Time-dependent nature in peptic hydrolysis of native bovine hemoglobin

Native bovine hemoglobin was digested with pepsin at pH 4.5 in a batched stirred tank reactor. The resulting peptides in the time-course samples were separated and identified by RP-HPLC coupled with ESI-MS/MS. The secondary structure of bovine hemoglobin was not significantly changed after acid-treatment at pH 4.5 based on far-UV circular dichroism spectra. The alpha-helix contents of peptic hydrolysates decreased gradually with time according to 'Cyone-by-one' mechanism. MS/MS analysis enabled unambiguous identification of 31 and 15 peptides released respectively from alpha-chain and beta-chain, which resulted in their sequence coverage of 100 and 76%. The discrimination of peptic susceptibility between different protein areas was compared in terms of the time-dependent release of peptides. At first, peptic cleavage sites were concentrated around N- and C-terminal regions of alpha-chain and beta-chain. Later, pepsin hydrolyzed the middle part of alpha-chain from N- to C-terminal, while little enzymatic cleavage occurred in the center region of beta-chain due mainly to their high hydrophilic nature. The release kinetics of peptides was discussed in relation to the hydrophobicity of amino acid residues of polypeptide chains.