the interaction of apoprotein from porcine high-density lipoprotein with dimyristoyl...

Eur. J. Biochem. 48, 583-594 (1974)

The Interaction of Apoprotein from Porcine High-Density Lipoprotein with Dimyristoyl Phosphatidylcholine Helmut HAUSER, Robert HENRY, Robert B. LESLIE, and J. Morriss STUBBS Biophysics Division, Unilever Research Laboratory, Colworth/Welwyn, Hertfordshire

(Received June 5, 1974)

The morphology and structure of lipoprotein complexes reconstituted from apoprotein from porcine high-density lipoprotein and dimyristoyl lecithin have been studied by a range of physical techniques. At pH 8.6 and ionic strength I 2 0.1, disc-shaped particles are observed with a radius of 4.0-4.5 nm and a thickness of 4.36 k 0.07 nm, the latter value being in good agreement with that of the average lipid bilayer thickness.

The values for the number, weight and Z-average particle weights are M , = 227000 daltons < M , = 325000 daltons < M , = 410000 daltons, respectively, and indicate the polydispersity of these complexes. The number-average particle weight is in good agreement with that of 236000 daltons determined from sedimentation and diffusion experiments. The latter particle weight is also consistent with the particle volume, determined from the dimensions of the disc, and the experimental partial specific volume of the complex, V z 5 = 0.917 ml/g. At the point where all the apoprotein is complexed the average lipid/protein molar ratio is 100 6. Particle size and weight analysis indicate that each lipoprotein disc of radius 4.0- 4.5 nm consists of 200 lipid and 2 apoprotein molecules.

If lecithin is present in excess of the complex described above, all the lipid may interact with apoprotein up to molar lipid/protein ratios of 200: 1 depending on the sonication conditions used.

The lipoprotein discs undergo an ionic-strength-dependent monomer-dimer equilibrium with low ionic strength leading to the side-by-side aggregation of discs. At an ionic strength I FZ 0.01 the disc radius is about twice that observed at I 2 0.1.

The results with dimyristoyl lecithin - apoprotein complexes are compared with those obtained with dilauroyl and dipalmitoyl lecithin - apoprotein complexes.

Lecithin and apoprotein interact to form a loose, disc-shaped complex in which the apoprotein is oriented at the lipid-water (hydrophobic-hydrophilic) interface such that all the polar groups of both lipid and protein are readily accessible to the aqueous medium.

Most of the lipid is present as a bilayer, however the cooperative motion of the lipid is significantly reduced due to the interaction with apoprotein. The latter shows a significant increase in a helical content from 50 :( to about 60- 65

The dispersing (solubilizing) effect which the apoprotein has on unsonicated multilayers of lecithin is explained in terms of its surface activity and conformational properties which may allow for the regional separation (accumulation) of opposite charges.

when complexed with lecithin.

In recent publications the considerable structural similarities of the human and porcine high-density lipoproteins and their constituent apoproteins have been documented [l - 31. Two major proteins (vari- ously referred to as fraction 111, apo LP-Gln-I or apo A-I and fraction IV, apo LP-Gln-11 or apo A-11) account for more than 90% of the total apoprotein of human high-density lipoprotein, the former being present in roughly three times the concentration of

the latter. The major apoprotein of porcine high- density lipoprotein has similar chemical, immuno- logical, and physical properties to human apo LP- Gln-I [2,3], but the relative amount of a protein corresponding to human apo LP-Gln-I1 appears reduced. The complete primary sequence of human apo LP-Gln-I1 is now available [4] and rapid progress is currently being made in the isolation and definition of the lipid-binding site(s) in apo LP-Gln-I [ 5 ] and

Eur. J . Biochem. 48 (1974)

584 Lecithin - Apoprotein Interaction

apo LP-Gln-I1 [6]. The relative importance, and possible synergistic effects of apo LP-Gln-I and I1 in determining the overall structure and morphology of native and reassembled high-density lipoproteins remains obscure [5]. The special attributes of the apoproteins which are responsible for their lipid- binding ability are completely unknown. The possible importance of a conformationally determined "bi- modality" and/or the role of (appropriately oriented) dipoles have been suggested [5,7,8]. To further elucidate the relative roles played by the lipid and apoprotein we have investigated in this work the interaction of phosphatidylcholines of defined chain length with the total apoprotein fraction derived from porcine high-density lipoprotein (density I .070 - 1.21 g,/ml). The emphasis will be on the interaction of dimyristoyl phosphatidylcholine (dimyristoyl lecithin) with the porcine apoprotein ; the gross morphology and some physical properties of the reconstituted lipoprotein complex as well as of the constituents will be presented here while forthcoming papers will discuss these lipoprotein complexes on a molecular scale and attempt to relate this information to natural serum lipoproteins.

MATERIALS AND METHODS

Muter i d s

Dilauroyl-~-3-phosphatidylcholine was prepared by acylation of L-a-glycerylphosphorylcholine - cad- mium chloride with lauroyl chloride [9]. Dimyristoyl- L-3-phosphatidylcholine and dipalmitoyl-~-3-phosphatidylcholine were purchased from Fluka AG (Buchs, Switzerland) and, if necessary, purified by silicic acid chromatography [lo]. The purity was checked by thin-layer chromatography and the fatty acid composition by gas-liquid chromatography.

Apoprotein from porcine high-density lipoprotein was prepared according to the method of Scanu [ l l ] using ethanol - ether (3 : 2, by vol.) as the delipidation solvent. To compare the effect of different lipid sol- vents, in some preparations the lipids were extracted with chloroform-methanol(2: 1, by vol.) [3].

Bio-Gel A50m was purchased from Bio-Rad Labo- ratories (Richmond, California) ; all other chemicals were AR grade.

Sample Preparation

Phospholipid dispersion were prepared as described before [12-141 and sonicated with a 150-watt probe-type ultrasonic disintegrator (from M.S.E., Crawley, Surrey, U. K.) using either l-ml samples and a microtip (diameter z 0.3 cm) or 3-ml samples

and a cylindrical tip (diameter = 1 cm) under similar experimental conditions to those described before [14]. Dilauroyl and dimyristoyl lecithins (the gel .+ liquid crystal transition temperature T, of these phospholipids are = 3 "C and 23 "C, respectively) were dispersed in water or buffer (0.01 M Tris pH 8.3 - 8.6, 1 mM EDTA, 0.02% NaN,, the ionic strength was adjusted by adding NaCl) at 25 "C, dipalmitoyl lecithin (T', = 41 "C) was dispersed at 50 "C. 3 ml phospholipid dispersions were sonicated 10 - 20 min and 1 ml dispersions were sonicated more gently for 3 x 2-min with 1 -min intervals. After sonication an aliquot of the lipid yas checked for purity by thin- layer chromatography. No lysophosphatidylcholine or other degradation products were detected when applying 300 pg as a spot. The lipoprotein complexes were prepared by adding dry apoprotein to the sonicated phospholipid dispersion and, unless other- wise stated, sonicating the mixture for a further 2 or 5 min. Any titanium released during sonication was removed by centrifugation at 3000 rev./min. Lipo- protein complexes were also prepared without sonication by incubating unsonicated phospholipid dispersions with apoprotein solutions and allowing several hours for equilibrium to be reached. Phospholipid concentrations were determined by phosphorus analysis according to the method of Chen et al. [15]; protein concentrations were determined either by the method of Lowry et al. [16] as modified by Schacterle and Pollack [17] or with ninhydrin according to Moore and Stein [18].

Gel-Permeation Chromatography

Gel permeation chromatography was carried out at 25 "C using mainly Sepharose 4B as described before [12,14]. Ackers' treatment [19] was used to calculate the Stokes' radius r from the elution volume V, of the peak fraction.

Y = U, + 6, ERFC-' (T (1)

where ERFC-' (T is the inverse error function comple- ment of 0 and

where V,, Vt are the void and total volumes, respectively. Using marker proteins as described before [12] and plotting their Stokes' radii as a function of ERFC- ' 0, straight line relationships were obtained with both the Sepharose 4B and the Bio-Gel A50m column. The intercepts and slopes are the column constants a, and b,, respectively, which have been determined for the different buffer systems used in this work (see Table 1).

Eur. J. Biochem. 48 (1974)

H. Hauser, R. Henry. R. B. Leslie, and J. M. Stubbs 585

Table 1. Column constants ,for the Sepharose 4B and the Bio- Gel A50nz columns High ionic strength buffer was 0.1 M NaCl, 0.01 M Tris pH 8.6, 1 mM EDTA, 0.02'%, NaN,. Low ionic strength buffer was 0.01 M Tris pH 8.6, 1 mM EDTA, 0.02% NaN,

Column, buffer vo v, a0 ho

ml ~~~ ~ _ _ _ _ ~

Sepharose 4B, high

Sepharose 4B, low

Bio-Gel ASOm, high

ionic strength 51.0 162.4 -12.8 165.1

ionic strength 50.1 163 -10.0 150

ionic strength 15.0 41.6 2.0 225.6

Electron microscopy [14], density-gradient centrifugation [3] and analytical ultracentrifugation [12] have been described before. Molecular weights were determined by the low-speed equilibrium method [29] using interference optics. Diffusion coefficients were determined in the analytical ultracentrifuge at a speed of 5200 rev./min using schlieren optics [28].

Density Measurements and Determination of Partial Specific Volumes

Densities were measured in the digital precision density [22] meter DMA 02C (A. Paar K.G., Graz, Austria) in an air-conditioned room with the density meter being enclosed in a transparent plastic hood. Measurements were carried out at 20 "C ? 0.003 "C. This accuracy in the temperature control was achieved with an ultrathermostat built in our own workshop and a Tronac precision temperature controller (model PTC-1000, with a standard probe, from Tronac, Inc., University Station Provo, Utah, U.S.A.). The temperature of the water in the ultrathermostat was kept constant within ? 0.001 "C of the set temperature. Densities were measured with an accuracy of & 1 pg/ ml and the accuracy in the partial specific volume of phospholipids was better than & 0.0004ml/g and that of proteins was & 0.0002 ml/g.

RESULTS

Gel-Permeation Chromatogvuphy

Fig. 1 A is the column chromatogram of a sonicated dimyristoyl lecithin dispersion in 0.1 M NaC1, 0.01 M Tris pH 8.6, 1 mM EDTA, 0.02% NaN, (henceforth referred to as high ionic strength buffer). A similar elution pattern was obtained with the same Tris buffer in the absence of NaCl (henceforth referred to as low ionic strength buffer). Some lipid was eluted in the void

volume (arrow) indicating that some multilamellar structures were present besides single shelled vesicles eluted at an average elution volume V, = 90 f 3 ml. Inserting into Eqn (1) the average Stokes' radius for single dimyristoyl lecithin vesicles was calculated as r = 9.5 f 0.4 nm [13,14]. Reducing the time and/or intensity of sonication caused the fraction in the void volume (Stokes radius > 30.0 nm) to increase (Fig. IB). Fig. 1A also shows that at 25 "C the dimyristoyl lecithin vesicles retained most but not all of the 22Nat incorporated in their internal compart- ment (c$ [14,25]). The fact that the radioactivity between the phospholipid peak and the 22NaC1 fraction at V, = 156 ml does not return to background level indicates that some 22Na+ was leaking out from the vesicles during chromatography.

Fig. 1 C and D are the column chromatograms of apoprotein from porcine high-density lipoprotein obtained with Sepharose 4B, in buffer of low and high ionic strength respectively. The elution patterns are characteristic of a protein undergoing reversible aggregation. The elution volumes of the main peaks in Fig. 1 C and 1 D depend on the ionic strength of the buffer, low ionic strength enhancing the aggregation of the apoprotein. The elution volumes V, were V, = 105 f 5 ml and V, = 127 f 3 ml at low and high ionic strength, respectively. Using these elution volumes and Eqn (1) the Stokes' radii obtained for low ionic strength varied from r = 8.0-9.5 nm (average: 8.8 & 0.2 nm) whilst at high ionic strength r = 3.5 f 0.4 nm. The elution pattern and thus the aggregation of the protein was related to the delipidation procedure. The chromatogram of apoprotein delipidated with chloroform - methanol (2 : 1, by vol.) differed from that of our standard preparation which was delipidated with ethanol-ether (3 : 2, by vol.). The former gave (at high ionic strength) a major fraction consisting of two peaks of elution volumes V, = 119 ml and V, = 125.6 ml, respectively, and a small proportion of probably monomeric protein molecules eluted at V , > 135 ml. The Stokes' radii corresponding to the elution volumes V, = 125.6 ml and V, = 119 ml were 3.6 nmand4.5 nm, respectively.

Fig. 1 E and F shows the column chromatograms of a lipoprotein complex consisting of dimyristoyl lecithin and apoprotein [weight (molar) ratio of lipidiprotein = 2.5: 1 (100: 1) using 27000 [2,3] for the molecular weight of the apoprotein] at low and high ionic strength, respectively. In both cases single, rather sharp, symmetric peaks were obtained indicative of a rather homogeneous particle size distribution. The homogeneity of the peaks varied from sample to sample, depending on the method of sample preparation, and the sonication time in particular appeared to effect the homogeneity. The effluent


A

0

Ve (m0

Y

V, (ml)

C- 0 2C 40

Lecithin - Apoprotein Interaction

3

T D G.4

k-1 1 160 180 200 220 240

160 180 200 220 240

140 160 180 200 220 240 V, (ml)

Fig. 1 . Gel-/,cr,i?ii.citioii clzr-omatogr-uplij OM Sepharose 4 B (33 x 2.5 cm). (A) 3 ml dimyristoyl lecithin dispersion (8 mg/ ml) in buffer (0.1 M NaCl containing "Na+, 0.01 M Tris pH 8.6, 1 mM EDTA, 0.02% NaN,) sonicated for 10 min. (o--o) Radioactivity in arbitrary units; ( x --x) phosphorus analysis. (B) As A except that the dispersion was sonicated for 6 min ( x - x ). For comparison, the chromatogram of a dispersion sonicated for 10 min is included (0-0). The arrows indicate the void volume V,. (C) Por- cine apoprotein prepared according to the method of Scanu el al. [ l l ] . 3 ml of the protein solution (2.2 mg/ml) in low

A --J _. 0 l a 160 180 200 220 210

ionic strength buffer (0.01 M Tris p l i 8.6, 1 mM EDTA, 0.02';; NaN,) were sonicated for 5 min and applied to the column. (D) As C except that high ionic strength buffer containing 0.1 M NaCl was used. (E) Dimyristoyl lecithin- apoprotein complex (lipid/protein weight ratio 2.5: 1) in low ionic strength buffer (as in C) application: 3 ml containing 12 mg lipoprotein/ml were prepared as described in Methods. (F) As E except that the high ionic strength buffer was used. (-0) Phosphorus analysis, ( x - x ) protein analysis. The dashed curve represents the lipidlprotein weight ratio calculated for each fraction from the lipid and protein analysis


H. Hauser, R . Henry, R. B. Leslie, and J. M. Stubbs

150- . Y 1

c .~ 100-

L a

50

587

-

was analyzed for both lipid and protein and at the above 1ipid:protein ratio the lipid and protein were eluted together (Fig. 1 E and F). Inserting the average elution volumes V, = 95 4 ml at low ionic strength and V, = 120 f 1.5 ml at high ionic strength into Eqn (1) the average Stokes’ radii thus obtained were r = 8.4 ?C 0.6 nm and r = 4.4 & 0.2 nm, respectively. At low ionic strength greater variations were observed, e.g. the elution volumes V, = 87 - 98 ml, corresponding to Stokes’ radii of about 8.0- 10.0 nm. If the time and/or intensity of sonication were sufficiently reduced, a minor fraction in the void volume was obtained in addition to the main peak and this fraction contain- ed both lipid and protein. For comparison the Stokes’ radius of the same lipoprotein complex (weight ratio lipid/protein 2.5: 1) in buffer of high ionic strength was determined on a Biogel A50m column and a value of about 5.0 nm was obtained.

The elution volume V, of the dimyristoyl lecithin - apoprotein complex (weight ratio of lipid/protein = 2.5: 1) was determined as a function of the ionic strength. Fig. 2 shows how the elution volume V, increases and the average Stokes’ radius decreases with NaC1-concentration, both relationships ap- proaching a constant value at concentrations > 0.1 M.

The reproducibility and reversibility of the ionic strength effect on the size of the lipoprotein complex was demonstrated as follows. The above dimyristoyl lecithin - apoprotein complex was prepared in buffer of low ionic strength and its elution volume V, = 93 ml was determined (cf Fig. 1E). Fractions corresponding to V, = 80- 105 ml were collected, and an aliquot of the pooled fractions was rechromatographed on the same column giving an identical elution volume V, = 93 ml. The second aliquot was dialyzed against buffer of high ionic strength and rechromatographed after the column had been equilibrated with the high ionic strength buffer. This time the elution volume was V, = 119 ml consistent with that shown in Fig. 1F. Similarly, when fractions V, = 100- 130 ml of the column chromatography shown in Fig. I F were collected and an aliquot of the pooled fractions rechromatographed on the column equilibrated with the low ionic strength buffer, a chromatogram similar to that of Fig. 1 E was obtained.

When the lipid/protein weight ratio of 2.5 : 1 (molar ratio 100: 1) was either increased or decreased, i .e. when either lipid or protein was present in excess of the lipoprotein complex eluted as a single peak (Fig. 1 F) a more complex elution pattern was observed (Fig. 3). With excess lipid the phosphorus positive peak was asymmetric and the peak width at half height about twice that of the peak in Fig. IF. The protein was eluted as two peaks, the first one at V, z 95 ml and the second one at V, z 119 ml. These

I 3 O 1 1 9’0

[NaCI] (MI

Fig. 2. Elution volume of the dimyristoyl lecithin- upoprotein complex (lipidlprotein weight ratio 2.5.1) as a function of NaCl concentration in the Tris bufler. Buffer was 0.01 M Tris, pH 8.6, 1 mM EDTA, 0.02% NaN,. At each NaCl concentration a single peak similar to that of Fig. 1 F was obtained. ( x __ x ) Elution volume, V,: (--O) Stokes’ radii calculated from V, using Eqn (1)

o b L . . - L L L . _ I - I _ i U _ - L

0 20 40 60 RC 10C 120 140 160 180 200 220 24C G (mi )

Fig. 3. Gel-permeution clzromatogruphy on Sephurose 4B qf a dimyristoyl lecithin - upoprotein complex containing excess lipid. 24 mg lipoprotein (lipidlprotein weight ratio 5 : 1) in 3 ml buffer (0.1 M NaCI, 0.01 M Tris pH 8.6, 1 mM EDTA, 0.02% NaN3) were applied. The dashed curve in Fig. 3 is the lipidjprotein weight ratio calculated for each fraction from the lipid and protein analysis. (0 -~--O) Protein analysis; ( x -- x ) phosphorus analysis

V, values correspond well to those of single-shelled dimyristoyl lecithin vesicles and of the lipoprotein complex (weight ratio 2.5: 1, Fig. 1 F), respectively. The details of the elution pattern depended on the time and/or intensity of sonication. By intensifying the sonication most of the excess lipid, even at a lipid/ protein weight ratio of 5 : 1 was incorporated into lipoprotein complexes and only a small proportion



Fig. 4. Electron micrographs of negatively stainedpreparations. (A) An aqueous dispersion of dimyristoyl lecithin (10 mg in 3 ml) sonicated for 10 min. The arrow indicates a large particle resulting from the aggregation and fusion of single-shelled

vesicles. (B) Porcine apoprotein delipidated with chloroform - methanol ( 2 : 1, v/v). (C) Porcine apoprotein delipidated with ethanol- ether (3 : 1, v/v). (D) Dimyristoyl lecithin- apo- protein complex (lipid/protein weight ratio 2.5 : 1)

was present as free lipid as evident from density gradient centrifugation (see below). The elution pattern of Fig. 3 shows that the peak at V, = 95 ml is only partly due to free lipid particles and that two lipoprotein complexes are formed, a lipid-rich one at V, = 95 ml and one at V, = 119 ml as discussed before. Reducing the sonication time and/or intensity caused the proportion of free lipid at V, = 95 ml to increase and, consistent with this, density gradient centrifugation of such preparations revealed the presence of free lipid. On the other hand, the chromatogram of lipid/protein mixtures containing excess protein (lipid/protein weight ratio 1 : 1) did not show any free lipid. The main fraction consisted of a single lipid peak, and a single protein peak which did not overlap completely, indicative of the presence of some free apoprotein. Lipid/protein complexes prepared in buffer containing 22Na+ were free of any radioactivity which was recovered completely in the total volume V , indicating that no "Na' was trapped within or bound to the lipoprotein complex.

Electron Microscopy Fig. 4A is a negatively stained preparation of

single-shelled dimyristoyl lecithin vesicles eluted in the peak fraction V, = 89 ml (Fig. 1A). The vesicle diameter ranged from 15.0- 50.0 nm (average 25.4 f 1.0 nm) and there was no negative stain in the internal cavities of the vesicles. The negative staining procedure can also lead to the aggregation and/or fusion of vesicles (see arrow in Fig. 4A). These latter structures, the size of which usually exceeds 100 nm, are artefacts of the negative staining procedure because column chromatography on Sepharose 4 B (with recoveries > 90 %,) failed to reveal a significant proportion of particles with diameter > 50.0 nm.

Fig. 4B is an electron micrograph of a negatively stained preparation of apoprotein showing spherical particles with diameters ranging from 12.0- 23.0 nm. Statistical analysis gave a mean diameter of 16.8

0.4 nm with a relatively small standard deviation of the mean indicative of a rather homogeneous particle size distribution. The aggregation of the apo-


H. Hauser, R. Henry, R. B. Leslie, and J. M. Stubbs 589

protein molecules (mol. wt 27000) [2,3] seems to depend on the extraction method of the lipid. This is illustrated in Fig. 4C which is an electron micrograph of a negatively stained sample of apoprotein delipidated with ethanol-ether = 3:2 (v/v). In contrast to the apoprotein in Fig. 4B which was freed from lipid with chloroform- methanol = 2 : 1 (v/v) this preparation showed an increased tendency to aggregate to elongated, thread-like structures. Comparison of Fig. 4C with the column chromatograms of Fig. 1C and D shows that particles of the size of the thread- like structures are not present in either buffer used in gel permeation chromatography. Since the recovery of the protein from the Sepharose 4B column exceeded 95%, the retention to any significant extent of the protein on the column bed can be ruled out. The spherical unit constituting the threads in Fig. 4C has an average diameter of 16.3 5 1.0 nm similar to that of the particles in Fig. 4B and consistent with the average thickness of the threads.

Fig. 4 D is an electron micrograph of a negatively stained sample of a dimyristoyl lecithin - apoprotein complex (lipid/protein weight ratio 2.5 : 1). The lipid and protein molecules associate to disc-shaped particles some of which lie flat on the grid and are viewed face on, and some stand up and are viewed edge on (see also [20,21]). The majority of discs, however, are aggregated and form stacks consisting of a few to several dozens of discs separated by layers of stain. The stacks may be arranged side by side which can lead to the fusion of discs in contact with each other. Statistical analysis of the disc-shaped particles gave the following average values for the diameter and the thickness of the discs; the average diameter was 15.3 f 0.8 nm ranging from 8.0- 22.0 nm. This is in good agreement with the average length measured from discs which are viewed edge on: 14.3 f 0.6 nm ranging from 7.0 to 24.0nm. The average thickness of the discs was 4.36 ? 0.07 nm in good agreement with the average thickness of dimyristoyl lecithin bilayers as measured with negatively stained preparation of unsonicated dimyristoyl lecithin dispersions : 4.33 0.1 nm. When instead of potassium phosphotungstate, uranyl acetate pH 4.3 was used as the negative stain the aggregation of the disc into stacks was prevented. Single discs, either face on or edge on were observed and the dimensions derived from these electron micrographs were similar to those shown in Fig. 4D.

The final dimension of the lipoprotein discs after attaining thermodynamic equilibrium did not depend on whether or not sonication was used to mix the lipid and the protein. When unsonicated dispersions of dimyristoyl lecithin were incubated with the apoprotein the same disc-shaped lipoprotein particles were obtained as when both phospholipid dispersions

and protein solutions were mixed and sonicated. In the latter case, however equilibrium was reached almost instantaneously while the incubated sample took at least 12 h to equilibrate. The column chromatogram of a complex obtained by incubating dimyristoyl lecithin bilayers with apoprotein for about 12 h differed only in the width of the peak which was broader than that obtained with sonicated preparations (Fig. 1 F) indicative of a wider particle size distribution.

Density-Gradient Centrifugation

Using sodium bromide density gradients in 10 mM Tris buffer pH 8.3 containing 1 mM EDTA information about the composition of the lipoprotein complexes was obtained. Fig. 5A shows the density gradient analysis of different lipid/protein mixtures. As the lipid/protein weight ratio of the mixtures increases the area (height) of the protein peak at an uncorrected mean density of 1.267 f 0.003 g/ml decreases and simultaneously the area (height) of the lipoprotein peak at an uncorrected mean density of 1.123 k 0.004 g/ml increases. Fig. 5B summarizes the results of the density gradient centrifugations giving the peak height of the apoprotein fraction as a function of the lipid/protein weight ratio in the original mixture. The composition of the lipoprotein complex was obtained by extrapolating the straight line to zero peak height which gives a lipid/protein weight (molar) ratio of 2.68 (107: 1). Alternatively, the lipoprotein peaks at an uncorrected mean density of d = 1.123 0.004 g/ml obtained by density gradient centrifugation were analyzed for lipid and protein. The average lipid/protein weight ratio thus obtained was 2.5 k 0.2. The average lipid/protein ratio of the dimyristoyl lecithin - apoprotein complex was also determined by lipid and protein analysis of each fraction of the column chromatogram shown in Fig. I F . The dashed line shows how this ratio varies across the lipoprotein peak. Excluding values with V, < 95 ml and V, > 135 ml the lipid/protein ratio averaged across the peak was 2.41 f 0.1 (see Table 3). When lipid and protein were mixed in ratios exceeding the “stoichiometric” value of about 2.5 the excess lipid was either present as free lipid or incorporated into the lipoprotein complex depending on the sonication condition. By increasing the time and/or intensity of the sonication more lipid was taken up and lipid- rich complexes with a buoyant density < 1.123 g/ml were produced (Fig. 6). This figure shows that up to lipid/protein weight ratios of 4: 1 all the lipid was complexed with the apoprotein, and as more and more lipid is incorporated the density of the complex decreases linearly.



" 0 5 10 15 20 25 30

Fraction number

1.30

35 _I 1.00 40

Fig. 5. Properties qf the dimyistoyl lecithin - apoprotein coni- plex. (A) Density gradient centrifugation of dimyristoyl lecithin - apoprotein mixtures of different composition. The lipid/protein weight ratios were 0.5 ( O o ) , 1 ( x ~ x ) and 1.5 (A-A), the total amount of lipid + protein was z 10 mg. The apoprotein distribution on the NaBr gradient after centrifugation for 24 h at 400000 x g was monitored at 280 nm. The band at a density of z 1.12 g/ml represents

\i\ 1.08 1 \

\ -- 1.07 I 0 1 2 3 4 5

L ip id /protein weight ra:ic

Fig. 6. Uncorrected densities obtained porn density-gradient centrifugation of vurious dimyristoyl lecithin - apoprotein mixtures. The horizontal line represents the average density of the lipoprotein complex formed in mixtures up to a lipid/protein weight ratio of 2 2.6

DISCUSSION

Size of the Lecithin - Apoprotein Complexes

Table 2 summarizes the results obtained with dimyristoyl lecithin, apoprotein and lipoprotein complexes. For comparison the values obtained with complexes of dilauroyl and dipalmitoyl lecithin with apoprotein are also included in the table. Inspection of the table shows that the disc radii of the complexes derived from electron micrographs of negatively stained preparations are consistent with the Stokes' radii observed in buffer of low ionic strength. Discs

2013

1

3 L

Lipid /protein weight rat io

the lipidlprotein complex of weight ratio 2.7, the band at a density of z 1.27 g/ml is the pure apoprotein. The straight line gives the density (from refractive index measurements) along the gradient. (B) The peak height of the protein fraction of density z 1.27 g/ml (from A) as a function of lipid/protein weight ratio. The line is fitted to the experimental points by least squares analysis and is given by the equation: peak height = 15.6- 5.8 (lipid/protein weight ratio)

of about 4.0 nm radius consistent with the Stokes' radius of 4.4 nm observed by gel filtration of dimyristoyl lecithin - apoprotein complexes (weight ratio 2.5: 1) in buffer of high ionic strength are only rarely observed with negatively stained preparations. This suggests that the processes involved in the preparation of negatively stained samples induce aggregation and/or fusion (cr [26]). Decreasing the ionic strength of the sample during the rinsing procedure does not seem to be the cause of the aggregation because the use of potassium phosphotungstate dis- solved in 0.1 M NaCl (pH 7.0) to maintain a constant ionic strength also failed to preserve the smaller particles. Several authors [20,21,23] have observed disc-shaped structures often aggregated into stacks when various apoproteins are either incubated or sonicated together with egg lecithin, but the real significance of these structures has not yet been decided.

The apoprotein itself as well as both the dilauroyl and the dimyristoyl lecithin - apoprotein complexes show similar aggregation behaviour as a function of the ionic strength with low salt concentrations facilitat- ing aggregation (Table 2). In contrast to this the size of lecithin vesicles is independent of ionic strength, at least over the concentration range (0.01 - 0.1 M NaC1) studied in this work. This suggests that the polymerisa- tion of the lipoprotein complex is controlled by the protein. Fig. 2 shows that as the ionic strength is raised dissociation of the lipoprotein complex into


H. Hauser, R. Henry, R. B. Leslie, and J. M. Stubbs 59 1

Table 2. Size of the lecithin- apopwtein complexes Stokes' radii were determined by gel permeation chromatography on Sepharose 4B. The figures represent the average k standard deviation of the mean; sometimes the range of the experimental results are given. The second column (ionic strength) applies only to gel permeation chromatography. Particle radii were obtained from negatively stained electron micrographs. Compound 2 was apoprotein extracted with ethanol-ether (3 : 2, by vol.); compound 3 was apoprotein extracted with chloroform-methanol (2: 1 , by vol.). For compound 6, the column chromatography was carried out at 2 4 "C, well below the gel + liquid crystal transition temperature of the dipalmitoyl lecithin. The peak corresponding to Y = 11 .O nm was only detected in the effluent by monitoring the absorbance at 280 nm, the main peak detected by ultraviolet, phosphorus and protein analysis corresponds to a Stokes' radius of Y = 7.6 nm. While phosphorus analysis of the effluent gave a single, broad asymmetric peak, protein analysis showed two distinct peaks, one corresponding to a Stokes' radius of Y = 7.6 nm and the other to Y = 5.0 nm

No. Compound Ionic Stokes' radius Particle radius strength

nm

1 Dimyristoyl lecithin 0.01 9.5 f 0.4 0.1 9.5 f 0.4

12.7 f 0.5 range: 7.5-25

2 Apoprotein 0.03 8.8 k 0.2 range: 8.0- 9.5 8.2 f 0.2 range: 6.0-11.5 0.1 3.5 k 0.4

3 Apoprotein 0.1 range: 3.6- 4.5 8.1 f 0.5 range: 6.0-12.0

4 Dimyristoyl lecithin-apoprotein 0.01 8.4 f 0.6 range: 7.8- 10.0 7.7 f 0.4" range: 4.0-11.0 complex, lipid/protein weight 0.1 4.4 f 0.2 7.2 f 0.3b range: 3.5-12.0 ratio 2.5: 1

(Negative stain - uranyl acetate) 9.2 f 0.2" 7.5 f 0.4b

5 Dilauroyl lecithin- apoprotein 0.01 8.5 f 0.3 complex, lipid/protein weight 0.1 3.5" ratio 1 : 1

8.2 f 0.4 range: 5.0-50

6 Dipalmitoyl lecithin - apoprotein 0.1 11.0 complex, lipidlprotein weight ratio 1 : 1 5.0

7.6 (main peak) 10.5 & 0.3" range: 5.0-17.0 10.9 f 0.4b

Diameter of flat discs (top view). Length of discs viewed edge on. For comparison gel permeation chromatography on Biogel gave a Stokes' radius of 3.1 nm.

smaller particles of the same composition occurs. Under the experimental conditions used the limiting size of the lipoprotein complex, i.e. the structural unit of the complex with a Stokes' radius of 4.0- 4.5 nm, is obtained at an ionic strength I % 0.1. A further increase in the ionic strength does not affect the particle size significantly. The observation of a single peak at an intermediate ionic strength indicates that the ionic-strength-dependent association-dissociation is rapid on the time-scale of the column chromatography. With dilauroyl lecithin the structural unit of the lipoprotein complex is smaller with a Stokes' radius of 3.0-3.5 nm at an ionic strength I 2 0.1. With the dipalmitoyl lecithin - apoprotein complexes no conclusion can be drawn with respect to the size and the salt dependence. Gel permeation chromatography on Sepharose 4B shows that aggregation of the lipoprotein complex occurs even at 0.1 M


NaCl (Table 2). However, these experiments have been carried out at temperatures below the gel + liquid crystal transition temperature, T,, of the phospholipid and at that temperature the aggregation properties may be dominated by the aggregation of the dipalmitoyl lecithin bilayers.

Weight and Shape of the Lecithin - Apoprotein Complexes

If the discs observed by electron microscopy are a true representation of the shape of the lipoprotein complex, then the consistency in the size derived from gel chromatography at low ionic strength and electron microscopy suggests that the Stokes radius derived from gel chromatography is the actual radius of the disc. In low ionic strength buffer the average disc radius of 8.4 0.6 nm is then about twice that


Table 3. L@id/apoprotein ratios at which no j r e e protein is detected Density gradient I was using the extrapolation procedure shown in Fig. 5B; I1 was using lipid and protein analysis. Note, the average weight ratio is thus 2.5 0.15 corresponding to a molar ratio of 100 (* 6)

Experimental technique Weight ratio Molar ratio

Density gradient I 2.7: 1 107: 1

Gel permeation Density gradient I1 2.5:l 100: 1

chromatography 2.4: 1 96: 1

observed in high ionic strength buffer ( I 2 0.1). This suggests that the average disc observed in Fig. 4D is the product of a side-by-side aggregation of the small discs (radius z 4.4 nm) present in high ionic strength buffer. Consistent with this interpretation is the finding that the thickness of the discs varying in radius between 3.5- 12.0 nm is 4.38 5 0.07 nm independent of the disc radius. Thus the average volume of the smaller discs (radius z 4.4 nm) can be calculated as 2.7 x cm3 and the volume ratio of the average disc present in low and high ionic strength respectively, as 4 : 1. For comparison the minimum weight of the lipoprotein complex calculated from the experimental lipid/protein molar ratio = 1OO:l (Fig. 5 , Table 3) and the molecular weights of 678 and 27000 for dimyristoyl lecithin and apoprotein, respectively, is 95 000 daltons. With the experimentally determined partial specific volume of that complex ijZ5 = 0.917ml/g this gives a minimum volume of 1 . 4 ~ cm3. Thus, by comparison, the disc with a radius of z 4.4 nm consists of two such units (100 lipid molecules + 1 protein molecule) containing a total of 200 lipid and 2 apoprotein molecules. A similar calculation is carried out for the dilauroyl lecithin- apoprotein complex. In contrast to the dimyristoyl lecithin - apoprotein complex, the average lipid/protein weight ratio (determined from density gradient centrifugation by a similar extrapolation procedure as shown in Fig. 5B) is 1 (molar ratio 43.5: 1) and the minimum weight is 54000 daltons. With the measured partial specific volumes for that complex U25 = 0.859 ml/g a minimum volume of 7.7 x x cm3 is obtained. The Stokes’ radius of 3.5 nm (see Table 2) and the average thickness of 4.1 nm obtained from statistical analysis of the discs observed by electron microscopy give a volume of 1.58 x x cm3. Thus, as with the dimyristoyl lecithin- apoprotein complex the dilauroyl lecithin - apoprotein complex formed in buffer of 1 2 0.1 contains two units each containing 44 lipid and 1 protein molecule, i.e. a total of about 90 lipid and 2 apoprotein molecules.

Direct Determination of the Weight of the Disc-Shaped Particles

For comparison the particle weights were determined by ultracentrifugal analysis. The peak fraction (Fig. 1 F) containing 0.9 mg lipoprotein per ml high ionic strength buffer gave an uncorrected sedimentation coefficient of s20 = 3.84 S, and an uncorrected diffusion coefficient D,, = 5.24 cmz/s. Inserting these values in the Svedberg equation

(3)

gives an apparent particle weight of 236000 daltons. Applying the conventional sedimentation equilibrium method [29] to the same sample the apparent number, weight and Z-average particle weights are obtained [27]. M , (number average) = 227000 daltons, M , (weight average) = 325 000 daltons, M , (Z-average) = 410000 daltons. A4, > M , > M,, and the deviation from linearity of the plot of log c versus X 2 (i.e. the upward curving of the line in Fig. 7) are indications of the polydispersity of the lipoprotein complex. The particle weight as a function of X 2 is computed from Eqn (4)

2RT 2.3 d log c ~ _ _ _ _ _ _ ~ M =

(1 - GQ) d ( X 2 ) (4)

and is also shown in Fig, 7. The number average particle weight and the weight determined from the ratio s,o/D,, are in good agreement with that of a disc- shaped particle consisting of 200 lipid molecules and 2 protein molecules: z 200000 daltons (see above). Some aggregation of this “unit” particle takes place even in high ionic strength leading to the higher values of M , and M,.

“Stoichiometry”

The average lipid/protein ratios (at the point of complete complexation of the apoprotein, i.e. the “stoichiometric” ratio) derived from various techniques are in good agreement (see Table 3). Lipid- protein mixtures of such a composition are always eluted as a single peak (see Fig. 1F). Fig. I F also shows that the composition of the complex can vary within wide limits and the value of 2.4 represents the average of a broad distribution. The high and low values observed for the weight ratio at elution volumes V, < 95 ml and V, > 135 ml, respectively, probably reflect the presence of small quantities of residual free lipid and protein, respectively. When lipid is present in excess of a lipid/protein weight ratio of z 2.5 the lipid is more or less incorporated depending

Eur. J . Biochem. 48 (1974)

H. Hauser, R. Henry, R. B. Leslie, and J. M. Stubbs

0.6

2 0.5 2 0.4

593

-

-

-

46 47 48 49 50 51

x 2 (m2)

Fig. I. Plot of log c ( x -- x ) and apparent weight average particle weight (o---) of the dimyristoyl lecithin-apoprotein complex (weight ratio zz 2.5) , ,fractionated on Sephu- rose 4B, as a junction of X2. The peak fraction containing 0.9 mg lipoprotein/ml of the column chromatogram shown in Fig. 1 F was used. c = lipoprotein concentration expressed in terms of fringe numbers, X = distance between rotor centre and the point in the cell at which the particle weight was determined. The bars give the errors due to the uncertainty in the determination of d log c/d ( X 2 )

on the sonication conditions used. Fig. 3 shows that under these conditions several lipid-protein complexes differing in the lipid/protein ratio can be formed. In addition to the normal complex with a lipid/protein weight ratio E 2.5, lipid-rich complexes with a higher lipid/protein weight ratio and a lower buoyant density are observed (cfi Fig. 6). Our experiments do not warrant any conclusion as regards the structure of those lipid-rich complexes.

Structure and Aggregation o j the Lecithin - Apopvotein Complex

From the results presented here and experiments with other physical techniques which will be described elsewhere it is possible to derive some molecular details of the structure of these reconstituted lipoproteins. The discussion which follows is confined to dimyristoyl lecithin - apoprotein complexes. While sonicated dispersions of dimyristoyl lecithin form spherical vesicles surrounded by a single bilayer, the lipoprotein (lipid/protein weight ratio E 2.5 : 1) forms discs. On the grounds of no 22Na+ being trapped within the lipoprotein complex the encapsulation of apoprotein in phospholipid vesicles and the presence

of internal cavities in the lipoprotein structure are ruled out. Electron micrographs of negatively stained preparations of the lipoprotein and unsonicated dimyristoyl lecithin enabled us to compare the thickness of the discs with that of dimyristoyl lecithin bilayers. Statistical analysis of several hundred particles shows that the values agree with each other within experimental error. Similar results are obtained with dilauroyl and dipalmitoyl lecithins suggesting that the phospholipid bilayer structure is retained in some modified form in these lipoprotein complexes. Differ- ential scanning calorimetry (Phillips, M. C., to be published), electron-spin-resonance spin-label experiments and fluorescence polarisation using probe molecules for both the lipid and the protein support this interpretation (see accompanying paper). All three techniques show that with the dimyristoyl lecithin - apoprotein complex the chain melting transition at T, z 23 "C characteristic of dimyristoyl lecithin bilayers still occurs though broadened out over a larger temperature range. The broadening effect of the protein on the lipid transition as probed by spin and fluorescence labels is greater the closer the probe is located near the glycerol group of the phospholipid. Thus the 200 dimyristoyl lecithin molecules of the complex with apoprotein appear to form a bilayer disc in which the cooperative motion is reduced due to the interaction with the apoprotein. As regards the location of the apoprotein the spin label and fluorescence polarisation experiments sug- gest that the protein backbone is oriented at the lipid- water (hydrophobic-hydrophilic) interface. Our results support this suggestion that the protein is oriented with its polar groups readily accessible to the aqueous environment since this accessibility of the protein explains the ionic-strength-dependence of the disc radius which has been shown to be solely a property of the apoprotein. This orientation of the protein also allows for the polar groups of the lecithin being readily available to the aqueous environment. Evidence for this is obtained from nuclear magnetic resonance experiments (Finer, E. G., to be published). All protons of the choline group of dimyristoyl lecithin are shifted in the presence of paramagnetic Fe(CN)i- .The upfield shifts are in the order N(CH& > N(CH2) > CH,OP indicating that most of the phospholipid polar group is available to the Fe(CN)i- ion and not affected by the interaction of the phospholipid with the apoprotein as would be expected if the protein forms two layers sand- wiching the phospholipid bilayer. High resolution nuclear magnetic resonance experiments with dimyristoyl lecithin - apoprotein complexes (in the absence of paramagnetic ions) show that the signals from the polar group are the only ones of the lecithin spectrum which show changes in chemical shift when lecithin


594 H. Hauser, R. Henry, R. B. Leslie, and J. M. Stubbs: Lecithin-Apoprotein Interaction

is complexed with apoprotein. This finding is again consistent with the orientation of the protein described above.

Spectroscopic evidence also suggests that the lipid and the protein form a loose complex with all the polar groups exposed to the aqueous environment and with the hydrophobic amino acids of the protein penetrating into the hydrophobic part of the lipid bilayer. Consistent with the interpretation of a loose complex is the finding that the partial specific volume of the complex U = 0.917 ml/g is only z 0.8 larger than that calculated from the partial specific volume of the constituents of the complex. Similar results apply to apoprotein complexes with dilauroyl and dipalmito yl lecithin.

Similar to the results of Lux et ul. [24] with apoprotein from human high-density lipoprotein circular dichroism of the peptide and aromatic chromophores of apoprotein from porcine high-density lipoprotein shows that the CI helical content increases significantly from about 50 :d to 60- 65 ”/, when the apoprotein is complexed with dimyristoyl lecithin.

As discussed the ionic-strength-dependent monomer-dimer equilibrium of the dimyristoyl lecithin - apoprotein complex is a property of the apoprotein rather than of the phospholipid. More work is required to explain this observation in greater detail. The aggregation at low ionic strength probably involves electro- static forces and could be explained by postulating that the conformation of the apoprotein in the complex provides for the necessary charge distribution on the apoprotein. A regional separation of oppositely charged groups, and the surface activity of the apoprotein [7] are probably responsible for the dispersing action the apoprotein has on lecithin bilayers as evident from the “solubilization” of the large, multilamellar lecithin bilayer structures (diameter upto 20 pm) when incubated with the apoprotein.

Future work and the accompanying article by Barratt et al. will deal, on a molecular level, with the structure and the mechanism of aggregation of lecithin-apoprotein complexes. It is to be hoped that a detailed study of this type of complexes will contri- bute to our understanding of naturally occurring lipoproteins, particularly that it will throw light on the structural and functional role of the cholesteryl esters and non-polar lipids present in natural lipoproteins.

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H. Hauser’s present address: Laboratorium fiir Biochemie dcr E.T. H., UnivcrsitBtsstrasse 16, CH-8006 Zurich, Switzerland R. Henry, R. B. Leslie, and J . M. Stubbs, Unilever Research Laboratories, The Frythe, Welwyn (Herts), Great Britain


the interaction of apoprotein from porcine high-density lipoprotein with dimyristoyl...

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