(652c) Supercharging Hemoglobin for Stability and Improved in-Vitro Behavior.

Protein engineering requires precise control over bioactivity, structural integrity, and functional stability due to the intrinsic complexity of proteins. While experimental and computational strategies have significantly advanced the field, challenges remain—particularly in maintaining protein stability and activity while minimizing toxicity and immune responses. Previous work on supercharging has demonstrated potential, but its impact on quaternary proteins such as hemoglobin remains underexplored.

Objective

This study presents an experimental approach to enhance the functionality and stability of complex quaternary proteins, using hemoglobin as a model due to its intricate structure and known challenges in preserving bioactivity during modification. The primary objective is to apply rational design principles to modify hemoglobin surfaces using succinic anhydride, thereby evaluating the effects of increased negative surface charge on protein stability and biocompatibility.

Method

By varying succinic anhydride concentrations, distinct degrees of hemoglobin surface modification will be achieved, characterized using quadrupole time-of-flight mass spectrometry (Q-TOF), and visualized with native PAGE. Structural stability will be assessed via high-performance liquid chromatography (HPLC). Given the inherent toxicity and potential for organ damage associated with acellular hemoglobin, this study further investigates how surface modification influences cytotoxicity. Cytotoxic effects will be evaluated using human umbilical vein endothelial cells (HUVECs), with PrestoBlue and LDH assays. Additionally, as hemoglobin's binding to nitric oxide (NO) can induce vasoconstriction, NO-binding behavior post-modification will also be assessed.

This comprehensive methodology aims to advance protein engineering by offering valuable insights for both academic research and industrial applications—particularly for proteins that pose complex design challenges, such as hemoglobin.