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 degrees C of acellular and liposome-encapsulated hemoglobin (LEH) crosslinked between alpha chains with bis (3,5-dibromosalicyl) fumarate (alphaalphaHb). 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 alphaalphaHb and LEH samples, respectively. Similar oxidation measurements with alphaalphaHb 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 alphaalphaHb 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 for the encapsulated hemoglobin, indicating a perturbation of this low-spin complex. We suggest that lipid-associated perturbations are responsible for the enhancement of the oxidation observed with the LEH samples compared to the unencapsulated material.
Blood Research Detachment, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, USA.
O O
OO
Abugo
C
C
Balagopalakrishna
J M
JM
Rifkind
A S
AS
Rudolph
J R
JR
Hess
V W
VW
Macdonald
D001241:Aspirin / Q000031:analogs & derivatives / Q000494:pharmacology; D001802:Blood Substitutes / Q000737:chemistry / Q000378:metabolism*; D004347:Drug Interactions; D004578:Electron Spin Resonance Spectroscopy; D006454:Hemoglobins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D010084:Oxidation-Reduction; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
D001802:Blood Substitutes; D006454:Hemoglobins; D008081:Liposomes; C053936:hemoglobin XL99alpha; D001241:Aspirin