Direct measurements of hemoglobin interactions with liposomes using EPR spectroscopy

Area of Science:

• Biochemistry
• Biophysics
• Materials Science

Background:

• Hemoglobin (Hb) is a potential oxygen carrier, but its stability and oxidation rates are critical for applications.
• Liposome encapsulation (LEH) can improve Hb properties, yet its effect on autoxidation kinetics requires precise measurement.
• Conventional spectroscopy faces challenges with liposome turbidity, necessitating alternative methods.

Purpose of the Study:

• To compare the autoxidation rates of acellular crosslinked hemoglobin (alphaalphaHb) and liposome-encapsulated hemoglobin (LEH) at 37°C.
• To evaluate Electron Paramagnetic Resonance (EPR) spectroscopy as a method for studying LEH oxidation kinetics.
• To investigate the influence of liposome encapsulation on hemoglobin's electronic structure and oxidation mechanisms.

Main Methods:

• Utilized Electron Paramagnetic Resonance (EPR) spectroscopy to monitor autoxidation of alphaalphaHb and LEH.
• Employed ultraviolet-visible (UV-vis) spectroscopy for comparative measurements on acellular alphaalphaHb.
• Assessed indirect oxidation measurement via chloroform extraction, identifying its limitations.

Main Results:

• EPR determined autoxidation rate constants: 0.039/h for alphaalphaHb and 0.065/h for LEH.
• UV-vis measurements on alphaalphaHb yielded comparable oxidation rates to EPR.
• EPR detected spectral shifts (g value) and intensity changes in the bis-histidine low-spin B complex for LEH, indicating structural perturbations.

Conclusions:

• Liposome encapsulation significantly enhances the autoxidation rate of hemoglobin.
• EPR spectroscopy is a viable and advantageous method for quantifying LEH oxidation kinetics, overcoming liposome turbidity issues.
• Lipid-associated perturbations within the liposome environment are suggested as the cause for accelerated LEH oxidation.