formation. The presence of PP5-like peptides of 20 kDa 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 24 h 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. [Copyright &y& Elsevier]

Bovine αS2-casein was purified by anionic exchange and hydrophobic interaction chromatographies. The purified αS2-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 αS2-casein in solution was proposed. [Copyright &y& Elsevier]