Peptides with Angiotensin I-Converting Enzyme (ACE) Inhibitory Activity from Defibrinated, Hydrolyzed Bovine Plasma

Abstract

Defibrinated bovine plasma (DBP) was treated with the microbial protease Flavourzyme to obtain protein hydrolysates with various degrees of hydrolysis (DH). The angiotensin I-converting enzyme (ACE) inhibiting activity of the hydrolyzed protein was assessed with hippuryl-His-Leu as the substrate. The amount of hippuric acid released, due to uninhibited ACE activity, was determined by high-performance liquid chromatography. ACE inhibiting (ACEI) activity was found to increase with increasing DH; the 43% DH hydrolysate exhibited the highest activity and had an IC₅₀ of 1.08 mg/mL. Peptide fractions with high ACEI activity were isolated using size exclusion chromatography. The fraction that possessed the highest ACEI activity contained peptides with GYP, HL(I), HPY, HPGH, L(I)F, SPY, and YPH sequence motifs, as determined by reversed-phase liquid chromatography−tandem mass spectrometry using a novel immonium precursor-ion scanning technique. Some of these motifs correspond to sequences found in bovine serum albumin, a potential source of ACEI peptides in bovine plasma. Keywords: Angiotensin I-converting enzyme inhibitors; bioactive peptides; immonium precursor-ions; LC-MS; MALDI-TOF MS; plasma protein; peptide sequencing; protein hydrolysis