Evaluation of the Binding Affinity of Nitrosylcobalamin to Intrinsic Factor as a Predictive Model for Cobalamin Binding Protein Interactions: A Comparative Study with Hydroxocobalamin

Abstract

Background: Intrinsic factor (IF) is a glycoprotein crucial for cobalamin (vitamin B12) absorption in the human body. This study aimed to evaluate the binding affinity of nitrosylcobalamin (NO-Cbl), a cobalamin analog, to recombinant human IF derived from plants, using hydroxocobalamin (OH-Cbl) as a comparative standard. Methods: Surface plasmon resonance (SPR) was employed to assess the kinetic parameters of NO-Cbl and OH-Cbl interactions with plant- derived IF across various concentrations. Results: SPR analysis demonstrated that NO-Cbl and OH-Cbl exhibited high binding affinities to IF, with equilibrium dissociation constant (KD) values in the picomolar range. OH-Cbl showed a slightly stronger binding affinity (KD = 4.79 × 10-11 M) than NO-Cbl (KD = 8.58 × 10-11 M). Despite NO-Cbl and OH-Cbl both being bound to IF, differences in binding affinity and stability were observed, particularly at higher concentrations. Conclusion: Variations in IF binding between NO-Cbl and OH-Cbl may be attributed to the saturation of binding sites or recognition issues specific to plant-derived IF. This study underscores the potential of NO-Cbl as a targeted therapeutic agent capable of leveraging natural cobalamin uptake pathways. These results also highlight the suitability of using recombinant plant-derived IF as a model for predicting the biological activity of cobalamin analogs despite the nuanced differences from native human IF.

Keywords

• nitrosylcobalamin (no-cbl)
• hydroxocobalamin (oh-cbl) vitamin b12
• cobalamin
• intrinsic factor (if)
• nitric oxide (no)
• surface plasmon resonance (spr)