Effects of ionization on chemical shifts of the surrounding protons of bromelain inhibitor VI

Abstract

Proton nuclear magnetic resonance (NMR) titration shifts are a very sensitive indication of proton dissociation from carboxyl groups and of hydrogen-bond formation between carboxylate groups and backbone amide protons. As a model protein, we selected bromelain inhibitor VI (BI-VI), which is a unique double-chain molecule (M _r = 5888), and characterized almost all the proton resonances in the pH range of 1.5-9.9 by two-dimensional NMR. In this study, we first examined the ionization effects of Asp13^H(β-CO₂H) and Glu24^H(γ-CO ₂H) (superscript letters H and L indicate heavy and light chains, respectively). It was found that their ionization affected the chemical shifts of the surrounding protons through a maximum of seven consecutive covalent bonds, but scarcely through non-covalent bonds. Furthermore, it was revealed that the formation of two hydrogen bonds of Ser4^L(NH)-Asp9^H(β-CO ₂H) and Lys19^H(NH)-Asp32^H(β-CO₂H) influenced not only the chemical shifts of the amide protons of Ser4^L and Lys19^H but also those of the alpha protons of Asp9^H and Asp32^H.


(Communicated by Masanori OTSUKA, M.J.A.)