Biphasic protonation of hydrophilic cargo agents in unilamellar phospholipid vesicles: implications about cargo location.

The commonly used cargo agents for liposome entrapment, chromate and 5(6)-carboxyflourescein (CF), have been sequestered in small unilamellar vesicles composed of dipalmitoylphosphatidylcholine through preparations involving either sonication or extrusion methods. Once loaded, these water-soluble chromophoric cargo agents have been exposed to small quantities of externally applied acid solution, which decreases the pH from neutral to approx. 6. By monitoring photometrically the time profile of the protonation of the sequestered chromophores, it is evident that the uptake of protons by each cargo agent is biphasic. An immediate spectral change is followed by further change over 10-40 min, where the extent of protonation occurring in each time frame is approximately equal. The vesicles themselves are unaffected by the induced pH change. The leakages of both chromate and CF from loaded sonicated vesicles were monitored at both 25 degrees C and 45 degrees C. Overall, the leakage processes exhibited a deceleration over time. The biphasic protonation and decelerating leakage phenomena are together interpreted in terms of a mechanism of cargo loading involving an intercalation of the water-soluble agent along with water into the vesicle bilayer, rather than involving internal capture of the cargo inside the vesicles, or through electrostatic interactions with the bilayer surfaces. In addition, the measured extents of cargo loading are more consistent with calculated estimates of loading through bilayer intercalation than with those for internal capture.

The interaction of oxymyoglobin with hydrogen peroxide: a kinetic anomaly at large excesses of hydrogen peroxide.

The reaction of oxymyoglobin (MbO2) with H2O2 has been examined at pH 7.2 and 20(+/- 2) degrees C for reactant ratios of [H2O2]:[MbO2] greater than approximately 15:1. Under the conditions of large excesses of H2O2, the reaction is characterized by an increase in the rate of loss of MbO2 as [H2O2] is increased, for which a value of k(MbO2 + H2O2) approximately 3 M-1 s-1 is obtained. This kinetic behavior contrasts the saturation kinetics observed previously at lower values of [H2O2]. The change in kinetics at increasing excesses of H2O2 is accompanied by a progressive tendency toward the direct formation of ferrimyoglobin at the expense of ferrylmyoglobin formation. A mechanism is proposed in which an initially formed intermediate produces the ferryl derivative in competition with the formation of ferrimyoglobin through the interaction of further H2O2. Overall, the H2O2 is catalytically decomposed by the MbO2. This mechanism is integrated with that determined previously at low excesses of H2O2 into a complex general scheme that applies over the entire studied range of [H2O2]:[MbO2]. No evidence is obtained for the conversion of ferrylmyoglobin to oxymyoglobin by the large excesses of H2O2, regardless of whether the ferryl derivative is the product of the reaction of H2O2 with the oxy or ferri derivative of myoglobin.

The interaction of oxymyoglobin with hydrogen peroxide: the formation of ferrylmyoglobin at moderate excesses of hydrogen peroxide.

The reaction of oxymyoglobin with H2O2 has been examined at pH 7.2 and 20(+/-2) degrees over a range of [H2O2] up to an initial excess of 25:1. The reaction is characterized by a direct conversion of oxymyoglobin to ferrylmyoglobin without the intermediacy of the ferri derivative. The initial rate of loss of oxymyoglobin is first-order with respect to [oxymyoglobin], and exhibits saturation kinetics with increasing [H2O2]. In addition, the stoichiometric relationship between the reactants varies as [H2O2] increases. A complex non-Michaelis-Menten mechanism is proposed in which an intermediate, produced upon the initial interaction of the reactants, regenerates oxymyoglobin by reaction with further H2O2, in competition with the formation of the ferryl derivative. In this way, oxymyoglobin catalytically decomposes excess H2O2. Deoxygenated ferromyoglobin is substantially more reactive with H2O2 in producing the transient intermediate than the oxy analog. Some fundamental similarity is noted between the catalytic mechanism and that of catalase activity. From a detailed examination of the probable nature of the intermediate, conventional Fenton reactivity is rejected for the reaction of H2O2 with oxymyoglobin.

Interaction of radiation-generated radicals with myoglobin in aqueous solution. III. Effects of oxygen and catalase on the product distribution in solutions of ferrimyoglobin containing N2O.

The gamma-radiolysis of aqueous solutions of ferrimyoglobin in the presence of N2O at pH 7.3 has been examined as a function of added catalase and oxygen. Changes in the nature of the heme group have been monitored by visible absorption spectrophotometry and analysed quantitatively by a multiple wavelength method based on Beer's Law. Simple chemical analyses have been used to confirm qualitative identification of the product derivatives. As observed previously, the ferriheme is reduced by indirect globin-mediated action initiated by OH/H. The yield of reduced product decreases as [O2] increases. Conversion to ferrimyoglobin through the participation of H2O2 derived from irradiated water and from protein-mediated processes in oxygenated solution, is eliminated by the presence of catalase. Formation of a hemichrome form of ferrimyoglobin is apparent at higher doses in the presence of O2. These results demonstrate that oxygen plays an important role in controlling the nature and extent of redox that manifests ultimately on the heme group of ferrimyoglobin as a result of the initial interaction of OH/H.