- Email: [email protected]
EiJ
Journal of Dairy Research (1974), 41, 9-17
The influence of lysolecithin on the complex formation between /?-lactoglobulin and K-casein By 0. KORVER AND H. MEDER Unilever Research, Vlaardingen, The Netherlands (Received 23 March 1973) Ultraviolet difference spectral measurements and some circular dichroism data are reported on the systems (/Mactoglobulin (BLG)/soya-lysolecithin (sll)), (K-casein (KC)/sll) and (BLG/KC/sll). Sll destabilizes the native conformation of BLG and KC and promotes the interaction between BLG and KC. The influence of sll: protein ratio and time of heating on the complex formation has been investigated. SUMMARY.
The heat-induced complex formation between /Mactoglobulin (BLG) and ^-casein (KC) has been studied thoroughly because of its importance for the properties of milk and milk products. The present knowledge in this field has been recently reviewed (Sawyer, 1969) and can be summarized as follows: 1. Complex formation does not occur below 65 °C. It is known that heating of BLG above 65 CC causes a partly reversible conformational change (Dupont, 1965). KC has only a small amount of ordered structure so that disruption by heat cannot take place (Herskovits, 1966; McKenzie, 1967). It is, therefore, reasonable to assume that BLG must be partly denatured before complex formation occurs. 2. There is still controversy about the mechanism of the complex formation. It is known that the primary thermo-denaturation of BLG involves disulphide aggregation. It may be that only in this aggregated form is BLG capable of complex formation with KC in a non-specific manner. On the other hand, there is a possibility that in the presence of KC, denaturation of BLG will result in disulphide bond formation between the 2 proteins. It seems that the experimental evidence is in favour of the first possibility. 3. The degree of interaction reaches a maximum at 85 °C. Heating of a 1:1 mixture of BLG and KC for 20 min at pH 6-5 produces the following amounts of BLG reacting with KC: 3-4% at 65 °C, 8 3 % at 85 °C and 76% at 99 °C (Kresheck, Van Winkle & Gould, 1964). During an investigation of protein/phospholipid interactions by spectroscopic techniques, we found that lysolecithins have a destabilizing influence on the native conformation of both BLG and KC. This led us to investigate the influence of lysolecithins on the complex formation between these 2 proteins. The results of this work and the results of (BLG/soya-lysolecithin (sll)) and (KC/sll) interaction studies are discussed in this paper. The technique used was ultraviolet difference spectroscopy (UVDS). Some circular dichroism (CD) experiments were performed on the (BLG/sll) interaction.
Buffer
a%BLG/c% KC/6% sll -<
1
I-
1
2c%KC
f
c%KC/6%sll
1
4a% BLG
a% BLG/c% KC/6% sll
»~
l
Reference
Sample
t
t
«%BLG'c%KC
1
sll
KC
BLG
t
Reference
sll
KC
sll
1
Reference Sample
t
sll
KC
BLG
t
Reference
sll
KC
sll
1
t
Buffer
KC
sll
BLG
t
Sample
sll
sll
KC
BLG
Actual measurements
BLG
Measurements
BLG
sll
KC
BLG
t
Sample
sll
KC
sll
BLG
Baseline
I Buffer
2o%BLG/2c%KC
4c%KC
>~
i Buffer
26% sll
46% sll
1 Buffer
a%BLG/6%sll
26% sll
|
6% sll
«
Buffer
Dilution
2a% BLG/26% sll
4a % BLG
6% sll
v
Fig. 1. Dilution scheme. Stock solutions of /S-lactoglobulin (BLG), soya-lysolecithin (sll) and K-casein (KC) must contain 4a, 46 and 2c% respectively in method I, and 4a, 26 and 4c % respectively in method II. The composition of the starting solution depends on the desired concentration of the end solution. In every dilution step, equal volumes are mixed.
II
I
Method
o
p
fi-Lactoglobulin K-casein complex formation
11
sll:BLG = 0-2
300
325
350
sll:KC=02
250 Fig. 2. Difference spectra of the systems: (o) (BLG/sll) v. BLG + sll; (6) (KC/sll) v. KC + sll; and (c) (BLG/KC/sll) v. (BLG/sll) + (KC/sll) after 45 min at 40 °C. EXPERIMENTAL
Ultraviolet difference spectral measurements. The UVDS measurements were carried
out with a Unicam SP 800 spectrophotometer (Pye Unicam Ltd, Cambridge, England) using a 20-fold scale expansion and energy programme 8 E. For the (BLG/sll) and (KC/sll) UVDS measurements the procedures as described by Herskovits & Laskowski (1962) were used. Protein concentration was 0-2%. For the measurements on the system (BLG/KC/sll) 2 methods were applied (see Fig. 1). These procedures guaranteed the most accurate measurements for the weak signals. All solutions were clear. Stock solutions of BLG, KC and sll (noted in Fig. 1) were diluted by means of one and the same pipette which was blown out each time after use to increase reproducibility. For control all mixtures were weighed after pipetting. Maximum pipetting and weighing errors were smaller than the uncertainty in the spectrometric measurement. For a ratio BLG:sll:KC = a:b:c, the stock solutions contained 4a, 46 and 2c % respectively in method I, and 4a, 26 and 4c % respectively in method II. Phosphate buffer pH 7-2 and ionic strength 0-25 was used as solvent. The final mixtures were introduced into tandem cells and a baseline run. To obtain reproducible results the baseline was run with the same amount of absorbing material in both beams as was used in the final measurement (see Fig. 1). The measurements were made between
12
0 . KOBVER AND H . MEDEB
014
0
Sample
012 010
Molar ratio sll: BLG 16 20 24
12
Reference
BLG sll
-
BLG
sll
28
32
36
008 006 004 002
0
0-2
0-4
0-6
0-8
10
Weight ratio sll:BLG Fig. 3. Variation of the ultraviolet difference spectrum intensity at 291 nm for the system (BLG/sll) with the ratio slhBLG at 40 °C.
350 and 250 nm. In the 350-320 nm region the spectrum coincided with the baseline. For temperature control a Tamson thermostat was used. Both sample and reference cells were placed in a copper block to minimize temperature differences. Circular dichroism measurements. CD measurements were made on 0-2 % solutions in 1-cm and 0*1-mm cells, with a Jouan Dichrograph 18511 (Jouan Que'tin, Paris, France). CD data were given as Ae values. Ae = SMdjlc, where 8 = scale factor of the instrument, M = mean residue molecular weight (taken as 115 for KC and BLG), d = deflexion in mm; I = cell length in cm; c = concentration in g/1. Materials. BLG was obtained from Serva (Heidelberg, Germany) and purified by gel filtration on Sephadex G 100. The product was dialysed against water and freeze-dried. KC was prepared according to McKenzie & Wake (1961). Sll was prepared from soya-lecithin by enzymic action of phospholipase A 2 (pancreatin, from Merck AG, Darmstadt, Germany). Free fatty acids were not present. The fatty acid composition was: 16:0 (30-9%); 18:0 (12-0%); 18:1 (18-0%); 18:2 (34-7%); 18:3 (4-4%). Unless stated otherwise, all measurements were carried out in phosphate buffer pH 7-2, ionic strength 0-25. BESTJLTS
The interaction offi-lactoglobulinwith soya-lysolecithin. The difference spectrum produced by the interaction of sll with BLG is shown in Fig. 2(a). The influence of the ratio slhBLG on the UVDS intensity after 10 and 20 h is shown in Fig. 3. After
fi-Lactoglobulin K-casein complex formation
13
Table 1. Influence of heating at 70 °G for 10 min on circular dichroism intensities measured at 40 °C of the system (sll/BLG) (A = 291 n m is a negative maximum relating to a tryptophan chromophore; A = 215 nm is the negative maximum ascribed to the ^-conformation.) — Ae291 x 103 Weight ratio sllrBLG
Not heated
10 min a t 70 °C
Not heated
10 min at 70 °C
0 0-25 0-50 1-00
21 20 20 18
17 15 13
1-61 1-65 1-65 1-76
1-78 1-88 2-04 2-17
Molar ratio sll:KC 10 15 20 25 30
35 40
012 010
g 008 < 006 004 002 0
2
4
6
8
10 12 14 16 18 20 22 24 Time, h
Fig. 4 Variation for t h e ultraviolet difference spectrum intensity a t 290 nm with time for the system (KC/sll) at 40 °C with the ratio sll:KC = 0-5.
0 01 0-2 0-3 0-4 0-5 0-6 0-7 0-8 Weight ratio sll:KC Fig. 5. Variation of the ultraviolet difference spectrum intensity at 290 nm for the system (KC/sll) with the ratio sll:KC at 40 °C.
10 h a distinct maximum occurred at 291 nm, indicating involvement of a tryptophan residue. CD results on the system (sll/BLG) are shown in Table 1. The interaction of K-casein with soya-lysolecithin. The difference spectrum of the system (KC/sll) (Pig. 26) shows maxima at 290 and 281 nm (presumably arising from transitions in the trytophan and tyrosine chromophores respectively) and a strong negative band at about 260 nm. At 40 °C the difference spectrum remained constant for about 2 h after which its intensity increased (Fig. 4). On 2 h incubation at 40 °C, the UVDS intensity at 290 nm was independent of the sll:KC ratio (Fig. 5), but at 10 and 20 h incubation at 40 °C the intensity increased strongly when the ratio sll:KC was increased from 0-1 to 0-25 (by weight) or from 4 to 10 (molar ratio). When the ratio was further increased, the intensity increase was proportionately much smaller. The interaction of /3-lactoglobulin with K-casein in the presence of soya-lysolecithin. UVDS measurements carried out on the system (BLG/KC) did not give a difference spectrum at 40 °C; at 70 °C, however, positive bands appeared at 292 and 283 nm.
14
0 . KORVEB AND H . MEDEB
002
40° KC Sample""1**^ — BLG sll sll KC BLG Reference — sll sll
0-06 001
/
0-04 002
/ i
0-5
1-5
i
i
i
i
i
t
i
i
10 12 14 16 IX 20 Time, h Fig. 7. Variation of the ultraviolet difference spectrum intensity at 292 nm with time for the system (BLG/KC/sll) v. (BLG/sll) and (KC/sll) at 40 °C (weight ratios BLG:KC = 1, sll:BLG = sll-.KC = 0-3). 8
Time, h Fig. 6. Variation of the ultraviolet difference spectrum intensity a t 292 n m with time for the system (BLG/KC) v. BLG and K C a t 70 °C (weight ratio BLG:KC = 1 ) .
20 1-6 BLG KC sll BLG KC sll
< 0-8 0-4 0
2
4
6
Sample Reference
8 10 12 14 16 18 20 Time, h
Fig. 8. Variation of the ultraviolet difference spectrum intensity at 292 nm with time for the system (BLG/KC/sll) v. (BLG/KC) and sll at 40 °C (weight ratios BLG:KC = 1, sll:BLG = sll:KC = 0-3).
The intensity of these bands initially increased, but decreased again after 30 min (Fig. 6). When soya-lysolecithin (sll) was added to all cell compartments, a difference spectrum (method I, see Experimental) was obtained at 40 °C (Fig. 2c). A decrease in intensity was observed after 8 h (Fig. 7). The maximum occurring in the curves of Figs 6 and 7 (weight ratio sll:BLG = sll:KC = 0-3) may be explained in the following way. The complex formation between BLG and KC (and between BLG, KC and sll) produces an increase in absorption in the aromatic region. Thermal denaturation of BLG and KC and interaction between sll and BLG or KC also give positive difference spectra. Because these interactions take place in the reference cell a decrease in the intensity of the signals occurs. Apparently the complex formation between BLG and KC (and between BLG, KC and sll) is a much faster process than the thermal denaturation of BLG and KC or the complex formation between sll and BLG or KC. If the complex formation between BLG and KC (and between BLG, KC and sll) is much faster than the processes taking place in the reference cells or, in other words if the UVDS intensity produced by the reference cell solutions is much smaller than
jS-Lactoglobulin K-casein complex 0-32
0
4
formation
15
Molar ratios sll:KC = sll:BLG 8 12 16 20 24 28 32 36 40 20 h
0-28 0-24
lOh
0-20 0-16
o-n 008 004 0 0-1 0-2 0-3 0-4 0-5 0-6 0-7 0-8 0-9 1-0 11 Weight ratios sll:KC=sll:BLG Fig. 9. Variation of the ultraviolet difference spectrum intensity at 292 nm for the system (sll/BLG/KC) with the ratios sll:BLG and sll:KC at 40 °C.
that arising from the sample solutions, it is justified to measure the UVDS according to method II (see Experimental). Plots of &E v. time obtained in this way are shown in Fig. 8. The influence of the sll:protein ratio is shown in Fig. 9. In all experiments, curves similar to those of Fig. 8 were obtained. DISCUSSION
The reaction betweenfi-lactoglobulinand soya-lysolecithin. Circular dichroism (CD) results on BLG have been discussed in previous work on the optical rotatory dispersion and CD spectra of BLG (Timasheff, Townend & Mescanti, 1966; Townend, Kumosinski & Timasheff, 1967). One conclusion of this work was the following. When the protein is denatured (for instance by raising the pH) the CD spectrum changes in consequence of the conformational transition from /^-conformation to unordered conformation. Because of the presence of overlapping bands the dichroic intensity at 215 nm first increases and then decreases. Denaturation by heat gives similar CD curves. The intensity of the aromatic dichroic peaks between 300 and 250 nm decreases if the ordered arrangement of the aromatic side chains is intimately related to the secondary structure of the protein. It has been demonstrated that this is the case with BLG (Timasheff et al. 1966; Townend et al. 1967). Both alkaline denaturation (which gives unordered conformation) and alcohol denaturation (which produces a-helices) cause a decrease and eventually the disappearance of the aromatic Cotton effects. The CD results (Table 1) indicate that sll produces changes in the BLG conformation similar to those caused by heat denaturation or increase in pH, i.e. a decrease in the 291 nm intensity and an increase in the 215 nm intensity. The simultaneous effect of sll and heat enhances the effects. The destabilization of the BLG native conformation by sll may be explained by assuming that the changed environment (more apolar because of sll) makes it less unfavourable for hydrophobic regions of the proteins to be at the outside. The positive difference spectrum intensity at 292 nm of the system (sll/BLG) is in agreement with tryptophan chromophores being 2
DAR 41
16
0 . KOBVER AND H . MEDBB
shifted to a more apolar environment on contact with sll. The influence of the sll: BLG ratio on the spectrum intensity (see Fig. 3) shows a type of curve often found in protein interaction studies and has a similar explanation to that given for the system (KC/sll) (see below; Steinhardt & Reynolds, 1969). The reaction between K-casein and soya-lysolecithin. The positive difference spectrum in the aromatic region for the system (KC/sll) may also be ascribed to a shift of the chromophores to a more apolar environment and to a dissociation of KC aggregates (Cheeseman & Knight, 1970). It is reasonable to expect a hydrophobic interaction between the alkyl chain of sll and apolar amino-acid residues. Such a hydrophobic interaction has also been found in nuclear magnetic resonance (NMR) and electron spin resonance (ESR) studies (Barratt & Rayner, 1972). The interaction is accompanied by disaggregation or a loosening of the KC structure, as shown by the similarity of the effects on the NMR spectrum of lysolecithin and urea addition (Barratt & Rayner, 1972). Fig. 2(6) clearly shows that the positive peaks at 290 and 281 nm are superposed on a negative band at about 260 nm. It is known (Talbot & Waugh, 1970) that disulphide bridges play a role in KC-aggregation. Therefore, this band may contain a contribution (Donovan, 1969) from the cystine chromophore. Barratt & Rayner (1972) state that the interaction between sodium dodecyl sulphate (SDS) and caseins is similar to that between lysolecithin and caseins. In this respect it is noteworthy that the difference spectrum of the system (SDS/KC) as given by Cheeseman & Knight (1970) shows negative bands at 293 and 281 nm in contrast with our findings on the (sll/KC) system. It is not clear whether this discrepancy is caused by a fundamental difference in interaction or from differences in measuring technique. For instance, our measurements were carried out in phosphate buffer; Cheeseman & Knight's in tris buffer. Direct interaction between tris and proteins has been demonstrated (Rawitch & Gleason, 1971). Fig. 4 shows that heating at 40 °C produces a further loosening of KC-structure. The extent of this effect is dependent on the sll:KC ratio as shown in Fig. 5. The sharp increase in intensity between weight ratios 0-1 and 0-2, and the flattening of the curve at higher ratios, may be explained by assuming that there are several stages of binding, i.e. between ratios 0-1 and 0-2 disaggregation makes other binding sites accessible to sll. This is a normal situation for proteins. The reactions betweenfi-lactoglobulin,K-casein and soya-lysolecithin. The difference spectrum of the system (BLG/KC/sll), (Fig. 2c) is qualitatively similar to that of the system (KC/sll), (Fig. 26). It is, therefore, very likely that what are actually observed in the measurements of the mixtures are signals originating from KC. If the positive difference spectrum in the system (KC/sll) is interpreted as indicating dissociation of the KC aggregates, sll plays a dual role in the (BLG/KC) complex formation i.e. it destabilizes the native conformation of BLG, and it promotes dissociation of KC. The following scheme is in agreement with the experimental data obtained so far: i BLGnatIve -
II
, BLGr p a r t l y unfolded 1. sll 2. heat
+
-"^dissociated
£ = =
1. sll 2. heat
| Fast
(BLG/KC) c o m p l e x (sll)
fi-Lactoglobulin K-casein complex formation 17 The equilibria I and II lie far to the left and to the right respectively under 'native conditions'. The association with sll promotes the exposure of hydrophobic regions of the protein, either by unfolding (BLG) or by dissociating (KC) the protein. Once the partly unfolded BLG and the dissociated KC have been formed, they associate rapidly to form the complex. It is not possible to decide from the present experiments whether the sll is actually present in the complex or serves only as a catalyst. Further NMR measurements might shed light on this question. The scheme implies that the concentrations of partly unfolded BLG and dissociated KC may be very small, depending on the equilibrium positions. In some instances (sll:protein molar ratio < 5) the positive signals in the systems (BLG/sll) and (KC/sll) are very weak or virtually absent, although the signal in the (BLG/KC/sll) system is still reasonably strong. This is an indication that what is observed is the spectrum originating from the complex formation. The data of Fig. 9 clearly show that on addition of sll to the (BLG/KC) mixture there is a relatively rapid increase of A.E with increasing sll:protein ratio. After the increase a plateau is reached and subsequently a new increase. Similar results have already been mentioned and explained for the (KC/sll) and (BLG/sll) interactions. From the present work it can be concluded that sll facilitates the complex formation between BLG and KC. If the proposed mechanism is correct, it can be concluded that, in general, compounds that more effectively promote the unfolding of BLG should accelerate the rate of complex formation more. The same applies to the influence of phospholipids on the KC dissociation. Little is known about this process at present. It will be necessary to obtain more information by NMR and UVDS measurements. Possibly ultracentrifuge experiments may also be of use. REFERENCES BARRATT, M. D. & RAYNER, L. (1972). Biochimica et Biophysica Ada 255, 974. CHEESEMAN, G. C. & KNIGHT, D. J. (1970). Journal of Dairy Research 37, 259. DONOVAN, J. W. (1969). In Physical Principles and Techniques of Protein Chemistry, Part A, p. 138. (Ed. S. J. Leach.) New York: Academic Press. DUPONT, M. (1965). Biochimica et Biophysica Ada 102, 500. HERSKOVITS, T. T. (1966). Biochemistry 5, 1018. HERSKOVITS, T. T. & LASKOWSKI, M., J R (1962). Journal of Biological Chemistry 237, 2481. KRESHECK, G. C , VAN WINKLE, Q. & GOULD, I. A. (1964). Journal of Dairy Science 47, 126.
MCKENZIE, H. A. (1967). Advances in Protein Chemistry 22, 55. MCKENZIE, H. A. & WAKE, R. G. (1961). Biochimica et Biophysica Ada 47, 240. RAWITCH, A. B. & GLEASON, M. (1971). Biochemical and Biophysical Research Communications 45, 590. SAWYER, W. H. (1969). Journal of Dairy Science 52, 1347. STEINHARDT, J. & REYNOLDS, J. A. (1969). Multiple Equilibria in Proteins. New York: Academic Press. TALBOT, B. & WAUGH, D. F. (1970). Biochemistry 9, 2807. TIMASHEFF, S. N., TOWNEND, R. & MESCANTI, L. (1966). Journal of Biological Chemistry 241, 1863.
TOWNEND, R., KTJMOSINSKI, T. F. & TIMASHEFF, S. N. (1967). Journal of Biological Chemistry 242, 4538.
Printed in Oreat Britain
The influence of lysolecithin on the complex formation between /?-lactoglobulin and K-casein By 0. KORVER AND H. MEDER Unilever Research, Vlaardingen, The Netherlands (Received 23 March 1973) Ultraviolet difference spectral measurements and some circular dichroism data are reported on the systems (/Mactoglobulin (BLG)/soya-lysolecithin (sll)), (K-casein (KC)/sll) and (BLG/KC/sll). Sll destabilizes the native conformation of BLG and KC and promotes the interaction between BLG and KC. The influence of sll: protein ratio and time of heating on the complex formation has been investigated. SUMMARY.
The heat-induced complex formation between /Mactoglobulin (BLG) and ^-casein (KC) has been studied thoroughly because of its importance for the properties of milk and milk products. The present knowledge in this field has been recently reviewed (Sawyer, 1969) and can be summarized as follows: 1. Complex formation does not occur below 65 °C. It is known that heating of BLG above 65 CC causes a partly reversible conformational change (Dupont, 1965). KC has only a small amount of ordered structure so that disruption by heat cannot take place (Herskovits, 1966; McKenzie, 1967). It is, therefore, reasonable to assume that BLG must be partly denatured before complex formation occurs. 2. There is still controversy about the mechanism of the complex formation. It is known that the primary thermo-denaturation of BLG involves disulphide aggregation. It may be that only in this aggregated form is BLG capable of complex formation with KC in a non-specific manner. On the other hand, there is a possibility that in the presence of KC, denaturation of BLG will result in disulphide bond formation between the 2 proteins. It seems that the experimental evidence is in favour of the first possibility. 3. The degree of interaction reaches a maximum at 85 °C. Heating of a 1:1 mixture of BLG and KC for 20 min at pH 6-5 produces the following amounts of BLG reacting with KC: 3-4% at 65 °C, 8 3 % at 85 °C and 76% at 99 °C (Kresheck, Van Winkle & Gould, 1964). During an investigation of protein/phospholipid interactions by spectroscopic techniques, we found that lysolecithins have a destabilizing influence on the native conformation of both BLG and KC. This led us to investigate the influence of lysolecithins on the complex formation between these 2 proteins. The results of this work and the results of (BLG/soya-lysolecithin (sll)) and (KC/sll) interaction studies are discussed in this paper. The technique used was ultraviolet difference spectroscopy (UVDS). Some circular dichroism (CD) experiments were performed on the (BLG/sll) interaction.
Buffer
a%BLG/c% KC/6% sll -<
1
I-
1
2c%KC
f
c%KC/6%sll
1
4a% BLG
a% BLG/c% KC/6% sll
»~
l
Reference
Sample
t
t
«%BLG'c%KC
1
sll
KC
BLG
t
Reference
sll
KC
sll
1
Reference Sample
t
sll
KC
BLG
t
Reference
sll
KC
sll
1
t
Buffer
KC
sll
BLG
t
Sample
sll
sll
KC
BLG
Actual measurements
BLG
Measurements
BLG
sll
KC
BLG
t
Sample
sll
KC
sll
BLG
Baseline
I Buffer
2o%BLG/2c%KC
4c%KC
>~
i Buffer
26% sll
46% sll
1 Buffer
a%BLG/6%sll
26% sll
|
6% sll
«
Buffer
Dilution
2a% BLG/26% sll
4a % BLG
6% sll
v
Fig. 1. Dilution scheme. Stock solutions of /S-lactoglobulin (BLG), soya-lysolecithin (sll) and K-casein (KC) must contain 4a, 46 and 2c% respectively in method I, and 4a, 26 and 4c % respectively in method II. The composition of the starting solution depends on the desired concentration of the end solution. In every dilution step, equal volumes are mixed.
II
I
Method
o
p
fi-Lactoglobulin K-casein complex formation
11
sll:BLG = 0-2
300
325
350
sll:KC=02
250 Fig. 2. Difference spectra of the systems: (o) (BLG/sll) v. BLG + sll; (6) (KC/sll) v. KC + sll; and (c) (BLG/KC/sll) v. (BLG/sll) + (KC/sll) after 45 min at 40 °C. EXPERIMENTAL
Ultraviolet difference spectral measurements. The UVDS measurements were carried
out with a Unicam SP 800 spectrophotometer (Pye Unicam Ltd, Cambridge, England) using a 20-fold scale expansion and energy programme 8 E. For the (BLG/sll) and (KC/sll) UVDS measurements the procedures as described by Herskovits & Laskowski (1962) were used. Protein concentration was 0-2%. For the measurements on the system (BLG/KC/sll) 2 methods were applied (see Fig. 1). These procedures guaranteed the most accurate measurements for the weak signals. All solutions were clear. Stock solutions of BLG, KC and sll (noted in Fig. 1) were diluted by means of one and the same pipette which was blown out each time after use to increase reproducibility. For control all mixtures were weighed after pipetting. Maximum pipetting and weighing errors were smaller than the uncertainty in the spectrometric measurement. For a ratio BLG:sll:KC = a:b:c, the stock solutions contained 4a, 46 and 2c % respectively in method I, and 4a, 26 and 4c % respectively in method II. Phosphate buffer pH 7-2 and ionic strength 0-25 was used as solvent. The final mixtures were introduced into tandem cells and a baseline run. To obtain reproducible results the baseline was run with the same amount of absorbing material in both beams as was used in the final measurement (see Fig. 1). The measurements were made between
12
0 . KOBVER AND H . MEDEB
014
0
Sample
012 010
Molar ratio sll: BLG 16 20 24
12
Reference
BLG sll
-
BLG
sll
28
32
36
008 006 004 002
0
0-2
0-4
0-6
0-8
10
Weight ratio sll:BLG Fig. 3. Variation of the ultraviolet difference spectrum intensity at 291 nm for the system (BLG/sll) with the ratio slhBLG at 40 °C.
350 and 250 nm. In the 350-320 nm region the spectrum coincided with the baseline. For temperature control a Tamson thermostat was used. Both sample and reference cells were placed in a copper block to minimize temperature differences. Circular dichroism measurements. CD measurements were made on 0-2 % solutions in 1-cm and 0*1-mm cells, with a Jouan Dichrograph 18511 (Jouan Que'tin, Paris, France). CD data were given as Ae values. Ae = SMdjlc, where 8 = scale factor of the instrument, M = mean residue molecular weight (taken as 115 for KC and BLG), d = deflexion in mm; I = cell length in cm; c = concentration in g/1. Materials. BLG was obtained from Serva (Heidelberg, Germany) and purified by gel filtration on Sephadex G 100. The product was dialysed against water and freeze-dried. KC was prepared according to McKenzie & Wake (1961). Sll was prepared from soya-lecithin by enzymic action of phospholipase A 2 (pancreatin, from Merck AG, Darmstadt, Germany). Free fatty acids were not present. The fatty acid composition was: 16:0 (30-9%); 18:0 (12-0%); 18:1 (18-0%); 18:2 (34-7%); 18:3 (4-4%). Unless stated otherwise, all measurements were carried out in phosphate buffer pH 7-2, ionic strength 0-25. BESTJLTS
The interaction offi-lactoglobulinwith soya-lysolecithin. The difference spectrum produced by the interaction of sll with BLG is shown in Fig. 2(a). The influence of the ratio slhBLG on the UVDS intensity after 10 and 20 h is shown in Fig. 3. After
fi-Lactoglobulin K-casein complex formation
13
Table 1. Influence of heating at 70 °G for 10 min on circular dichroism intensities measured at 40 °C of the system (sll/BLG) (A = 291 n m is a negative maximum relating to a tryptophan chromophore; A = 215 nm is the negative maximum ascribed to the ^-conformation.) — Ae291 x 103 Weight ratio sllrBLG
Not heated
10 min a t 70 °C
Not heated
10 min at 70 °C
0 0-25 0-50 1-00
21 20 20 18
17 15 13
1-61 1-65 1-65 1-76
1-78 1-88 2-04 2-17
Molar ratio sll:KC 10 15 20 25 30
35 40
012 010
g 008 < 006 004 002 0
2
4
6
8
10 12 14 16 18 20 22 24 Time, h
Fig. 4 Variation for t h e ultraviolet difference spectrum intensity a t 290 nm with time for the system (KC/sll) at 40 °C with the ratio sll:KC = 0-5.
0 01 0-2 0-3 0-4 0-5 0-6 0-7 0-8 Weight ratio sll:KC Fig. 5. Variation of the ultraviolet difference spectrum intensity at 290 nm for the system (KC/sll) with the ratio sll:KC at 40 °C.
10 h a distinct maximum occurred at 291 nm, indicating involvement of a tryptophan residue. CD results on the system (sll/BLG) are shown in Table 1. The interaction of K-casein with soya-lysolecithin. The difference spectrum of the system (KC/sll) (Pig. 26) shows maxima at 290 and 281 nm (presumably arising from transitions in the trytophan and tyrosine chromophores respectively) and a strong negative band at about 260 nm. At 40 °C the difference spectrum remained constant for about 2 h after which its intensity increased (Fig. 4). On 2 h incubation at 40 °C, the UVDS intensity at 290 nm was independent of the sll:KC ratio (Fig. 5), but at 10 and 20 h incubation at 40 °C the intensity increased strongly when the ratio sll:KC was increased from 0-1 to 0-25 (by weight) or from 4 to 10 (molar ratio). When the ratio was further increased, the intensity increase was proportionately much smaller. The interaction of /3-lactoglobulin with K-casein in the presence of soya-lysolecithin. UVDS measurements carried out on the system (BLG/KC) did not give a difference spectrum at 40 °C; at 70 °C, however, positive bands appeared at 292 and 283 nm.
14
0 . KORVEB AND H . MEDEB
002
40° KC Sample""1**^ — BLG sll sll KC BLG Reference — sll sll
0-06 001
/
0-04 002
/ i
0-5
1-5
i
i
i
i
i
t
i
i
10 12 14 16 IX 20 Time, h Fig. 7. Variation of the ultraviolet difference spectrum intensity at 292 nm with time for the system (BLG/KC/sll) v. (BLG/sll) and (KC/sll) at 40 °C (weight ratios BLG:KC = 1, sll:BLG = sll-.KC = 0-3). 8
Time, h Fig. 6. Variation of the ultraviolet difference spectrum intensity a t 292 n m with time for the system (BLG/KC) v. BLG and K C a t 70 °C (weight ratio BLG:KC = 1 ) .
20 1-6 BLG KC sll BLG KC sll
< 0-8 0-4 0
2
4
6
Sample Reference
8 10 12 14 16 18 20 Time, h
Fig. 8. Variation of the ultraviolet difference spectrum intensity at 292 nm with time for the system (BLG/KC/sll) v. (BLG/KC) and sll at 40 °C (weight ratios BLG:KC = 1, sll:BLG = sll:KC = 0-3).
The intensity of these bands initially increased, but decreased again after 30 min (Fig. 6). When soya-lysolecithin (sll) was added to all cell compartments, a difference spectrum (method I, see Experimental) was obtained at 40 °C (Fig. 2c). A decrease in intensity was observed after 8 h (Fig. 7). The maximum occurring in the curves of Figs 6 and 7 (weight ratio sll:BLG = sll:KC = 0-3) may be explained in the following way. The complex formation between BLG and KC (and between BLG, KC and sll) produces an increase in absorption in the aromatic region. Thermal denaturation of BLG and KC and interaction between sll and BLG or KC also give positive difference spectra. Because these interactions take place in the reference cell a decrease in the intensity of the signals occurs. Apparently the complex formation between BLG and KC (and between BLG, KC and sll) is a much faster process than the thermal denaturation of BLG and KC or the complex formation between sll and BLG or KC. If the complex formation between BLG and KC (and between BLG, KC and sll) is much faster than the processes taking place in the reference cells or, in other words if the UVDS intensity produced by the reference cell solutions is much smaller than
jS-Lactoglobulin K-casein complex 0-32
0
4
formation
15
Molar ratios sll:KC = sll:BLG 8 12 16 20 24 28 32 36 40 20 h
0-28 0-24
lOh
0-20 0-16
o-n 008 004 0 0-1 0-2 0-3 0-4 0-5 0-6 0-7 0-8 0-9 1-0 11 Weight ratios sll:KC=sll:BLG Fig. 9. Variation of the ultraviolet difference spectrum intensity at 292 nm for the system (sll/BLG/KC) with the ratios sll:BLG and sll:KC at 40 °C.
that arising from the sample solutions, it is justified to measure the UVDS according to method II (see Experimental). Plots of &E v. time obtained in this way are shown in Fig. 8. The influence of the sll:protein ratio is shown in Fig. 9. In all experiments, curves similar to those of Fig. 8 were obtained. DISCUSSION
The reaction betweenfi-lactoglobulinand soya-lysolecithin. Circular dichroism (CD) results on BLG have been discussed in previous work on the optical rotatory dispersion and CD spectra of BLG (Timasheff, Townend & Mescanti, 1966; Townend, Kumosinski & Timasheff, 1967). One conclusion of this work was the following. When the protein is denatured (for instance by raising the pH) the CD spectrum changes in consequence of the conformational transition from /^-conformation to unordered conformation. Because of the presence of overlapping bands the dichroic intensity at 215 nm first increases and then decreases. Denaturation by heat gives similar CD curves. The intensity of the aromatic dichroic peaks between 300 and 250 nm decreases if the ordered arrangement of the aromatic side chains is intimately related to the secondary structure of the protein. It has been demonstrated that this is the case with BLG (Timasheff et al. 1966; Townend et al. 1967). Both alkaline denaturation (which gives unordered conformation) and alcohol denaturation (which produces a-helices) cause a decrease and eventually the disappearance of the aromatic Cotton effects. The CD results (Table 1) indicate that sll produces changes in the BLG conformation similar to those caused by heat denaturation or increase in pH, i.e. a decrease in the 291 nm intensity and an increase in the 215 nm intensity. The simultaneous effect of sll and heat enhances the effects. The destabilization of the BLG native conformation by sll may be explained by assuming that the changed environment (more apolar because of sll) makes it less unfavourable for hydrophobic regions of the proteins to be at the outside. The positive difference spectrum intensity at 292 nm of the system (sll/BLG) is in agreement with tryptophan chromophores being 2
DAR 41
16
0 . KOBVER AND H . MEDBB
shifted to a more apolar environment on contact with sll. The influence of the sll: BLG ratio on the spectrum intensity (see Fig. 3) shows a type of curve often found in protein interaction studies and has a similar explanation to that given for the system (KC/sll) (see below; Steinhardt & Reynolds, 1969). The reaction between K-casein and soya-lysolecithin. The positive difference spectrum in the aromatic region for the system (KC/sll) may also be ascribed to a shift of the chromophores to a more apolar environment and to a dissociation of KC aggregates (Cheeseman & Knight, 1970). It is reasonable to expect a hydrophobic interaction between the alkyl chain of sll and apolar amino-acid residues. Such a hydrophobic interaction has also been found in nuclear magnetic resonance (NMR) and electron spin resonance (ESR) studies (Barratt & Rayner, 1972). The interaction is accompanied by disaggregation or a loosening of the KC structure, as shown by the similarity of the effects on the NMR spectrum of lysolecithin and urea addition (Barratt & Rayner, 1972). Fig. 2(6) clearly shows that the positive peaks at 290 and 281 nm are superposed on a negative band at about 260 nm. It is known (Talbot & Waugh, 1970) that disulphide bridges play a role in KC-aggregation. Therefore, this band may contain a contribution (Donovan, 1969) from the cystine chromophore. Barratt & Rayner (1972) state that the interaction between sodium dodecyl sulphate (SDS) and caseins is similar to that between lysolecithin and caseins. In this respect it is noteworthy that the difference spectrum of the system (SDS/KC) as given by Cheeseman & Knight (1970) shows negative bands at 293 and 281 nm in contrast with our findings on the (sll/KC) system. It is not clear whether this discrepancy is caused by a fundamental difference in interaction or from differences in measuring technique. For instance, our measurements were carried out in phosphate buffer; Cheeseman & Knight's in tris buffer. Direct interaction between tris and proteins has been demonstrated (Rawitch & Gleason, 1971). Fig. 4 shows that heating at 40 °C produces a further loosening of KC-structure. The extent of this effect is dependent on the sll:KC ratio as shown in Fig. 5. The sharp increase in intensity between weight ratios 0-1 and 0-2, and the flattening of the curve at higher ratios, may be explained by assuming that there are several stages of binding, i.e. between ratios 0-1 and 0-2 disaggregation makes other binding sites accessible to sll. This is a normal situation for proteins. The reactions betweenfi-lactoglobulin,K-casein and soya-lysolecithin. The difference spectrum of the system (BLG/KC/sll), (Fig. 2c) is qualitatively similar to that of the system (KC/sll), (Fig. 26). It is, therefore, very likely that what are actually observed in the measurements of the mixtures are signals originating from KC. If the positive difference spectrum in the system (KC/sll) is interpreted as indicating dissociation of the KC aggregates, sll plays a dual role in the (BLG/KC) complex formation i.e. it destabilizes the native conformation of BLG, and it promotes dissociation of KC. The following scheme is in agreement with the experimental data obtained so far: i BLGnatIve -
II
, BLGr p a r t l y unfolded 1. sll 2. heat
+
-"^dissociated
£ = =
1. sll 2. heat
| Fast
(BLG/KC) c o m p l e x (sll)
fi-Lactoglobulin K-casein complex formation 17 The equilibria I and II lie far to the left and to the right respectively under 'native conditions'. The association with sll promotes the exposure of hydrophobic regions of the protein, either by unfolding (BLG) or by dissociating (KC) the protein. Once the partly unfolded BLG and the dissociated KC have been formed, they associate rapidly to form the complex. It is not possible to decide from the present experiments whether the sll is actually present in the complex or serves only as a catalyst. Further NMR measurements might shed light on this question. The scheme implies that the concentrations of partly unfolded BLG and dissociated KC may be very small, depending on the equilibrium positions. In some instances (sll:protein molar ratio < 5) the positive signals in the systems (BLG/sll) and (KC/sll) are very weak or virtually absent, although the signal in the (BLG/KC/sll) system is still reasonably strong. This is an indication that what is observed is the spectrum originating from the complex formation. The data of Fig. 9 clearly show that on addition of sll to the (BLG/KC) mixture there is a relatively rapid increase of A.E with increasing sll:protein ratio. After the increase a plateau is reached and subsequently a new increase. Similar results have already been mentioned and explained for the (KC/sll) and (BLG/sll) interactions. From the present work it can be concluded that sll facilitates the complex formation between BLG and KC. If the proposed mechanism is correct, it can be concluded that, in general, compounds that more effectively promote the unfolding of BLG should accelerate the rate of complex formation more. The same applies to the influence of phospholipids on the KC dissociation. Little is known about this process at present. It will be necessary to obtain more information by NMR and UVDS measurements. Possibly ultracentrifuge experiments may also be of use. REFERENCES BARRATT, M. D. & RAYNER, L. (1972). Biochimica et Biophysica Ada 255, 974. CHEESEMAN, G. C. & KNIGHT, D. J. (1970). Journal of Dairy Research 37, 259. DONOVAN, J. W. (1969). In Physical Principles and Techniques of Protein Chemistry, Part A, p. 138. (Ed. S. J. Leach.) New York: Academic Press. DUPONT, M. (1965). Biochimica et Biophysica Ada 102, 500. HERSKOVITS, T. T. (1966). Biochemistry 5, 1018. HERSKOVITS, T. T. & LASKOWSKI, M., J R (1962). Journal of Biological Chemistry 237, 2481. KRESHECK, G. C , VAN WINKLE, Q. & GOULD, I. A. (1964). Journal of Dairy Science 47, 126.
MCKENZIE, H. A. (1967). Advances in Protein Chemistry 22, 55. MCKENZIE, H. A. & WAKE, R. G. (1961). Biochimica et Biophysica Ada 47, 240. RAWITCH, A. B. & GLEASON, M. (1971). Biochemical and Biophysical Research Communications 45, 590. SAWYER, W. H. (1969). Journal of Dairy Science 52, 1347. STEINHARDT, J. & REYNOLDS, J. A. (1969). Multiple Equilibria in Proteins. New York: Academic Press. TALBOT, B. & WAUGH, D. F. (1970). Biochemistry 9, 2807. TIMASHEFF, S. N., TOWNEND, R. & MESCANTI, L. (1966). Journal of Biological Chemistry 241, 1863.
TOWNEND, R., KTJMOSINSKI, T. F. & TIMASHEFF, S. N. (1967). Journal of Biological Chemistry 242, 4538.
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