Structural Analysis of New Antihypertensive Peptides Derived from Cheese Whey Protein by Proteinase K Digestion

Abstract Whey protein was digested with one of seven kinds of proteases at 37°C (trypsin, proteinase K, actinase E, thermolysin, or papain) or at 25°C (pepsin or chymotrypsin) for 24h. The digested samples were assayed for the inhibitory activity of angiotensin-converting enzyme and for changes in the systolic blood pressure caused in spontaneously hypertensive rats after gastric intubation. The strongest depressive effect on the systolic blood pressure (−55mm Hg) was observed at 6h after gastric intubation of the whey protein that was digested by proteinase K. Finally, six peptides were chromatographically isolated from the proteinase K digest by a combination of hydrophobic reversed-phase HPLC and gel filtration. The amino acid sequences and their origins were clarified as follows: Val-Tyr-Pro-Phe-Pro-Gly [β-casein (CN); f 59–64], Gly-Lys-Pro ( β 2 -microglobulin; f 18–20), Ile-Pro-Ala ( β-lactoglobulin; f 78–80), Phe-Pro (serum albumin; f 221–222; β-CN, f 62–63, f157–158, and f 205–206), Val-Tyr-Pro ( β-CN; f59–61), and Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu- Gln-Pro ( β-CN; f 80–90). Chemical synthesis of these six peptides confirmed that all peptides, except an undecapeptide, have antihypertensive activity in spontaneously hypertensive rats. The synthetic tripeptide Ile-Pro-Ala, originating from β-lactoglobulin, showed the strongest antihypertensive activity (–31 mm Hg).