DIRECT MEASUREMENTS OF HEMOGLOBIN INTERACTIONS WITH LIPOSOMES USING EPR SPECTROSCOPY

Electron paramagnetic resonance (EPR) spectroscopy was used to compare the rates of autoxidation at 37°C of acellular and liposome-encapsulated hemoglobin (LEH) crosslinked between alpha chains with bis (3,5-dibromosalicyl) fumarate (ααHb). This method avoids the difficulties inherent in using conventional ultraviolet-visible (UV-vis) spectroscopy caused by the high turbidity of liposome suspensions. Rate constants of 0.039/h and 0.065/h were obtained for the ααHb and LEH samples, respectively. Similar oxidation measurements with ααHb using UV-vis spectroscopy gave a rate constant comparable to that obtained with EPR spectroscopy. Indirect measurement of the oxidation kinetics of LEH utilizing extraction of ααHb with chloroform from partially oxidized LEH samples was unreliable because the amount of extractable hemoglobin was inversely proportional to the degree of oxidation. EPR measurements showed a shift in the g value and substantial enhancement in the intensity of the bis-histidine low-spin B complex...