The influence of the alkyl chain length of lecithins and lysolecithins on their interaction with αsl-casein

Biochimica et Biophysics Acta, 348 (1974) 126-135 0 EtsevierScientificPublishing Company, Amsterdam

- Printed in The Netherlands

BBA 56424

THE INFLUENCE OF THE ALKYL CHAIN LENGTH OF LECITHINS AND LYSOLECITHINS ON THEIR INTERACTION WITH a,,-CASEIN

M. D. BARRAlT,

J. P. AUSTIN and R. J. WHITEHURST*

Biophysics Division, L&lever Research Laboratory Colworthl Welwyn, The Frythe, Welwyn, Herts. (Gent Britain) (Received October 8th, 1973)

SUMMARY

I. The interactions between casein fractions and synthetic lecithins were examined by density gradient centrifugation as a function of the lipid alkyl chain length. Dicapryl (C,,) and dilauroyl (C,,) lecithin both formed lipid-protein complexes with or,,-casein but not with ,f%casein; also dicapryl lecithin did not interact with rc-casein. Dimyristoyl (C,,) lecithin, however, showed no interaction with a,,-casein. 2. The interactions between synthetic lysolecithins (C,O-Czo) and a,,-casein were also studied by density gradient ultracentrifugation. The results showed that the short-chain lysolecithins (C,,, CIJ formed complexes which had a tendency to dissociate under the experimental conditions used. C14, Ci6, and C,, lysol~ithins formed stable lipid-protein complexes whilst C,, 1ysolecithin formed complexes with some difficulty. 3. The experimental data have been correlated with the hydrophobic free energies of transfer of lipid monomer from water to lipid micelle (or bilayer).

INTRODUCTION In previous work we have examined the hydrophobically-stabilised lipid-protein complexes formed between egg lysolecithin and a,,-casein [I, 21. We have recently found that there is no complex formation when egg lecithin and tisl-, fi- or #-casein are dispersed or even sonicated together. When significant amounts of egg lysolecithin are added to egg lecithin, however, the mixtures interact with cr,,-casein with incorporation of lecithin into the resultant lipoproteins [3]. This observation led us to ask whether similar interactions would occur using synthetic lecithins with shorter alkyl chains. In this paper we present the results from density gradient ultracentrifugation and gel electrophoresis of mixtures of Q- casein with lecithins and lysol~ithins of different alkyl chain lengths. The results are used to suggest a the~odyna~c framework applicable to lipid-protein interactions in general. Abbreviations: (diC&lecithin, I$-dicapryl-sn-glycero-3-phosphorylcholine; (C&lysolecithin, monopalmitoyl-~~-glycero-3-phosphorylchol~ne. * Present address: British Soya Products Ltd, Pnckeridge, Ware, Her% Great Britain.

127 MATERIALS AND METHODS

a,,-Casein was prepared by the method of Thompson and Kiddy [4] and purified by chromatography on DEAE-cellulose in 3.3 M urea, 0.01 M imidazole buffer (pH 7.0). j?-Casein was prepared by the method of Hipp et al. [5] and rc-casein by the method of Zittle and Custer [6]. Synthetic lecithins were prepared by the acylation of r_-a-glycerylphosphorylcholine-cadmium chloride complex. Myristoyl (C,,), palmitoy1 (C,,) and arachidoyl (C,,,) lysolecithins were prepared from the corresponding lecithins by treatment with phospholipase A (ex Crotalus adamanteus venom-Koch Light Ltd) [7]. Capryl (C,,), 1auroyl (C,,), stearoyl (C1szO)and oleoyl (C,,:,) lysolecithins were prepared by Dr A. F. Van Dam of U.R.L. Vlaardingen. Density gradient ultracentrifugation

Density gradients were prepared by successively layering decreasing concentrations of sodium bromide (NaBr) in IO-~ M Tris, IO-~ M EDTA, 0.02 % NaN, buffer at pH 7 in 5.5 ml cellulose nitrate or polyallomer centrifuge tubes. The NaBr solutions used were usually in the range 60-20 % for lysolecithin-casein mixtures and 50-10 % for lecithin-casein mixtures. The phospholipid and casein were dispersed (dissolved) in Tris-EDTA-NaN, buffer (I ml) before the addition of NaBr (IOO mg in the case of the lysolecithins and 50 mg with the lecithins). This formed the uppermost layer of the gradient. Centrifugation was carried out at 58000 rev./min (240000 x g) for 24 h in a Beckmann L2-65 ultracentrifuge using an SW-65 rotor. Gradients were cut into approximately 20 fractions. A o. r-ml aliquot was taken from each fraction, diluted with 1.0 ml of water and its absorbance at 280 nm was measured. The refractive index of each fraction was measured using an Abbe refractometer. Densities were estimated from refractive indices. Refractive indices were not corrected for contributions due to the presence of protein and phospholipid. Protein was determined by the method of Lowry et al. [B], and lipid phosphorus by the method of Chen et al. [9]. This was corrected for the phosphorus content of the casein present. Gel electrophoresis

Gel electrophoresis was carried out using a Shandon disc electrophoresis tank equipped with 8 Precibore tubes (SAE-2735). The gel and buffer compositions were: A, 16 ml of IM HCl, 12. I g Tris, o.og ml TEM,ED (N,N,N’,N’-tetramethyl ethylenediamine), water to 100 ml. B, 15.0 g acrylamide, 0.9 g N,N’-methylbisacrylamide, water to 100 ml. C, 0.028 g ammonium persulphate in 5 ml water. Three parts of A were mixed with four parts of B, degassed, and then mixed with one part of C. I ml of the mixture was used for each rod. Buffer: Tris 6 g, glycine 28.8 g, sodium azide 0.4 g, water to 2 1 (giving pH 8.3). Samples for electrophoresis were prepared as follows : 0.5 mg of each lysolecithin was dissolved in 0.5 ml of buffer. The C1s and CzO samples were sonicated briefly (low power) at 45 “C to ease dispersion. 0.5 ml of a stock solution of a,,-casein (I mg/ml) in buffer was added to each sample. The samples were incubated at 40 “C for 24 h before use. Glycerol (0.1 ml) was added to each sample to facilitate layering on

128 the gel rods. A sample volume of 20 p-1was used and bromophenol blue was added to the top tank of the apparatus as a marker. A current of 8 mA was applied for 30 min after which it was increased to 32 mA for the remainder of the experiment (approx. 45 min). The gel rods were stained with amido black (I % in 7 % acetic acid) for 2 h. Destaining was carried out in 7 % acetic using a Quickfit destainer. RESULTS AND DISCUSSION

The density gradient profiles of 1,2-~cap~l~~n-glycero-3-phosphorylcholine ((diC,,)lecithin) with cr,,-, p- and K-caseins are shown in Fig. I. This figure illustrates that (diClO)lecithin forms a lipoprotein complex with a,,-casein but not with fl- or rc-caseins. We found that (diC,,)lecith.in also complexed to a certain extent with c[,~casein, but not with &casein (Davis, M. A. F., personal co~uni~ation). Extending the fatty acid chain length to (diC,,)lecithin, however, we found no complex formation with cc,,-casein even after co-sonication of the lipid and protein. As mentioned earlier, egg lecithin, which represents a further increase in the fatty acid chain length, also failed to form complexes with a,,-casein.

DiC,oFC/Casein

Ratio of I*O(%)

occasein -

1.5

1.3

f4casein -L-a-

A nm

Kcasein

P

4-a-

I

0

0

6"

7" Refractometer

Fig.

I.

8" -ding

10"

Density gradient plot of E,~-, b- an d rc-caseins with (diC,O)lecithin (DiC&PC).

If mere protein hydrophobicity was important in lipid binding, one would expect fi- and rc-casein to have a greater tendency than a,,-casein to interact to form lipoproteins. On the Bigelow scale [IO], /3-and rc-caseins are more hydrophobic than a,,-casein [I1-131, and /Lcasein is rated as one of the most hydrophobic proteins by surface balance techniques [143. The most important feature in favour of a,,-casein

129

as a potential lipid-binding protein in our opinion, however, is that it is the most structured of the three main casein fractions, as shown by NMR spectroscopy [IS] and intrinsic viscosity measurements [16], existing under the experimental conditions used in this work, mainly as a tetramer [ 171(mol. wt 4 x 23 616 [I I]) with a low degree of a-helical conformation. It is possible that the a-helical content of a,,-casein may increase under the influence of lipid-protein interactions, c.f. the apoprotein of serum high-density lipoprotein [IS, 191. a,,-Casein possesses the ability to change its ahelical content, e.g. it is reported to form 50% a-helix in 2-chloroethanol solution

Pal. (b) as,-Casein-lysolecithin interactions (i) Density gradient centrifugation The density gradient profiles of different ratios of monopalmitoyl-sn-glycero-3phosphorylcholine ((C,,)lysolecithin) with cc,,-casein are shown in Fig. 2. The estimated buoyant densities of the complexes plotted against the initial lipid-protein ratios (w/w) are shown in Fig. 3. Also included in Fig. 3 is the calculated density curve assuming IOO% complexing of the lipid and protein. (This calculated curve assumes hydrated densities of 1.31 and 1.08 g/ml, respectively, for a,,-casein and (C,,)lysolecithin). The binding of (C,,)lysolecithin to cc,,-casein proceeds in a series of discrete steps. The first few molecules of lysolecithin added (estimated at approx. 5 per monomer of a,,-casein) are shared equally by all the a,,-casein molecules present. This is seen as a shift of the a,,-casein peak to a slightly lower density (d 1.30 g/ml). Addition of subsequent lysolecithin results in a deaggregation of the a,,-casein tetramer into two dimers as described previously [2]. This deaggregation of the protein presumably

Cb Lysa PC +a s,-casein

Refmctometer

Fig.

2.

reading

Density gradient ultracentrifugation plot of a,i-casein with (Ci,Jlysolecithin (C,,LPC!).

130

l-32-

I 1*30-

Fig. 3. Plot of protein density (H) and lipoprotein density ( l) of aSl-casein-(Cl,)lysolecithin (CMLPC) gradients versus initial lipid protein ratio (w/w). The solid line represents the calculated density assuming all lipid and protein is complexed.

exposes lipid-binding sites of a greater affinity than those initially present with the result that as more lysolecithin is added, the peak at d 1.30 g/ml decreases in intensity and the discrete lipoprotein peak at d 1.24-1.25 g/ml correspondingly increases in intensity. (Equal binding of lysolecithin to all protein molecules would have resulted in the observation of a single peak moving to lower density with increasing lysolecithin). The Last stage in the process consists of the lipoprotem (d 1.24-1.25 g/ml) binding a few additional molecules of lysolecithin and moving to its final density of about I .23 g/ml at a lipid : protein ratio of 0.6 (w/w). (Addition of further lysolecithin results in no further change in the lipoprotein density). The added lipid :protein ratio (0.6, w/w) corresponds to a ratio of aB(C,,)lysolecithin molecules per a,,-casein molecule, the density of the complex (1.23 g/ml) from the calculated density curve corresponds to a molar ratio of 25 to I compared with the experimentally determined lipid: protein ratio of about 35 to I. All of the lysolecithins tested for their interaction with cl,,-casein, formed soluble complexes. (C,,), (C,,), (C,,), (C1szO)and (C,s:,)-lysolecithins all interacted readily at room temperature. (C,,)lysolecithin, when tested by density gradient ultracentrifugation immediately after mixing, showed no interaction. A sample (initial 1ipid:protein (w/w) = 1.0) allowed to stand for 24 h at room temperature, however, showed about 70 % conversion to a lipid-protein complex of density I .22 g/ml. The approximate buoyant densities and measured molar ratios of the complexes formed by a range of lysolecithins are shown in Table I. (C&Lysolecithin was found to have a chain melting temperature of about 44 “C. Lipid-protein complexes could be formed by incubating (C,o)lysolecithin with a,,-casein at 45 “C for 24 h. The complexes so formed dissociated progressively with time in the density gradient. The effect of centrifuging the (C,,)lysolecithin-a,,-casein complex for 24,48 and 72 h at room temperature is shown in Fig. 4. Under no conditions were we able to observe cr,,-casein converted quantitatively to a(C,,)lysolecithina,,-casein complex.

I31 TABLE I BUOYANT

DENSlTlES

Lipid (C1,)Lysolecithm (C1a)Lysolecithii (C~~)L~ol~it~n (CQLysolecithin (CQLysolecithin (C,~)Lysol~i~in (C,,)Lysolecithin (CiJLysolecithin (C,s,o)Lysolecithin (C~s:*)Ly~lecithin . . (C1s:l)LysolecIthm

AND MOLAR R.ATKlS OF LIPID-PROTEIN Measured L/P

Initial L/P ratio (w/w)

Lipoprotein density (g/ml)

(moWmole)

1.0

I.22 I.24 1.26 I.24 1.23 I.24 1.23 I.24 I.22 I.22 1.24

Not determined Not determined 24 f 5 30 6 5 64 f IO 37 f 5 35 i 5 35 l 5 22 f 5 32 f 5 Not determined

1.0 0.4 0.6 1.0 0.4 0.6 0.8 0.6

O-9 0.6

COMPLEXES

ratio

Go l_ysoPC~as;casein L/P Ratio l*O@@

280 nm o-2

Od-

5

Fig. 4. Density gradient plot of o;l-casein-(C&lysolecithin 48 and 72 h at room temperature.

(C&LPC) complex centrifuged

for 24,

cl,,-Casein appears to bind about 3o-35 molecules of lysolecithin (C,,-C,,) iude~ndently of the lipid chain length. We had ~~~~~ in obtaining consistent analytical results for the lipid-protein ratios (cf. Morrisett et al. fir]). One of the problems was the poor resolution between the lipoprotein and lipid bands in the gradients, particularly with the shorter chain lengths. (ii) Gel eiectrophoresis

The results from the gel electrophoresis of lysolecithin-qcasein mixtures (containing an excess of 1ysoI~it~n) are shown in Fig. 5, As described previously [z], when egg lysolecithin forms complexes with g,-casein, the lipoprotein travels with approximately half the mobility of the q-casein. No complex formation could be de-

132

Fig. 5. Gel electrophoresis of iz,l-casein. I, cl,I-casein control, and G,-casein with: 2, (CIO)lysolecithin; 3. (CIz)lysolecithin; 4, (C&lysolecithin; 5, (C16)lysolecithin; 6, (C,s)lysolecithin; 7, (CzO)lysolecithin; 8, egg lysolecithin; all at lipid/protein ratio 1.0 (w/w).

tected with (C,,)- or (C~~)lysolecithin. (C&-, (C1&, and (C~s)lysol~ithin all appeared to interact in the same way as egg lysolecithin to form complexes whilst (C,,)lysolecithin interacted to only a small extent. Gel electrophoresis is a technique which uses the criterion of charge/size ratio to separate different species. Increasing the lengths of the alkyl chain of the lysolecithin is accompanied by a decrease in mobility of the lipoproteins as shown in Fig. 5. As the net charge of the complex is supplied by the casein, increasing the size of the lipid-protein complex results in a decrease in the charge/size ratio and hence a decrease in mobility. The gel electrophoresis results are apparently inconsistent with the density gradient results in that no interaction is observed between (Cc,,)- and (C,,)lysolecithin and a,,-casein using the former technique. Under the experimental conditions used, both techniques operate in a manner which perturbs the equilibrium. Gel electrophoresis appears to be more destructive of lipid-protein complexes than density gradient ultracent~fugation. This may be related to the high ionic strength used in the latter technique, which would tend to stabilise hydrophobic interactions. (c) General discussion of lipid-protein interactions

In a mixture of lipid and protein in water, complex formation can only occur

133

if the free energy lost by the lipid monomer in associating with the protein is greater than the free energy lost by its association with the lipid micelle or bilayer. Using the arguments presented by Tanford [22], the standard free energy of transfer of a lipid molecule from water to a micelle (or bilayer) is AG” = $(micelle) -$‘(water)

= RTln [Lipid,,,,,,,]

= - RTln K

(to a first approximation). Where the concentration of monomer is expressed in mole fraction units, and the lipid monomer is assumed to form an ideal solution. A corollary of this statement is that for binding of lipid to protein to take place the concentration of lipid monomer at equilibrium with the protein must be lower than the concentration of lipid monomer at equilibrium with the lipid micelle (or bilayer). This is

‘1 3 -

c,* LPC

-4 -

C,.$LPC

.-

Egg LPC

-5 -

CSLPC

4

..............

C,.LPC

..............

D,CaK

-10

&v

P-B

sss

asI-casein

k-casein

-, ........ .. . C,Lpc . . .... . . . ..

e 9

-15

DiC,.&

............

DiCUpC

. . ...... .... ..

EY,_

-

Df,&

10

_........._DiC,&

HDLapopmtein

-11

Fig. 6. Free energies of transfer of lipid monomers from water to lipid micelles (or bilayer). dGOtr.,,,rcr = RT ln[monomer], the monomer concentration is expressed in mole fraction units. Proteins will complex with lipids above them on the AG” scale but not with lipids below them. SDS, sodium dodecylsulphate; HDL, high density lipoprotein; EYL, egg-yolk lecithin.

expressed in a more recognisable form by saying that the binding constant of the lipidprotein interaction must exceed the binding constant of the lipid-lipid interaction in order that lipid-protein interaction may take place to a significant extent. In Fig. 6 we show an attempt to construct a semi-quantitative scale for the free energy of transfer (AGO) of monomer from water to lipid micelle or bilayer for a number of lecithins and lysolecithins. For micellar lipids, the free monomer concentrations in equilibrium with the micelles are taken to be equal to the critical micelle

I34

concentrations and the AG” values are calculated from these quantities. The solid lines refer to lipids where the critical micelle concentrations are known to us or reported in the literature*; the values indicated by broken lines are calculated by extrapolation. The chain length dependence of AG” for the lecithins was assumed to be the same as that of the lysolecithins, in the absence of evidence to the contrary. (The extrapolated values do not take into account chain melting transitions). We have also used Fig. 6 to rank several proteins according to their interactions with lipids. The proteins are located below lipids with which they form hydrophobically-stabilised,complexes and above lipids with which they do not interact. We have located as,-, /?-, K-caseins and also serum high density lipoprotein apoproteins on the free energy diagram according to their known interactions with phospholipids. We have already shown that a,,-casein interacts to form stable lipid-protein complexes with (diC,,)lecithin and with (C$,)lysolecithin (with some difficulty) but not with (diCl&cithin. We have therefore located cc,,-casein between (diC,& and (diCX4) lecithin in Fig. 6. /?- and xc-caseins interact readily with egglysolecithin but not with (diCl,Jlecithin. These proteins have been placed tentatively just above (diC,,,)lecithin in Fig. 6. High density lipoprotein apoprotein has been shown to interact with synthetic lecithins up to and including (diC,,)lecithin and also with egg-yolk lecithin [rg, 261. The high density lipoprotein apoprotein was thus located below the egg-yolk lecithin. The value of a free energy diagram such as Fig. 6, is that having located a protein on the diagram by its known interactions with one or two lipids under a standard set of conditions, we can predict that under the same conditions it will interact with lipids which lie above it on the diagram but not with lipids which lie below it. The diagram may also be used cautiously to predict the binding constants of lipids to proteins with the limitation that with shorter chain-length lipids, the hydrophobic contributions to the free energies of transfer of the molecules may become so small that other forces may dominate their behaviour. The observation that complex formation between the short-chain lysolecithins and cr,,-casein is not quantitative is consistent with the expectation that hydrophobic interactions with proteins should be weaker with shorter lipid alkyl chains. Indeed, the evidence is that hydrophobic interactions do increase with chain length, but only up to a certain point. The constraint is provided by the protein which presumably has a binding site of limited size or accessibility (cf. the binding of hydrocarbons to /?lactoglobulin) [26]. The free energy of transfer of lipid to protein will thus decrease with lipid chain length until the binding site is filled. After that, any further increase in lipid chain length merely results in lowering the free energy of the lipid phase with the ultimate result that lipid-protein interaction becomes unfavourable. We are currently attempting to determine experimentally the binding constants (and thus the free energies of binding) of lysolecithins to the various proteins in our scheme and thus refine the free energy values in Fig. 6. This, in turn, will enable us to judge the real predictive value of this approach. * Critical micelle concentrations of (Cl&, (C&, (C,& and (C&lysolecithin from Hayashi et al. [23], (diC&lecithin from Smith and Tanford [24], egg lysolecithin and sodium dodecylsulphate were measured in this laboratory. The value for egg-yolk lecithin was located in the lecithin range by analogy with the egg lysolecithin location in the lysolecithin range.

I35

ACKNOWLEDGEMENTS

The authors wish to thank C. E. Hill for assistance with the synthesis of phospholipids, S. Richardson for general technical assistance and D. Atkinson, R. B. Leslie and M. C. Phillips for helpful discussions. REFERENCES I Barratt, M. D. and Rayner, L. (1972) Biochlm. Biophys. Acta 255,974-980 Partrick, B., Barratt, M. D., Davis, M. A. F. and Rayner, L. (1972) Biochim. Biophys. Acta 255, 981-987 3 Whitehurst, R. J. and Barratt, M. D., unpublished. 4 Thompson, M. P. and Kiddy, C. A. (1964) J. Dairy Sci. 47, 626632 5 Hipp, N. J., Groves, M. L., Custer, I. H. and McMeekin, T. L. (1952) I. Dairy Sci. 35, 272-281 6 Zittle, C. A. and Custer, J. H. (1963) J. Dairy Sci. 46, 1183-1188 7 Hanahan, D. J. Rodbell, M. and Turner, L. D. (1954) J. Biol. Chem. 206, 431-441 8 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265275 9 Chen, W. S., Toribara, T. Y. and Warner, H. (1956) Anal. Chem. 28, 1756-1758 IO Bigelow, C. C. (1967) J. Theoret. Biol. 16, 187-211 II Mercier, J. C., Grosclaude, F. and Rlbadeau-Dumas, B. (1971) Eur. J. Biochem. 23,41-51 12 Ribadeau-Dumas, B., Brlgnon, G., Grosclaude, F. and Mercier, J. C. (1972) Eur. J. Biochem. 25, 505-514 13 Hill, R. 3. and Wake, R. G. (1969) Nature 221, 635-639 14 Phillips, M. C., Evans, M. T. A. and Hauser, H. (1972) Proc. 6th Int. Cong. Surface Activity., Zurich, Vol. II, pp. 381-391, Carl Hanser, Munich 15 Leslie, R. B., Irons, L. and Chapman, D. (1969) Biochim. Biophys. Acta 188, 237-246 16 Ho, C. and Chen, A. (1967) J. Biol. Chem. 242, 551-553 17 Schmidt, D. G. (1970) Biochim. Biophys. Acta 207, 130-138 18 Scanu, A. M. (1965) Proc. Natl. Acad. Sci. U.S. 54, 1699-1705 19 Lux, S. E., Him, R., Shrager, R. I. and Gotto, A. M. (1972) J. Biol. Chem. 247, 2598-2606 20 Herskovlts, T. T. (1966) Biochemistry 5, 1018-1026 21 Morrlsett, J. D., David, J. S. K., Pownall, H. J. and Gotto, A. M. (1973) Biochemistry 12, 1290I299 22 Tanford, C. (1972) J. Mol. Biol. 67, 59-74 23 Hayashi, M., Okazaki, M. and Hara, I. (1972) Proc. 6th Int. Cong. Surface Activity., Zurich, Vol. II, pp. 361-370, Carl Hanser, Munich 24 Smith, R. and Tanford, C. (1972) J. Mol. Biol. 67, 75-83 25 Davis, M. A. F., Hauser, H., Leslie, R. B. and Phillips, M. C. (1973) Biochlm. Biophys. Acta 317, 214-218 26 Wishnia, A. and Pinder, Jr, T. W. (1966) Biochemistry 5, 1534-1542 2