Hemoglobin is encapsulated in liposomes of different lipid composition. The resulting dispersion consists primarily of multilamellar liposomes (hemosomes) of a wide particle size distribution (diameter ranging mainly between 0.1 and 1 micron). The encapsulation efficiency is significantly larger with liposomes containing negatively charged lipids as compared to liposomes made of phosphatidylcholine. The integrity of the phospholipid bilayer is maintained in the presence of hemoglobin. The reaction rate of CO binding to encapsulated hemoglobin is reduced compared to that of free hemoglobin, but it is still greater than that observed in red blood cells. Hemoglobin encapsulated in liposomes made from negatively charged phospholipids is less stable than hemoglobin entrapped in isoelectric phosphatidylcholine. The instability of hemoglobin is due to the protein interacting with the negatively charged lipid bilayer. This interaction leads in turn to hemoglobin denaturation, possibly involving the dissociation of the heme group from the heme-globin complex. The nature of the negatively charged phospholipid is important in promoting the interaction with hemoglobin, the effect being in the order phosphatidic acid greater than phosphatidylinositol congruent to phosphatidylglycerol greater than phosphatidylserine. The presence of equimolar amounts of cholesterol in the phospholipid bilayer has a stabilizing effect on hemoglobin. This effect is pronounced with saturated phospholipids, but it is also observed, though to a lesser extent, with unsaturated ones, indicating that the bilayer fluidity has a modulating effect. The presence of cholesterol possibly interferes with secondary interactions following the binding of hemoglobin to the negatively charged lipid bilayer.

The effective magnetic moments for a number of human and carp methemoglobin derivatives were determined in solution at room temperature. The data permit us to confirm the dependence of the spin-state equilibrium of azide methemoglobin on the quaternary state of the hemoglobin and to demonstrate a similar dependence for both human and carp aquomethemoglobin.In addition, the pH dependence of the effective magnetic moment and the Soret spectrum of carp azidemethemoglobin are compared.In 1978 Perutz et al. [I] and Messana et al. [2] reported that the spin-state equilibria of derivatives of ferric hemoglobin were thermodynamically linked to the conformational equilibrium of the quaternary structure of the protein. This was most clearly demonstrated by studying the effect of the conversion of carp azidemethemoglobin from the R to the T quaternary state on the equilibrium between low-spin and high-spin forms of the derivative. Indications of the shift in spin-state equilibrium were found by a variety of spectroscopic techniques, but the most direct demonstration was obtained by measurements of magnetic susceptibility. These authors measured the molar magnetic susceptibilities of a number of ferric derivatives of carp and human hemoglobin in the R and T state. At that time the method for determination of the molar paramagnetic contribution to the susceptibility relied in part on measurements over an extended temperature interval in the frozen state. It is now possible to estimate the paramagnetic contribution to susceptibility in the liquid state without examining the temperature dependence. This allows us to define the properties of the system at physiological temperature with much greater precision than was previously possible. In addition, this method permits the examination of the effects of solution variables, such as pH, which are difficult to define in frozen systems.We furthermore wanted to reexamine the effect of quaternary structure on the magnetic properties of aquomethemoglobin. Perutz et al. reported [l] that the magnetic susceptibility of this hemoglobin derivative was unaffected by changes in the quaternary structure from R to T state. At the time, this finding was unexpected in view of the very significant change in the visible spectrum of this derivative, as a consequence of the conformational transition. This spectral change strongly suggested a change in spin-state equilibrium. In addition, recent reasonance Raman spectroscopic studiesAbbreviations. R, relaxed (high-affinity) state of hemoglobin; T, tense (low-affinity) state of hemoglobin; NMR, nuclear magnetic resonance; P6-inosito1, inositol hexakisphosphate; pB, Bohr magneton.of the ferric derivatives of carp and human hemoglobins carried out in the laboratory of Dr Dennis Rousseau suggest that in fluoromethemoglobin the R and T transition has no effect on spin-state equilibrium, but that for aquomethemoglobin a small but significant change is observed (E. R. Henry, D. Rousseau, S. Simon, J. J. Hopfield, and R. W. Noble, unpublished results). Si...

The effects of membrane phospholipid composition, surface charge and cholesterol content on the deteriorating interactions between hemoglobin (Hb) and phospholipid bilayers were studied. Hb was either encapsulated in multilamellar liposomes (hemosomes), or incubated with small unilamellar vesicles (SUV). Negatively charged phospholipids increased the rate of oxyHb decay in unsaturated lipid hemosomes. This effect was not linked to Hb-induced lipid peroxidation, since the latter process was inhibited in hemosomes with negative surface charge. Cholesterol decreased both the negative-charge elicited fall in oxyHb-level, and Hb-induced lipid peroxidation. In hemosomes prepared from synthetic, saturated phospholipids, negative surface charge (phosphatidic acid) elicited drastic denaturation (bleaching) of Hb, which effect was completely prevented by cholesterol. The experiments with SUV prepared from unsaturated lipids indicated intercalation of Hb into the bilayer due to hydrophobic interaction. This process was decreased by membrane cholesterol. Negative surface charge of the vesicles, through an electrostatic interaction with the positively charged heme, resulted in the displacement of heme relative to globin. This process was also decreased by cholesterol. With saturated, negatively charged SUV, the penetration of Hb into the bilayer was smaller, but the ionic interaction between the acidic lipids and the heme led to the detachment of the letter from globin. Cholesterol in such membrane increased the intercalation of Hb into the membrane, and at the same time completely prevented the loss of heme. The latter observations suggest that the fluid phase of the membrane favours the hydrophobic interaction with the protein, whereas the gel state promotes the partition of the heme into the bilayer. It is suggested that the effects of cholesterol are indirect, mediated by changes in membrane fluidity. By highlighting potentially harmful reactions between Hb and phospholipid bilayers, our findings may help the design of in-vitro stable hemosomes.

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