Application of at-line two-dimensional liquid chromatography–mass spectrometry for identification of small hydrophilic angiotensin I-inhibiting peptides in milk hydrolysates

A two-dimensional chromatographic method with mass spectrometric detection has been developed for identification of small, hydrophilic angiotensin I-inhibiting peptides in enzymatically hydrolysed milk proteins. The method involves the further separation of the poorly retained hydrophilic fraction from a standard C18 reversed-phase column on a hydrophilic interaction liquid chromatography (HILIC) column. The latter column is specifically designed for the separation of hydrophilic compounds. Narrow fractions collected from the HILIC column were analysed for their angiotensin I-converting enzyme (ACE) inhibiting potential in an at-line assay. Fractions showing significant inhibition of ACE were analysed by LC–MS for structure elucidation. With this method the main peptides responsible for ACE-inhibition in the hydrophilic part of a milk hydrolysate could be determined. The ACE-inhibiting peptides RP, AP, VK, EK, and EW explained more than 85% of ACE-inhibition by the hydrophilic fraction

Thermal unfolding of a llama antibody fragment : a two-state reversible process

Camelids produce functional heavy chain antibodies which are devoid of light chains and CH1 domains [Hamers-Casterman, C., et al. (1993) Nature 363, 446-448]. It has been shown that the variable domains of these heavy chain antibodies (the VHH fragments) are functional at or after exposure to high temperatures, in contrast to conventional antibodies [Linden van der, R. H. J., et al. (1999) Biochim. Biophys. Acta 1431, 37-44]. For a detailed understanding of the higher thermostability of these VHH fragments, knowledge of their structure and conformational dynamics is required. As a first step toward this goal, we report here the essentially complete 1H and 15N NMR backbone resonance assignments of a llama VHH antibody fragment, and an extensive analysis of the structure at higher temperatures. The H-D exchange NMR data at 300 K indicate that the framework of the llama VHH fragment is highly protected with a ¿Gex of >5.4 kcal/mol, while more flexibility is observed for surface residues, particularly in the loops and the two outer strands (residues 4-7, 10-13, and 58-60) of the ß-sheet. The CD data indicate a reversible, two-state unfolding mechanism with a melting transition at 333 K and a ¿Hm of 56 kcal/mol. H-D exchange studies using NMR and ESI-MS show that below 313 K exchange occurs through local unfolding events whereas above 333 K exchange mainly occurs through global unfolding. The lack of a stable core at high temperatures, observed for VHH fragments, has also been observed for conventional antibody fragments. The main distinction between the llama VHH fragment and conventional antibody fragments is the reversibility of the thermal unfolding process, explaining its retained functionality after exposure to high temperatures

Enhanced Lacto-Tri-Peptide Bio-Availability by Co-Ingestion of Macronutrients

Some food-derived peptides possess bioactive properties, and may affect health positively. For example, the C-terminal lacto-tri-peptides Ile-Pro-Pro (IPP), Leu-Pro-Pro (LPP) and Val-Pro-Pro (VPP) (together named here XPP) are described to lower blood pressure. The bioactivity depends on their availability at the site of action. Quantitative trans-organ availability/kinetic measurements will provide more insight in C-terminal tri-peptides behavior in the body. We hypothesize that the composition of the meal will modify their systemic availability. We studied trans-organ XPP fluxes in catheterized pigs (25 kg; n=10) to determine systemic and portal availability, as well as renal and hepatic uptake of a water-based single dose of synthetic XPP and a XPP containing protein matrix (casein hydrolyte, CasH). In a second experiment (n=10), we compared the CasH-containing protein matrix with a CasH-containing meal matrix and the modifying effects of macronutrients in a meal on the availability (high carbohydrates, low quality protein, high fat, and fiber). Portal availability of synthetic XPP was 0.08 ± 0.01% of intake and increased when a protein matrix was present (respectively 3.1, 1.8 and 83 times for IPP, LPP and VPP). Difference between individual XPP was probably due to release from longer peptides. CasH prolonged portal bioavailability with 18 min (absorption half-life, synthetic XPP: 15 ± 2 min, CasH: 33 ± 3 min, p<0.0001) and increased systemic elimination with 20 min (synthetic XPP: 12 ± 2 min; CasH: 32 ± 3 min, p<0.0001). Subsequent renal and hepatic uptake is about 75% of the portal release. A meal containing CasH, increased portal 1.8 and systemic bioavailability 1.2 times. Low protein quality and fiber increased XPP systemic bioavailability further (respectively 1.5 and 1.4 times). We conclude that the amount and quality of the protein, and the presence of fiber in a meal, are the main factors that increase the systemic bioavailability of food-derived XPP