Isolation of [hydroxyproline3]Lysyl-bradykinin formed by kallikrein from human plasma protein

[Hydroxyproline3]Lysyl-bradykinin ([Hyp3]Lys-BK), a new kinin was isolated, besides Lysyl-bradykinin (Lys-BK), from the reaction mixture of human plasma protein Cohn's fraction IV-4 with hog pancreatic kallikrein. The liberated kinins were isolated by procedures including ethanol extraction, Sephadex G-15, CM cellulose and reverse-phase high performance liquid chromatography and quantitated by radioimmunoassay. On HPLC, two peaks of immunoreactive kinins emerged. Peak 1, an unknown kinin proceeded to peak 2 which had an identical retention time to that of Lys-BK. The amino acid sequence of the unknown peak 1 proved to be Lys-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg, or [Hydroxyproline3]Lys-BK, and peak 2 Lys-BK. The ratio of the amounts of two kinins thus formed were [Hyp3]Lys-BK 25 +/- 4% and Lys-BK 75 +/- 4%. The existence of [Hyp3]Lys-BK suggests a presence of a new kininogen containing [Hyp3]Lys-BK in human plasma protein.