Related Experiment Video
Updated: Sep 16, 2026

Measurement of Heme Synthesis Levels in Mammalian Cells
Published on: July 9, 2015
Gentamicin Targeting Human Hemoglobin Induces Methemoglobin Formation and Decreases Oxygen Affinity: A Molecular
Peilin Shu1, Pengfei Wang1, Wencong Li1
1Air Force Medical Center, Air Force Medical University, Beijing 100142, China.
Abstract:
The nephrotoxicity and ototoxicity of Gentamicin have been extensively investigated; however, whether they induce hematotoxicity remains unclear. This study aimed to explore the binding of Gentamicin to adult hemoglobin (HbA) and its toxicological implications. The effect of Gentamicin on HbA oxidation was assessed by quantifying methemoglobin (MetHb) formation via a four-wavelength spectrophotometric method, and scavenger rescue assays were employed to elucidate the oxidative mechanism. Alterations in the oxygen-carrying capacity of both HbA and RBCs were evaluated through the acquisition of oxygen equilibrium curves and oxygen dissociation assays. The binding affinity between Gentamicin and HbA was determined by surface plasmon resonance (SPR). Furthermore, the influence of Gentamicin on the secondary and tertiary structures of HbA was examined by microfluidic modulation spectroscopy (MMS) and UV-visible absorption spectroscopy, respectively. Molecular docking was employed to predict the binding sites of Gentamicin on HbA. Molecular dynamics simulations verified the stability of the binding. Gentamicin promoted HbA autoxidation, elevating MetHb levels, and reduced the oxygen affinity of HbA. Gentamicin-promoted HbA autoxidation is primarily mediated by both direct heme-pocket perturbation and H2O2/iron-dependent amplification. SPR confirmed concentration-dependent specific binding between Gentamicin and HbA. MMS indicated no alteration in HbA secondary structure; however, UV-visible spectroscopy revealed that Gentamicin attenuated the Soret band, converted the oxyhemoglobin double-peak to a singlet, and generated a new band at 630 nm. Molecular docking predicted that Gentamicin binds β-chain residues via hydrogen, carbon-hydrogen, and hydrophobic interactions. This study reveals that Gentamicin exerts hematological effects in vitro. By binding to specific sites on HbA, Gentamicin affects the tertiary structure of HbA, promotes heme oxidation, ultimately increases MetHb content (oxidative damage), and decreases its oxygen-carrying capacity (functional impairment).
Related Concept Videos
Antifungal Agents
Drug Toxicity: Dose-Dependent Reactions
Determination of Multiple Dosing Parameters: Steady-State, Minimum and Maximum Concentrations
Oxygen Transport in the Blood
Hemoglobin
When all four heme groups are bound to oxygen, the resulting molecule is called oxyhemoglobin. As a result, arterial blood...
Bioactivation and Tissue Toxicity

