Development of an at-line method for the identification of angiotensin-I inhibiting peptides in protein hydrolysates

Abstract

A fast at-line method was developed for the identification of ACE inhibiting (ACEI) peptides in protein hydrolysates. The method consists of activity measurements of fractions collected from a two-dimensional HPLC fractionation of the peptide mixture followed by MS identification of the peptides in the inhibiting fractions. The inhibition assay is based on the inhibiting effect of ACEI peptides on the hydrolytic scission of the substrate Hippuric acid-His-Leu (HHL) during the ACE-catalysed hydrolysis reaction. A fast LC method was developed for the quantification of Hippuric acid (H) and Hippuric acid-Histidine-Leucine (HHL), allowing a large number of fractions to be analysed within a reasonable time period. The method is sensitive and uses only standard laboratory equipment. The limit of detection is 0.34 M for the known ACEI peptide IPP. This is sufficiently sensitive for the identification of only moderately active peptides and/or ACEI peptides present at low concentrations. The relative standard deviation of the inhibition assay was 12% measured over a time period of 2 months. The IC50 value of IPP measured with the assay was 5.6 M, which is comparable to the values of 5 M and 5.15 M reported in literature for the standard Matsui method. The assay was successfully applied in the identification of ACEI peptides in enzymatically hydrolysed caseinate samples. Two new, not earlier published ACEI peptides were identified; MAP (␤-casein f102-104) and ITP (␣-s2-casein f119-121) with IC50 values of 3.8 M and 50 M, respectively.

Comparative study of the structural and physicochemical properties of two food derived antihypertensive tri-peptides, Isoleucine-Proline-Proline and Leucine-Lysine-Proline encapsulated into a chitosan based nanoparticle system

“…The results of the bioactivity assessment of the peptides are in agreement with the findings of other groups; for example, Henda et al (2013) reported an IC 50 value of 0.0151 ± 0.005 µM for captopril and Stanton (2011) reported the IC 50 value of LKP as 0.32 µM. Different IC 50 values have been reported for IPP, varying from 1.89 to 5 µM (Siltari, Viitanen, Kukkurainen, Vapaatalo, & Valjakka, 2014;van Platerink, Janssen, & Haverkamp, 2007). It has been reported that peptides with specific amino acids at the N-and C-terminus have shown variable inhibition activity with ACE (Iwaniak, Minkiewicz, & Darewicz, 2014).…”

Modeling of the Relationship between Dipeptide Structure and Dipeptide Stability, Permeability, and ACE Inhibitory Activity

“…The ACE inhibition experiments were performed with an HPLC‐UV at‐line assay using the artificial substrate HHL as described previously (van Platerink and others 2007). Briefly, dipeptide samples (40 μL) at concentrations of 20 and 200 μM were pipetted into a 96‐well plate and 27 μL of the ACE solution (33.4 mU/mL in PBS, pH 7.4; ACE activity was checked before the start of the experiment) were added to each well and incubated for 5 min on a 96‐well plate mixer at 700 RPM.…”

The glycine deportation system and its pharmacological consequences

“…There is good evidence in the mammalian circulation that HA is metabolically stable. The tripeptides hippuryl-L-histidyl-L-leucine (van Platerink et al, 2007) and hippurylglycylglycine (Jafarian-Tehrani et al, 2000) are commonly employed as substrates for angiotensin converting enzyme (ACE; peptidyl-dipeptidase A; EC 3.4.15.1) and are hydrolyzed by dipeptidase activity leaving the stable HA. Circulating HAwill certainly be exposed to ACE since it is expressed mainly in lung, vascular endothelium and kidney (Femia et al, 2008).…”