Development of an at-line method for the identification of angiotensin-I inhibiting peptides in protein hydrolysates.
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 microM 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 microM, which is comparable to the values of 5 microM and 5.15 microM 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 (beta-casein f102-104) and ITP (alpha-s2-casein f119-121) with IC50 values of 3.8 microM and 50 microM, respectively.
van Platerink
Chris J
CJ
Advanced Measurement and Imaging, Unilever Research Laboratory Vlaardingen, The Netherlands. chris-van.platerink@unilever.com
Janssen
Hans-Gerd M
HG
Haverkamp
Johan
J
D000803:Angiotensin I / Q000037:antagonists & inhibitors*; D002853:Chromatography, Liquid; D006868:Hydrolysis; D008904:Miniaturization; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D010455:Peptides; D011506:Proteins; D000803:Angiotensin I