Micellar Complexes Research Articles

Hydrolytic Metalloenzyme Models. Micellar Effects on the Activation of the Hydroxyl Groups of N-Alkyl-2-(hydroxymethyl)imidazole Ligands by Cu2+ in the Transacylation of p-Nitrophenyl Picolinate

Abstract The activation of the hydroxyl groups of two N-methyl/dodecyl-2-(hydroxymethyl)imidazole ligands by complexation with Cu2+ was investigated kinetically by observing the rates of the release of p-nitrophenol in the transacylation of p-nitrophenyl picolinate (PNPP). The kinetics were carried out in aqueous buffers of pH ranging from 4.5 to 8.0 at 25 °C in the absence of surfactant micelles for a hydrophilic N-methyl ligand, and in the presence of micelles for a lipophilic N-dodecyl ligand. The kinetic analyses indicated that the 1:1 and 2:1 complexes of the ligand and Cu2+ are active nucleophiles with the N-methyl and N-dodecyl ligand, respectively. The pKa’s of the hydroxyl groups of these complexes were determined to be 7.00 in water and 6.41 in aqueous CTABr micelles, respectively, by analyses of the pH-rate profiles. The rate constants, which show the nucleophilic reactivities of ionized hydroxyl anions of complexes toward PNPP, were determined to be kN=1.11×104 and 1.25×106 mol−1 dm3 s−1 with N-methyl and N-dodecyl ligands, respectively, indicating that the latter micellar complex is 100-times more active than the former non-micellar counterpart.

Reversible disc-micellization of dimyristoylphosphatidylcholine bilayers induced by melittin and [Ala-14]melittin

The properties of melittin and a synthetic analogue, [Ala-14]melittin (P14A), in inducing reversible transitions between vesicles and micelles at the liquid-crystalline to gel phase transition temperature ( T m) in complexes with saturated phosphatidylcholines has been studied by deuterium NMR and freeze-fracture electron microscopy (EM). At concentrations between 3 and 5 mol% relative to lipid, each peptide causes reversible micellization of dimyristoylphosphatidylcholine (DMPC) bilayers when the temperature is lowered below T m. At concentrations of 5 mol% relative to lipid, the peptides induce macroscopic magnetic orientation of DMPC bilayers at temperatures around the centre of the lipid phase transition; at temperatures a few degrees above T m, magnetic orientation is lost. These effects suggest a progressive phase separation of peptide and lipid on cooling the complexes through the phase transition, resulting in increased vesicle deformability. The rates of gel phase micellization, and of bilayer reformation from micelles at temperatures above T m, are decreased by 100-fold in P14A: DMPC complexes compared with with melittin: DMPC complexes. Freeze-fracture EM indicates that P14A suppresses the formation of the gel phase in DMPC bilayers at temperatures below T m. EM observations of the time-dependence of the reformation of bilayers from micelles after incubating P14A: DMPC micellar complexes at temperatures above T m indicate that micelles fuse to form growing bilayer sheets from which multilameliar vesicles eventually form. The presence of intramembranous particles (IP) on the fracture faces of both melittin: DMPC complexes and P14A: DMPC complexes in the fluid phase indicates that under the conditions of the study (50 mM Tris-HCl (pH 7.5), 5 mM EDTA) the peptides are organized as discrete aggregates that penetrate deeply into the bilayer.

Interaction of Human Apolipoprotein A-I with Dipalmitoylphosphatidylcholine in Vesicular and Micellar Complexes

The interactions of human apolipoprotein A-I (apo A-I) with dipalmitoylphosphatidylcholine (DPPC) in vesicular complexes at low protein concentrations and in micellar complexes at high protein concentrations are compared. The C-terminal segment of this protein, with a relative molecular weight (Mr) of about 11,000, is protected on trypsin treatment of apo A-I-vesicle complexes. A segment within the sequence from Leu-189 to Arg-215 of apo A-I penetrates the hydrophobic interior of the membrane, as found in a hydrophobic labeling experiment involving 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID). No appreciable stretch of apo A-I in micellar complexes was found to be protected from the tryptic digestion. This indicates that the interactions of apo A-I with lipids in the vesicular and micellar complexes are different. The binding equilibrium of apo A-I as to DPPC vesicles at low protein concentrations, as studied by hydrophobic labeling of the bilayer-penetrating segment, is reached within about 1 h, while the formation of micellar complexes at high protein concentrations takes about 24 h at 42 degrees C. Time-dependent labeling studies involving photoreactive phosphatidylcholine (PC) with high apo A-I concentrations suggested an initial interaction with the head group region of the bilayer followed by interaction with the tail ends of the acyl chains of the lipid. A possible mechanism for the micellization process is discussed.

REACTIVITY OF CHLOROPHYLL a/b‐PROTEINS AND MICELLAR TRITON X‐100 COMPLEXES OF CHLOROPHYLLS a OR b WITH BOROHYDRIDE

Abstract—The reaction of several plant chlorophyll‐protein complexes with NaBH4has been studied by absorption spectroscopy. In all the complexes studied, chlorophyllbis more reactive than Chia,due to preferential reaction of its formyl substituent at C‐7. The complexes also show large variations in reactivity towards NaBH4and the order of reactivity is: LHCI > PSII complex > LHCII > PSI > P700 (investigated as a component of PSI). Differential pools of the same type of chlorophyll have been observed in several complexes.Parallel work was undertaken on the reactivity of micellar complexes of chlorophyllaand of chlorophyllbwith NaBH4to study the effect of aggregation state on this reactivity. In these complexes, both chlorophyll a andbshow large variations in reactivity in the order monomer > oligomer > polymer with chlorophyllbgenerally being more reactive than chlorophylla.It is concluded that aggregation decreases the reactivity of chlorophylls towards NaBH4in vitro,and may similarly decrease reactivity in naturally‐occurring chlorophyll‐protein complexes.

Substrate specificity for interfacial catalysis by phospholipase A 2 in the scooting mode

Action of pig pancreatic phospholipase A 2 on vesicles and micelles of homologous anionic phospholipids is examined in the absence of additives. As shown elsewhere (Jain et al. (1986) Biochim. Biophys. Acta 860, 435–447), hydrolysis of anionic vesicles occurs by interfacial catalysis in the scooting mode, i.e., the catalytic turnover is fast relative to the off-rate of the enzyme from the interface. When the rate of intervesicle exchange of the enzyme is negligibly slow, it hydrolyses only the substrate molecules in the outer monolayer of the vesicle to which it is bound. Interfacial catalysis in the scooting mode with a high processivity occurs on vesicles of anionic phospholipids, and under these conditions the dynamics and order of the substrate in the interface influences the catalytic turnover only moderately, i.e., about 2- to 10-fold. Similarly, anomalous kinetic effects of the thermotropic gel-fluid phase transition or of a change in the general disorder of the bilayer organization (fluidity) has a minor effect on the kinetics of hydrolysis in the scooting mode. Similarly, higher unsaturation and shorter acyl chains in the substrate modestly increase the rate of catalytic turnover by the low-calcium form of the enzyme without noticeably influencing the affinity of the enzyme for the interface. On the other hand, perturbation of the charge distribution in the substrate interface can shift the proportion of the bound enzyme by several orders of magnitude. For example, the membrane perturbing amphiphiles (e.g., mepacrine, indomethacin, compound 48/80, aristolochic acid, local anesthetics, and the products of hydrolysis) do not influence the catalytic turnover of the bound enzyme but the proportion of the bound enzyme. Short-chain anionic phospholipids are readily hydrolyzed by phospholipase A 2. Now no anomalous increase in the rate of hydrolysis is observed at the critical micelle as is the case with the zwitterionic analogs. This is because with anionic (but not with zwitterionic) substrates the enzyme forms an aggregated complex below the cmc of the monomer. The stability of these micellar complexes does not appear to change noticeably with the acyl chain length of the monomers. These observations show that the factors regulating the quality of interface substantially influence the binding of the enzyme, but not the catalytic turnover in the interface.

Apomyoglobin forms a micellar complex with phospholipid at low pH

Apomyoglobin was found to break down phosphatidylcholine vesicles to form a micellar complex at pH 4. Size reduction during this process was observed by measuring light scattering at 400 nm and by passing the complex through a gel-filtration column. Micellar complex formation was confirmed by electron microscopy.

Detection of a novel catalase in extracts of Mycobacterium avium and Mycobacterium intracellulare.

A novel class of catalase, which differs from the previously described M- and T-catalases of mycobacteria, was detected in strains of Mycobacterium avium and M. intracellulare. Designated A-catalase, this enzyme resisted inactivation at 68 degrees C, was inactivated by 3-amino-1,2,4-triazole (aminotriazole), and exhibited no peroxidase activity. All of these properties distinguished the enzyme from T-catalase. The A-catalase exhibited a Km of 70 mM H2O2, which is between the upper and lower extremes of the ranges reported for T- and M-catalases, respectively. The A-catalase appeared to be more hydrophobic than M-catalase and did not react with antiserum to a representative sample of this class. The banding patterns of T- and M-catalases seen by polyacrylamide gel electrophoresis (PAGE) were essentially unaffected by the incorporation of sodium dodecyl sulfate (SDS) into the PAGE system, whereas the single band of A-catalase seen by PAGE without SDS resolved into as many as five bands in the presence of SDS; these bands were all of slower mobility than the original band. The banding pattern seen with SDS appeared to be related more to counterion charge effects than to molecular size increases that could be attributed to SDS complexed to the protein. It remains to be determined whether the multiple A-catalase bands reflect different proteins or different SDS micellar complexes of a single protein.

Electrochemical Formation of Polymer Thin Films by Disruption of Micellar Complex

Abstract Polymer thin films of poly(4-vinylpyridine), and copolymer of methyl methacrylate and methacrylic acid were prepared by electrolysis of an aqueous solution containing a surfactant with ferrocenyl moiety and corresponding polymer. The film thickness increased linearly with the amount of electricity. The morphology of the films was investigated by scanning electron microscopy.

Physical properties of apoprotein B in mixed micelles with sodium deoxycholate and in a vesicle with dimyristoyl phosphatidylcholine.

Apoprotein B, the major apoprotein of normal human low density lipoprotein (LDL) was solubilized with sodium deoxycholate (NaDC). The protein was recombined with the phospholipid dimyristoyl phosphatidylcholine (DMPC) to produce a complex of DMPC-apoB (4:1 w/w). (Biochemistry. 22: 3170-3178. 1983). Carboxyfluorescein and [3H]dextran entrapment studies show the DMPC-apoB 4:1 (w/w) complex to encapsulate an aqueous volume of 0.17 microliter/mumol of DMPC. From the chemistry and morphology of the complex and the evidence that the complex possesses an encapsulated volume, the most appropriate structural model for this assembly is that of a phospholipid single bilayer vesicle into which apoB is incorporated. Differential scanning calorimetry (DSC) and circular dichroic spectroscopy (CD) were used to investigate the physical properties of apoB in the mixed micellar complex with NaDC and in the vesicular DMPC-apoB complex. CD studies of apoB in NaDC mixed micelles show that apoB exhibits a similar secondary structure as apoB of native LDL over the temperature range 5-30 degrees C. Reversible structural changes occur between 30 and 50 degrees C. However, above 50 degrees C, disruption of the micellar particle and endothermic protein unfolding and denaturation occur with a Tmax of 52 degrees C and an enthalpy of 0.22 cal/g apoB, as shown by DSC. The DMPC-apoB complex exhibits a reversible thermal transition centered at 24 degrees C (delta H = 3.34 Kcal/mol DMPC) which is associated with the order-disorder transition of the hydrocarbon chains of DMPC. An endothermic transition occurs over the range 53-70 degrees C (delta H = 2.09 cal/g apoB) which, as shown by CD and turbidity study, corresponds to protein unfolding-denaturation and particle disruption. CD shows that apoB in the vesicular environment undergoes a series of conformational changes. The major alterations occur over the temperature range of the order-disorder transition of the phospholipid. Between 37-60 degrees C, the conformation is similar to that observed in native LDL.

Immunoprevention of Friend Leukaemia Virus-induced Erythroleukaemia by Vaccination with Aggregated gp70

Vaccines prepared from Friend leukaemia virus envelope and core polypeptides were compared for their efficiency in preventing erythroleukaemia in mice. High doses (100 micrograms) of gp85, the micellar complex of the envelope polypeptides gp70 and p15E, completely protected STU mice. The same dose of purified gp70 still protected about 80% of the animals, while p15E did not affect the cumulative mortality. The internal viral polypeptide p30 was ineffective. Serological examination indicated that immunity against death from leukaemia was mediated by specific antibodies. These leukaemia-preventing antibodies were predominantly induced by immunization with the gp70 env gene product, since p15E showed only minor protection. Glycoprotein gp70, however, was more effective when given as the gp85 micellar complex. An even more potent vaccine was obtained when gp70 was coupled to keyhole limpet haemocyanin (KLH) by glutaraldehyde. Ten micrograms gp70 coupled to KLH was enough to save more than 90% of Friend leukaemia virus-infected mice from erythroleukaemia. KLH may also be a suitable experimental carrier for subunits of gp70 or synthetic oligopeptides for viral vaccines.

Phorbol ester binding and activation of protein kinase C on triton X-100 mixed micelles containing phosphatidylserine.

A mixed micellar assay for the binding of phorbol-esters to protein kinase C was developed to investigate the specificity and stoichiometry of phospholipid cofactor dependence and oligomeric state of protein kinase C (Ca2+/phospholipid-dependent enzyme) required for phorbol ester binding. [3H]Phorbol dibutyrate was bound to protein kinase C in the presence of Triton X-100 mixed micelles containing 20 mol % phosphatidylserine (PS) in a calcium-dependent manner with a Kd of 5 X 10(-9) M. The [3H]phorbol dibutyrate X protein kinase C . Triton X-100 . PS mixed micellar complex eluted on a Sephacryl S-200 molecular sieve at an Mr of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate. This conclusion was supported by molecular sieve chromatography of a similar complex where Triton X-100 was replaced with beta-octylglucoside. Phorbol dibutyrate activation of protein kinase C in Triton X-100/PS mixed micelles occurred and was dependent on calcium. The PS dependence of both phorbol ester activation and binding to protein kinase C lagged initially and then was highly cooperative. The minimal mole per cent PS required was strongly dependent on the concentration of phorbol dibutyrate or phorbol myristic acetate employed. Even at the highest concentration of phorbol ester tested, a minimum of 3 mol % PS was required; this indicates that approximately four molecules of PS are required. [3H]Phorbol dibutyrate binding was independent of micelle number at 20 mol % PS. The phospholipid dependencies of phorbol ester binding and activation were similar, with PS being the most effective; anionic phospholipids (cardiolipin, phosphatidic acid, and phosphatidylglycerol were less effective, whereas phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin did not support binding or activation. sn-1,2-Dioleoylglycerol displaced [3H]phorbol dibutyrate quantitatively and competitively. The data are discussed in relation to a molecular model of protein kinase C activation.

Micellar complexes of energy transfer

In micellar solutions of cationic surfactants the rate of oxidation and electrooxidation of luminol, quantum recovery of the fluorescence of anionic fluorescer, and the probability of electron excitation energy transfer are enhanced. Owing to these facts, the intensity of chemiluminescence and electrochemiluminescence of luminol in the presence of cetyltrimethylammonium bromide and fluorescein is from 10 to 100 times higher compared with that of pure solutions free of surfactants. The intensity of the emitted radiation is proportional to the concentrations of micellar complexes, which allows the introduction of the idea of a complex of electron excitation energy transfer in which the excited oxidation product of luminol (aminophthalate ion) plays the role of the donor and the fluorescer the role of the acceptor of energy. The substrate-binding constants are of an order of magnitude of 10 4 up to 10 5 M −1. From the formal point of view, the intensity of chemiluminescence obeys the same laws as the kinetics of enzymatic and micellar catalysis, respectively.

Intestinal absorption of a .BETA.-adrenergic blocking agent nadolol. III Nuclear magnetic resonance spectroscopic study on nadolol-sodium cholate micellar complex and intestinal absorption of nadolol derivatives in rats.

The stable micellar complex formation between nadolol and sodium cholate was studied. The proton nuclear magnetic resonance spectroscopic study showed that the signals of protons in the hydrophobic site of cholic acid, i.e., the methine signals of C-7 and C-12, and the methyl signals of C-18 and C-19. exhibited rather larger shifts than those of the other protons. The longitudinal relaxation times of the aromatic ring protons (C-4, C-6, C-7 and C-8) of nadolol were shortened by about 10% in the presence of sodium cholate, indicating that the aromatic ring protons were more immobilized upon complex formation. These results suggest that the hydrophobic protons of sodium cholate and nadolol interact with each other, and complex formation with the hydrophobic protons inside and the hydrohilic protons outside may occur in aqueous solution. The intestinal absorption of four nadolol derivatives was examined by the in situ ligated loop method in rat jejunum in the presence of sodium cholate. The absorption of the trans-2, 3-diol isomer was inhibited as effectively as that of nadolol. The absorption of the 2-and 3-monohydroxy derivatives and cis-2, 3-dimethoxy derivative was inhibited by sodium cholate at 1 h after dosing, but no inhibition was observed at 4 h. This absorption behavior of nadolol derivatives was well correlated with the apparent dissociation constants of their micellar complexes with sodium cholate. It is concluded that the stability of the complexes of β-adrenergic blocking agents with sodium cholate depends upon the steric features of both hydrophobic and hydrophilic parts of the drug molecules. The hydrophilic cis-2, 3-diol moiety of nadolol plays an important role in stabilizing the micellar complex with sodium cholate.

Intestinal absorption of a beta-adrenergic blocking agents nadolol. II. Mechanism of the inhibitory effect on the intestinal absorption of nadolol by sodium cholate in rats.

The mechanism of the inhibitory effect of sodium cholate and its taurine and glycine conjugates on the intestinal absorption of nadolol was investigated in rats. It was found that the uptake of nadolol into the intestinal membrane was inhibited by sodium cholate and its conjugates, but the membrane permeability to nadolol was not affected. The inhibitory effect on nadolol absorption can be interpreted in terms of loss of thermodynamic activity of nadolol due to stable micelle formation, resulting in a decrease of the uptake into the intestinal membrane. Other β-blocking agents also form micellar complexes with sodium cholate, but their micelles are ten times less stable than that of nadolol. It is concluded that the stability of the micellar complex may be an important factor in the inhibition of intestinal absorption of nadolol. The micellar stability seems to be associated with not only the chemical nature of the bile salts but also that of the drug.

Effect of temperature and low pH on structure and stability of matrix porin in micellar detergent solutions

Thermal and low pH stabilities of matrix porin (Omp F) solubilized in the micellar solutions of ionic (SDS) and nonionic detergents were investigated by the methods of circular dichroism, intrinsic fluorescence, light scattering and sedimentation velocity. The stability of porin structure in solution is much higher in the presence of β-octyl glucoside than with SDS. In the presence of SDS, sharp transitions were detected by all parameters measured, above 55°C at neutral pH and below pH 4.5 at 20°C. These transitions involve at least three concomitant processes: unfolding of protein, dissociation of trimers to monomers and the disruption of the protein-detergent micellar complexes, all events being irreversible in the presence of SDS. The nonionic detergent, β-octyl glucoside, increases the stability of porin in acidic conditions, since neither dissociation nor denaturation was observed in the pH region between 7.5 and 2.0. However, at pH < 3.5, small reversible changes in protein structure became evident. The thermal stability of porin is also increased by β-octyl glucoside as evidenced by a transition temperature 15–20°C higher as compared to SDS. A considerable degree of native porin structure was regained after heat treatment in the presence of β-octyl glucoside, though the reconstituted trimers were not identical to the native ones. The addition of lipopolysaccharide and divalent cations (Ca 2+, Mg 2+) to the experimental system did not improve the thermal reversibility.

Complex photophysics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex.

The fluorescence emission of the single tryptophan in porcine pancreatic phospholipase A2, its zymogen, and a micellar complex of the enzyme with the nonhydrolyzable substrate analogue n-hexadecylphosphocholine has been studied by both steady-state and time-resolved techniques. Stern-Volmer quenching studies with acrylamide indicate that, both in the enzyme and in the zymogen, the tryptophan is exposed to solvent. Similar studies with ionic quenchers show that there is appreciable ionic character to the tryptophan environment. Single photon counting fluorescence measurements were performed using a high repetition rate synchronously pumped dye laser as a light source. When tryptophan fluorescence is collected with a broad-band (80-nm) emission filter, the decay kinetics in the enzyme and the zymogen require at least three, and often four, exponential terms for a proper description. The decay kinetics can be adequately described by three exponential terms when the fluorescence is collected at specific wavelengths by using narrow (10-nm) band-pass filters. The lifetimes are approximately constant across the emission band, but the amplitudes vary with the fraction of the long lifetime increasing at longer emission wavelengths. Formation of a complex between phospholipase A2 and micelles of n-hexadecylphospohocholine produces large changes in the tryptophan emission that are associated with transfer to a hydrophobic environment. The decay kinetics of tryptophan in the enzyme/micelle complex appears to require only two exponential terms. This is the first reported instance of fluorescence data from a single tryptophan protein requiring more than double-exponential decay kinetics. The results are discussed in terms of the range of environments sampled by the tryptophan residue and the resulting distribution of lifetimes.

The interaction of apolipoprotein A-I with small unilamellar vesicles of L-alpha-dipalmitoylphosphatidylcholine.

The interaction between apolipoprotein A-I and small unilamellar vesicles of dipalmitoylphosphatidylcholine at the lipid phase transition resulted in complete release of vesicle contents at molar ratios of lipid to protein from 4000:1 down to 50:1. This indicated the existence of two types of stable complexes: a vesicular apo-A-I complex with a maximum of two to three apo-A-Is/vesicle, and a micellar complex (disc) with a stoichiometry of about 50 phosphatidylcholines/apo-A-I (mol/mol). We characterized the complexes by density gradient centrifugation, by gel filtration, and by immunoprecipitation using an anti-apo-A-I antibody. The morphology of the discs was similar to that of previously reported discs. Apo-A-I-induced release of vesicle contents was monitored by the relief of self-quenching of vesicle-encapsulated carboxyfluorescein. Using this assay we characterized the nature of the interaction between apo-A-I and phospholipid vesicles. The formation of complexes between vesicles and apo-A-I followed a two-step process; below or above the lipid phase transition temperature (Tc), apo-A-I bound to phosphatidylcholine vesicles but caused little leakage of contents. Kinetic analysis of the interaction between apo-A-I and dipalmitoylphosphatidylcholine vesicles below Tc indicated that about 1 in 500 collisions leads to a stable apo-A-I-vesicle complex. The second step involved passage of those complexes through Tc, which resulted in a very rapid transition into discs or vesicular complexes. Vesicular complexes contain apo-A-I which was no longer capable of interacting with pure lipid. Discs, on the other hand, interacted with vesicles at their phase transition.

Membrane cholesterol uptake by recombinant lipoproteins

Recombinant lipoproteins, prepared with apo A-I isolated from human high density lipoprotein (HDL) and various phospholipids (PLs), were compared with respect to their ability to remove cholesterol (Chol) from labelled erythrocyte ghost membranes. It was found that uptake of Chol was essentially complete following an 8 h incubation at 37°C. Quantitation of the amount of cholesterol taken up showed that recombinants prepared from bovine brain sphingomyelin (BBSM) or dipalmitoyl phosphatidylcholine (DPPC) acquired the highest proportion of Chol (80–140 mol/mol protein), whereas shorter chain phospholipids like dimyristoyl phosphatidylcholine (DMPC) acquired little or no membrane Chol. Chemical analysis of the incubation products indicated that this latter result was due to loss of PL, presumably to the membrane, with consequent disruption of the recombinant particle. Results with DPPC:A-I recombinants of differing PL/protein ratios and sizes showed that Chol uptake was fairly constant at 0.70 mol Chol/mol PL. It is concluded that discoidal, phospholipid-rich recombinant lipoproteins can effectively take up substantial amounts of Chol from physiological membranes, provided that the PLs utilized form micellar complexes which are capable of retaining their structural integrity during the incubation with the membranes.

Influence of the protein conformation on the interaction between α-lactalbumin and dimyristoylphosphatidylcholine vesicles

α-Lactalbumin is a globular protein containing helical regions with highly amphiphathic character. In this work, the interaction between bovine α-lactalbumin and sonicated dimyristoylphosphatidylcholine vesicles has been compared in different circumstances which influence the protein conformation i.e., pH, ionic strength, decalcification, guanidine hydrochloride denaturation. Above the isoelectric point the interaction is mainly electrostatic; improved electrostatic interaction results in better contact with the apolar lipid phase. Below the isoelectric point, hydrophobic forces dominate the interaction and the vesicles are solubilized. The mode of interaction is not determined to a great extent by the demetallization of the protein. However, by a more explicit unfolding of the globular structure with guanidine hydrochloride, micellar complexes can be formed with the lipid, even at neutral pH. From this study it is obvious that the presence or capability for formation of helices with high amphipathic character is not a sufficient condition for lipid solubilization by a globular protein. Also, the capability of a globular protein to unfold its tertiary structure seems to be a prerequisite for its capability to lipid solubilization.

Kinetics of phospholipid transfer between liposomes (neutral or negatively charged) and high-density lipoproteins: a spin-label study of early events

The kinetics of spin-labeled phosphatidylcholine transfer between vesicles and HDL particles exhibited a two-phase process, as seen by ESR spectroscopy. The results were analyzed by considering several possible steps in the overall transfer, whose aspects were also studied: (i) micellar complex formation after HDL apolipoprotein-vesicle mixture, (ii) the rate of PC transfer from the micellar complex to HDL, (iii) the rate of the reverse reaction between overloaded HDL particles and other particles such as HDLs, LDLs, and lipid vesicles. The results agree most convincingly with a mechanism in which the diffusion of phospholipids into the HDL-endogenous lipids is the limiting step, occurring as a two-step process. In addition, we observed a negative charge effect on the lipid transfer rates and yields.