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 24 h.The digested samples were assayed for the inhibitory activity of angiotensinconverting 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 (-55 mm Hg) was observed at 6 h 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 [b-casein (CN); f 59-64], Gly-Lys-Pro ( b 2 -microglobulin; f 18-20), Ile-Pro-Ala ( b-lactoglobulin; f 78-80), Phe-Pro (serum albumin; f 221-222; b-CN, f 62-63, f 157-158, and f 205-206), Val-Tyr-Pro ( b-CN; f 59-61), and Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Leu-Gln-Pro ( b-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 b-lactoglobulin, showed the strongest antihypertensive activity (-31 mm Hg). ( Key words: antihypertensive peptide, cheese whey, protease digestion, whey protein) Abbreviation key: ACE = angiotensin-converting enzyme, Hip-His-Leu = hippuryl-histidyl-leucine, IC 50 = 50% inhibitory concentration, RP = reversedphase, SBP = systolic blood pressure, SHR = spontaneously hypertensive rats, TFA = trifluoroacetic acid.