pubs.acs.org/Langmuir © 2010 American Chemical Society
Resistance of β-Casein at the Air-Water Interface to Enzymatic Cleavage Jhih-Min Lin, Joo Chuan Ang, and J. W. White* Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia Received October 1, 2010. Revised Manuscript Received November 3, 2010 X-ray reflectivity from an air-buffer interfacial β-casein monomolecular film placed on a solution of chymosin (renin) showed unexpectedly slow proteolytic cleavage. To understand this, the separate structures of β-casein and chymosin, the presentation of each molecule to the other at the air/liquid interface, and that of their mixtures is reported. At the air/solution interface, the hydrophobicity of the protein molecules causes orientation and some deformation of the conformation. When β-casein was presented to a chymosin monomolecular interfacial film, the chymosin was largely displaced from the surface, which was accounted for by the different surfactancy of the two molecules at 25 °C. There was no observable proteolysis. In the reverse experiment, a significant enzymatic degradation and the signature of hydrophobic fragments was observed but only at and above an enzyme concentration of 0.015 mg/mL in the substrate. For comparison, the air/solution interface of premixed β-casein with chymosin in phosphate buffer showed that the film was composed of β-casein proteolytic fragments and chymosin.
Introduction We have previously observed significant differences in the response of β-lactoglobulin to denaturation at the air-water interface and in bulk solution.1,2 There is very little denaturation of a preformed monomolecular film of β-lactoglobulin at the airwater interface when subjected to concentrated guanidinium hydrochloride (GHCl) using the “flow trough” method3,4 to look only at the response of the surface film. By contrast, the surface film structure that arises from a bulk mixture of the protein with 4 M GHCl is very different from that of the air-water interface film formed from a dilute solution of the protein.3 To the best of our knowledge, previous reports of enzymatic action on β-casein or κ-casein were adsorbed on solid-liquid interfaces. The action of chymosin on κ-casein adsorbed on polystyrene latex particles5 resulted in an initial decrease of 8-10 nm in the particle diameter, as measured by dynamic light scattering, before aggregation sets in. Another independent study of κ-casein adsorbed onto latex particles coated with milk fat globule membrane material6 also observed similar results in the transient reduction in the particle hydrodynamic radius before flocculation of the particles. The decrease in particle size due to chymosin cleaving the κ-casein is in good agreement with reported size changes for rennet-treated casein micelles.7 Similar proteolysis experiments using a different enzyme, endoproteinase Asp N, for β-casein adsorbed on a hydrophobic silicon substrate revealed that only the outermost layer exposed to the buffer solution was affected by the addition of Asp N in the absence of calcium ions.8 *Author to whom comments are to be sent. E-mail:
[email protected]. Tel: þ61 2 61253578. Fax: þ61 2 61254903.
(1) Perriman, A. W.; Henderson, M. J.; Holt, S. A.; White, J. W. J. Phys. Chem. B 2007, 111, 13527. (2) Perriman, A. W.; Henderson, M. J.; Evenhuis, C. R.; McGillivray, D. J.; White, J. W. J. Phys. Chem. B 2008, 112, 9532. (3) Lin, J.-M.; White, J. W. J. Phys. Chem. B 2009, 113, 14513. (4) Perriman, A. W.; McGillivray, D. J.; White, J. W. Soft Matter 2008, 4, 2192. (5) Anema, S. G. Int. Dairy J. 1997, 7, 553. (6) Su, J. H.; Everett, D. W. Food Hydrocolloids 2003, 17, 529. (7) Walstra, P.; Bloomfield, V. A.; Wei, G. J.; Jenness, R. Biochim. Biophys. Acta 1981, 669, 258. (8) Follows, D.; Holt, C.; Nylander, T.; Thomas, R. K.; Tiberg, F. Biomacromolecules 2004, 5, 319.
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This paper explores the resilience of an adsorbed monomolecular film of β-casein subjected to enzymatic attack by chymosin (renin) at room temperature and the inverse situation when a chymosin monomolecular film at the air-water interface is presented with a β-casein substrate using the “flow trough”.4 The flow trough method provides a way to prepare the isolated film at the air/solution interface and allows the subphase solution to be replaced by the other solution. The flow trough contains a stable pumping system to wash in/out subphase solutions, and the trough is designed to fit our reflectometer and antivibration system. In a separate experiment, where the chymosin solution was presented to a preformed oriented β-casein film at the airwater interface, the ratio of enzyme to casein was 1:100. These are compared with the time-dependence of the X-ray reflectivity from the air/water interface of mixed chymosin-β-casein solutions with different chymosin concentrations. All experiments were conducted at pH 7. The kinetics of the enzymatic proteolysis of bovine β-casein by chymosin at 30 °C and pH 6.2 with an analysis of the peptide fragments formed9 indicates that, for solutions of the two components below the protein critical micelle concentration and for a mole ratio of enzyme to substrate of 1:6000, the reaction proceeds almost to completion in about 40 min. All of the molecular species formed in the proteolysis can be identified by RP-HPLC chromatography. Other evidence9 indicates that the reaction proceeds at an optimum rate at a pH of about 3.5.10 In experiments on the reaction in solution, mole ratios of enzyme to β-casein have been varied from 1:1 to 1:100. Given the high mole ratios of enzyme to β-casein in the monomolecular film, fast changes were expected. The concentration of enzyme had to be progressively increased to see this reaction with the film.
Methods and Materials Sample Preparation. The commercial β-casein purchased from Sigma-Aldrich was dissolved in 50 mM phosphate buffer (Na2HPO4/NaH2PO4) at room temperature. We examined the purity of β-casein by using ion exchange chromatography. (9) Carles, C.; Ribardeau-Dumas, B. Biochemistry 1984, 23, 6839. (10) Foltman, B. Phil. Trans. R. Soc. London B 1970, 257, 147.
Published on Web 11/23/2010
DOI: 10.1021/la103943b
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Table 1. Fitting Parameters of the Reflectivity Profiles of the β-Casein Adsorbed Layer Conducted at the Periods of Adsorbed Layer Formation and after Washing out the Forming Solution adsorbed layer formation time/minute 1st sublayer 2nd sublayer 3rd sublayer
thickness/A˚ SLD/10-6 A˚-2 roughness/ A˚ thickness/A˚ SLD/10-6 A˚-2 roughness/ A˚ thickness/A˚ SLD/10-6 A˚-2 roughness/ A˚
0 21.9(0.1) 10.8(0.05) 2.5(0.08) 36.9(1.6) 10.0(0.06) 4.0
350 21.2(1.2) 11.01(0.06) 2.9(0.08) 26.9(1.7) 10.4(0.06) 4.0 42.0(2.7) 9.8(0.05) 6.0
washing out 1340 20.4 (1.5) 11.1(0.06) 2.9(0.08) 24.4(1.8) 10.6(0.06) 4.0 30.3(2.1) 9.9(0.06) 6.0
1440 18.5(2.1) 11.1(0.08) 2.85(0.1) 22.18(2.0) 10.6(0.07) 4.0 26.8(2.1) 9.9(0.06) 6.0
1770 21.3(1.3) 10.9(0.06) 2.8(0.08) 24.2(1.8) 10.4(0.06) 4.0 26.0(2.5) 9.8(0.06) 6.0
2790 21.8(1.1) 11.0(0.06) 2.8(0.08) 24.4(1.7) 10.4(0.06) 4.0 29.9(2.7) 9.8(0.06) 6.0
Table 2. Calculated Parameters of the Adsorbed Layer Formed from 0.1 mg/mL β-Casein Solution and after Washing out the Forming Solution adsorbed layer formation time/minute first 2 sublayers
whole layer
thickness/A˚ SLD/10-6 A˚-2 volume fraction surface excess/mg/m2 thickness/ A˚ SLD/10-6 A˚-2 volume fraction surface excess/mg/m2
0 58.8 10.3 0.31 2.38
The total content of β-casein was about 95%, and the other minor impurities were γ3-casein and κ-casein. On the other hand, the chymosin was manufactured by Chr. Hansen, and the concentration of this enzyme was 1.5 mg/mL, with 100% of it being recombinant chymosin. In the first part of the experiments, we used 0.1 mg/mL β-casein solution to form an adsorbed layer at the air/liquid interface. The adsorbed layers of chymosin were formed by three different concentrations. By using the “flow trough” the forming solution of either β-casein or chymosin was replaced by phosphate buffer to get an isolated adsorbed layer at the air/liquid interface. After the isolated adsorbed layer had been equilibrated on phosphate buffer, the chymosin or β-casein solution was introduced underneath. In the second part, “premixed” solutions of β-casein (0.1 mg/ mL -0.43 10-2 mM) and chymosin (0.0015-0.42 10-4 mM, 0.015-0.42 10-3 mM, and 0.15 mg/mL -0.42 10-2 mM) were mixed at 25 °C and allowed to stand for 10 min to react and to form adsorbed layers at the air/liquid interface before measuring the reflectivity. The proteolysis of chymosin to β-casein is wellknown and cleaves the β-casein in minutes at this temperature, particularly at the Ala-189-Phe-190 and Leu-192-Tyr-193 positions on the chain.9 The structure of the adsorbed layer composed of these small fragments and chymosin molecules was measured by X-ray reflectivity. X-ray Reflectivity. Structural measurements were made with the X-ray reflectometer at the Research School of Chemistry, Australian National University, and all the measurements were conducted at room temperature.9 For the data fitting, we chose a model to describe the protein film at the air/liquid interface that divided this surface film into several sublayers. The structure of each sublayer was represented by three parameters: the scattering length density (SLD), the thickness (τ), and the roughness. Besides these parameters, the SLD and the roughness of the subphase solution must be taken into account. The Motofit procedure11,12 in the Igor pro application was used to fit the data. For more quantitative analysis, a self-consistent approach to the protein volume fractions and surface excess was used to define the small changes in the film structures. (11) Nelson, A. J. Appl. Crystallogr. 2006, 39, 273. (12) Brown, A. S.; Holt, S. A.; Saville, P. M.; White, J. W. Aust. J. Phys. 1997, 50, 391.
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350 48.1 10.7 0.45 2.78 90.1 10.3 0.30 3.53
washing out 1340 44.8 10.8 0.49 2.83 75.1 10.4 0.36 3.55
1440 40.7 10. 8 0.49 2.61 67.5 10.4 0.37 3.2
1770 45.5 10.6 0.44 2.58 71.5 10.3 0.33 3.03
2790 46.2 10.7 0.45 2.68 76.1 10.3 0.32 3.17
In the fitting process, the two roughness parameters for sublayers 2 and 3 were constrained to give the best minimum set of seven parameters for a three-layer model. In the formula, eq 1, this model gives the Nbi and τi for each sublayer, but the sum is constrained by the average SLD of the protein film (Nbx), the volume fraction of the protein in the protein film (f) as well as the surface excess (Γ). To estimate the overall effects, eqs 2 and 3 were used iteratively. n P
Nbx ¼
i¼1
Nbx, i 3 τi n P i¼1
f ¼
τi
ð1Þ
Nbx - Nbx, base Nbx, protein - Nbx, base
ð2Þ
Γ ¼ Fprotein 3 f 3 τ
ð3Þ
Results and Discussion β-Casein and chymosin solutions were used to form the isolated adsorbed layers at the air/liquid interface at 25 °C using the flow trough technique. Due to the hydrophobicity of the molecules, they self-assemble at the interface. When the subphase solutions underneath the isolated adsorbed layers are replaced by the flow trough technique, only subtle fluctuations in their structures were observed.4 Isolated Adsorbed Layers of β-Casein and Chymosin at the Air/Liquid Interface. β-Casein Monolayers. The kinetics of formation for the isolated adsorbed layers from β-casein and chymosin solutions is different and indicates different surfactancy strength. In the flow trough experiments, the concentration of β-casein solution used to form the adsorbed layer was 0.1 mg/mL. A series of X-ray measurements to investigate the structural evolution of the adsorbed layer at the air/solution interface were conducted. The first profile of X-ray reflectivity Langmuir 2010, 26(24), 18985–18991
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Figure 3. The SLD profiles of the adsorbed layer formed from 0.0015 mg/mL chymosin solution. Table 3. Fitting Parameters and Calculated Surface Excesses of the Adsorbed Layer of 0.0015 mg/mL Chymosin Solution time/minute thickness/ A˚ SLD/10-6 A˚-2 volume fraction surface excess/mg/m2
0 13.51(4.54) 9.9(0.3) 2.56(0.34) 0.3
130 14.61(3.46) 9.9(0.2) 2.56(0.25) 0.32
265 15.85(2.68) 9.9(0.1) 2.49(0.19) 0.35
Figure 1. The real-space SLD profiles of the adsorbed layer formed from 0.1 mg/mL β-casein solution. (a) Two SLD profiles collected before washing out the forming solution at 0 and 1340 min after sample preparation. (b) After washing out the forming solution, the SLD profiles collected at 1440, 1770, and 2790 min, respectively.
Figure 4. The SLD profiles of the adsorbed layer formed from 0.015 mg/mL chymosin solution.
Figure 2. The surface excesses of the β-casein adsorbed layer formed by a 0.1 mg/mL solution. The β-casein solution was washed out after the film equilibrium (1340 min), and the first measurement after washing out β-casein solution commenced at 1440 min.
was collected immediately after pouring the β-casein solution into the trough. The fitting of the X-ray reflectivity data used a two-slab model in the early stage of adsorbed layer formation, and a three-slab model was employed for the intermediate and equilibrium states. The fitting parameters are shown in Table 1. The real-space SLD profiles are shown in Figure 1a before washing out the β-casein solution and in Figure 1b after washing Langmuir 2010, 26(24), 18985–18991
out the β-casein solution. The saturated adsorbed layer at the interface is composed of three sublayers. Although there were some subtle variations in SLD and thickness of each sublayer caused by washing out the β-casein solution, the principal structure of the adsorbed layer was invariant. Furthermore, we calculated the surface excess and volume fraction to decompose the contributions of each sublayer by eqs 1, 2 and 3 as shown in Figure 2. The black straight line shows that the surface excess of the uppermost sublayer in the β-casein adsorbed layer is nearly constant. Between the time of 1340 and 1440 min, the forming solution was replaced by the buffer solution, and there was a significant drop in surface excesses of the first two sublayers and the whole adsorbed layer. Thus the middle and bottom sublayers were affected by washing out the forming solution much more than the uppermost sublayer. We suppose that the bottom sublayer in contact with the subphase solution makes the effect of changing the subphase solution stronger. By contrast, the uppermost sublayer was protected, and no significant change of the uppermost sublayer in surface excess occurred. The surface area was saturated and occupied by β-casein molecules in about 350 min DOI: 10.1021/la103943b
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Lin et al. Table 4. The Fitting Parameters of 0.015 mg/mL Chymosin Solution adsorbed layer formation
time/minute 1st sublayer 2nd sublayer
0 16.3(2.7) 10.0(0.1) 2.54(0.2)
thickness/A˚ SLD/10-6 A˚-2 roughness/ A˚ thickness/A˚ SLD/10-6 A˚-2 roughness/ A˚
400 16.8(1.6) 10.3(0.1) 2.36(0.2) 60.0(4.5) 9.6(0.04) 4.0
Table 5. Calculated Parameters of the Adsorbed Layer Formed from 0.015 mg/mL Chymosin Solution adsorbed layer formation time/minute thickness/ A˚ Average SLD/10-6 A˚-2 volume fraction surface excess/mg/m2
0 16.33 10.03 0.22 0.46
400 76.83 9.76 0.12 1.21
1910 74.03 9.88 0.16 1.58
washing out 2010 75.66 9.82 0.14 1.40
3145 71.53 9.93 0.18 1.67
after sample preparation. The short equilibrium time shows that the β-casein molecule has strong surface activity. Chymosin Monolayers. To prepare isolated chymosin adsorbed layers, three different concentrations of chymosin solutions were used. First we used the concentration of 0.0015 mg/mL chymosin solution to form the adsorbed layer at the interface. In Figure 3, the SLD profile shows that the adsorbed layer is diffuse. The surface excess of the adsorbed layer is 0.35 mg/m2 at 265 min, and the fitting parameters are shown in Table 3. Compared with the normal surface excess of a protein layer at the interface, the surface excess of a chymosin layer from the chymosin solution of 0.0015 mg/mL is only 10% of the normal surface coverage. That means there are not enough molecules to cover the surface even with a long incubation time. When the concentration of chymosin solutions was increased from 0.0015 to 0.015 and eventually 0.15 mg/mL, more close packed films were formed. In the film from 0.015 mg/mL chymosin solution, the fitting of the X-ray reflectivity profiles used a single-slab model for the first measurement and a two-slab model for the following measurements. The SLD profiles are shown in Figure 4. The fitting results are summarized in Table 4, and the parameters calculated from the fitting results are shown in Tables 5, 6 and 7. In Figure 4 the profiles show that the structure of the adsorbed layer is composed of a protein-rich sublayer and a diffuse tail extending into the subphase solution. In the first measurement after sample preparation, the structure of the adsorbed layer is a single layer similar to the structure of the adsorbed layer formed from 0.0015 mg/mL chymosin solution. The surface excess is 0.46 mg/m2, and it is slightly higher than that of the adsorbed layer formed from the 0.0015 mg/mL chymosin solution. Compared with the adsorbed layer of β-casein at the interface, the surface excess of chymosin adsorbed layer is relatively low. We attribute this to the lower surface activity of chymosin compared to β-casein. In Figure 5, the plot of surface excess versus time also indicates that the protein-rich sublayer and the diffuse tail were influenced by the process of washing out the forming solution at the same time with different time constants. From Table 5 the parameters show that the adsorbed layer of chymosin is saturated with a surface excess of around 1.6 mg/m2. For the sample of 0.15 mg/mL chymosin solution the fitting model used was a two-slab model for the first two time periods after formation and a three-slab model for the following measurements. The SLD profiles show that the structure of the adsorbed 18988 DOI: 10.1021/la103943b
washing out 1910 14.0(1.9) 10.8(0.3) 2.77(0.3) 60.0(3.8) 9.7(0.04) 4.0
2010 15.7(1.5) 10.6(0.1) 2.43(0.2) 60.0(4.3) 9.63(0.04) 4.0
3145 13.3(2.0) 11.1(0.4) 2.85(0.4) 58.3(3.9) 9.67(0.04) 4.0
layer is different from that formed from the low concentration forming solutions. Between the top protein-rich sublayer and the bottom diffuse tail, there is an intermediate sublayer as shown in Figure 6. The surface excess of the adsorbed layer is 2.52 mg/m2, and the volume fraction is 0.26. In Figure 5, the surface is saturated at around 1500 min for the adsorbed layer of the 0.015 mg/mL chymosin solution. Furthermore, the surface excess is 1.6 mg/m2, and the volume fraction is 0.18 at 3145 min. The numbers are close to the parameters in the reflectivity of the adsorbed layer formed from 0.15 mg/mL conducted at 375 min. We suppose the structure formed from the 0.15 mg/mL solution is an isolated monomolecular adsorbed layer plus the additional sublayer located between 20 A˚ and 40 A˚ beneath due to excess molecules adhering in this strong solution. In Figures 5 and 7, the variations of the surface excesses are large after washing out the forming solution. We attribute the perturbation between points 3 and 4 of Figure 7 to the lower surface activity of chymosin and greater ease of washing out. β-Casein Insertion into the Chymosin Adsorbed Layer. Isolated chymosin adsorbed layers were prepared by 0.015 and 0.15 mg/mL of chymosin buffer solutions. The β-casein solution of 0.1 mg/mL was introduced underneath these two isolated adsorbed layers. On the basis of the results of previous section, the structures of the adsorbed layers from 0.015 and 0.15 mg/mL were different. As the β-casein solution was introduced underneath these chymosin adsorbed layers in the two different experiments, the responses were different. For the adsorbed layer formed from 0.015 mg/mL chymosin solution, the structure of the adsorbed layer was best represented by a film composed of four sublayers as shown in Figure 8. The red curve in Figure 8 is the measurement taken just after flowing in the β-casein solution, and the blue curve is the real-space SLD profile collected at equilibrium after 2795 min at 25 °C. In Figure 9 the real-space SLD profiles are the results of the same experiment for the 0.15 mg/mL chymosin sample. In Figures 8 and 9, the black curves are the real-space SLD profiles of chymosin adsorbed layers. For the chymosin adsorbed layer formed from 0.015 mg/mL solution, the SLD profile rises due to the β-casein molecule quickly penetrating the interface. We deduce that the structure of the chymosin layer from 0.015 mg/ mL was loose and that insertion is easy as a result. From the SLD profiles after flowing in the β-casein solution, there was a diffuse tail extending into the subphase solution. Because of the different surface activities of these molecules, the chymosin molecules were replaced by β-casein molecules at the interface. Compared with the SLD profile of β-casein adsorbed layer, the SLD of the uppermost sublayer is slightly higher than that for a pure β-casein adsorbed layer. This indicates that the replacement was not complete, and some residual chymosin molecules remain at the interface. The chymosin surface film made from a 0.15 mg/mL solution is more resistant to β-casein penetration. The first two sublayers of the adsorbed layer are similar to those of the pure chymosin Langmuir 2010, 26(24), 18985–18991
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Article Table 6. Fitting Parameters of 0.15 mg/mL Chymosin Solution adsorbed layer formation
time/minute 1st sublayer 2nd sublayer 3rd sublayer
0 14.8(2.0) 10.5(0.2) 2.61(0.2) 60.0(3.7) 9.7(0.06) 4.0
thickness/A˚ SLD/10-6 A˚-2 roughness/ A˚ thickness/A˚ SLD/10-6 A˚-2 roughness/ A˚ thickness/A˚ SLD/10-6 A˚-2 roughness/ A˚
375 15.6(1.5) 10.7(0.2) 2.58(0.2) 60.0(3.1) 9.7(0.04) 4.0
washing out 1385 20.5(0.8) 10.9(0.06) 2.91(0.08) 24.8(3.8) 9.9(0.06) 4.0 34.2(4.9) 9.7(0.05) 6.0
1485 15.0(5.7) 11.2(0.5) 3.16(0.5) 13.0(2.6) 10.25(0.3) 4.0 46.5(5.7) 9.7(0.05) 6.0
2650 15.6(3.8) 11.3(0.4) 3.35(0.4) 14.2(2.1) 10.3(0.2) 4.0 44.7(4.7) 9.7(0.05) 6.0
Table 7. Calculated Parameters of the Adsorbed Layer Formed from 0.15 mg/mL Chymosin Solution adsorbed layer formation time/minute thickness/ A˚ average SLD/10-6 A˚-2 volume fraction surface excess/mg/m2
0 74.79 9.85 0.15 1.47
375 75.57 9.91 0.17 1.71
1385 79.51 10.05 0.22 2.31
washing out 1485 74.47 10.10 0.24 2.34
2550 74.56 10.15 0.26 2.52
Figure 7. The surface excesses of the chymosin adsorbed layer formed from the solution of 0.15 mg/mL.
Figure 5. The surface excesses of the chymosin adsorbed layer formed from the solution of 0.015 mg/mL.
Figure 8. The real-space SLD profiles of isolated chymosin adsorbed layer formed from 0.015 mg/mL chymosin solution with flowing in β-casein solution of 0.1 mg/mL.
Figure 6. The SLD profiles of the adsorbed layer formed from 0.15 mg/mL chymosin solution.
formed from 0.15 mg/mL chymosin solution. This structure indicates that the chymosin layer is relatively dense compared to that from 0.015 mg/mL chymosin solution. Comparison of the surface excesses of the two chymosin solutions and of the chymosin films with β-casein solution flowed beneath indicates Langmuir 2010, 26(24), 18985–18991
stronger lateral binding between chymosin molecules in the dense adsorbed layer. These differences in the chymosin films0 responses for 0.015 and 0.15 mg/mL forming solutions are not apparent after long time equilibrium, indicating that the films are of the same packing and possibly conformation. The first two sublayers are mainly contributed by β-casein and the chymosin molecules are replaced and push to the bottom of the adsorbed layer. Thus even the dense chymosin film eventually responds to the stronger β-casein surfactancy. The Enzymatic Degradation of β-Casein Adsorbed Layer at the Air/Liquid Interface. With 0.0015 mg/mL chymosin solution flowed under a β-casein adsorbed layer from 0.1 mg/mL forming solution, there was only a small, but perceptable change in the interfacial film SLD after 29 h at 25 °C. For 10 times more (a 0.015 mg/mL chymosin solution), there were significant changes, as DOI: 10.1021/la103943b
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Figure 9. The real-space SLD profiles of isolated chymosin adsorbed layer formed from 0.15 mg/mL chymosin solution with flowing in β-casein solution of 0.1 mg/mL.
Figure 10. The reflectivity profiles of the β-casein adsorbed layer subjected to chymosin solution of 0.015 mg/mL.
shown in Figure 10, but not complete cleavage of the molecule. The SLD profiles show this (Figure 10) at the beginning of the buffer subphase replacment and at 2810 min after flowing in the chymosin solution. Surface excess changes in all the three sublayers were observable. The overall loss is 25% from the surface excess of the whole film, and the losses from the first and sublayers are 8.8%, 30%, and 18%, respectively. These decreases are attributed to degradation of the β-casein film by chymosin. The increased thickness of the bottom sublayer is also significant, indicating that chymosin could be attached underneath the remaining β-casein and the cleaved hydrophobic residues (190 to 209 of β-casein). The Reflectivity of Chymosin Digested β-Casein Solutions. For solutions of β-casein and chymosin in phosphate buffer premixed prior to pouring into the trough, the concentration of β-casein was fixed at 0.1 mg/mL and the concentrations of chymosin were varied from 0.0015 to 0.15 mg/mL. The profiles in Figure 11a,b,c show the SLD for premixed solutions with molar ratios of enzyme to casein of 1:1, 1:10, and 1:100; there were similar structures formed at the air/solution interface. The fitting data are given in Table 9. These differ from the SLD profile of the initial measurement of the phosphate buffer β-casein solution where the adsorbed layer was composed of two sublayers, with the upper sublayer SLD at 10.7 10-6 A˚-2 and the bottom sublayer SLD about 10.1 10-6 A˚-2. The profiles of Figure 11b,c are very similar both at the first measurement (ca. 2 h) and after equilibration (ca. 70 h). The reflectivity from the adsorbed layers needing three layers at 18990 DOI: 10.1021/la103943b
Figure 11. The SLD profiles of premixed solutions of β-casein and
chymsoin. The β-casein concentration was fixed at 0.1 mg/mL while varying the chymosin concentrations: (a) chymosin to β-casein mole ratio of 1:1; (b) chymosin to β-casein mole ratio of 1:10; (c) chymosin to β-casein mole ratio of 1:100. The solid lines are the measurements conducted as the premixed solutions were poured into the trough, and the dotted lines are the equilibrium state. Table 8. Fitting Parameters of the β-Casein Film Subjected to Chymosin Substrate Formed from 0.15 mg/mL Chymosin Solution washing in casein solution
time 1st sublayer 2nd sublayer 3rd sublayer
thickness/A˚ SLD/10-6 A˚-2 roughness/A˚ thickness/A˚ SLD/10-6 A˚-2 roughness/A˚ thickness/A˚ SLD/10-6 A˚-2 roughness/A˚
0 20.2(1.2) 10.9(0.06) 2.85(0.09) 22.8(1.8) 10.3(0.07) 4.0 32.5(3.0) 9.8(0.06) 6.0
2810 20.5(1.1) 10.8(0.06) 2.52(0.09) 21.4(2.6) 10.0(0.07) 4.0 40.6(4.6) 9.6(0.05) 6.0
the earliest observation times. Compared to the β-casein solution alone, the SLD of the uppermost sublayer is higher than that of β-casein adsorbed and there is also an additional sublayer. Langmuir 2010, 26(24), 18985–18991
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Article Table 9. Fitting Parameters for Premixed Chymosin-β-Casein Solution Reflectivity mols chymosin/casein 1/100
experimental parameter 1 2 3 4
thickness/A˚ SLD/10-6 A˚-2 roughness/A˚ thickness/A˚ SLD/10-6 A˚-2 roughness/A˚ thickness/A˚ SLD/10-6 A˚-2 roughness/A˚ thickness/A˚ SLD/10-6 A˚-2 roughness/A˚
beginning 18.6 (1.6) 11.1 (0.1) 2.8 (0.1) 22.1 (2.4) 10.1 (0.08) 6 45.4 (3.9) 9.7 (0.05) 6
equilibrium 17.5 (2.9) 11.0 (0.1) 3.1 (0.2) 18.2 (2.4) 10.4 (0.1) 5 44.1 (4.9) 10.0 (0.05) 6 52.6 (4.7) 9.8 (0.05) 6
The data at equilibrium resemble those of Figure 9, and so we propose that the first two sublayers of the β-casein/chymosin mixed system have β-casein molecules on top and chymosin molecules underneath to form the other two diffuse sublayers. The result indicates that, even for the 1:10 in the mixed solution, the cleavage reaction cannot out-compete the β casein adsorption at the interface and that enzymatic reaction under these conditions is slow. By contrast, the premixed solution of the mole ratio 1:1 (Figure 11a) is very different both after the first measurement (ca. 2 h) and equilibrium (>8 h). We attribute this to successful protein cleavage in the premixed solution. The interface still looks as if it is composed of β-casein and/or a β-casein like species, possibly with some chymosin molecules. The adsorbed layer is best fitted by only two sublayers, and the SLD of each sublayer was lower than that of the first two sublayers of all the other samples. The well-documented proteolysis of β-casein by chymosin in solution gives some clues to the species present. First the concentrations of the proteins, β-casein (0.1 mg/mL -0.43 10-2 mM) and chymosin (0.15 mg/mL -0.42 10-2 mM) suggest that the data of Carles et al.5 could be a guide. From the reversedphase high-performace liquid chromatography (RP-HPLC) elution pattern, the dominant component of the reacted mixture is β-casein itself. The second most dominant component, βI,13 consisted of the large pieces 1-189 and 1-192 beginning at the N terminus. Lacking the very hydrophobic C terminus fragment, these should be weaker surfactants than β-casein, as the hydropathy profile14 is dominated by hydrophilic peptides. The next strongest elution peaks are identified with the 190-192 and 193209 very hydrophobic C terminus fragments, which we suppose must also be present in the upper layer.
Conclusions The experiments illustrate the competitive surfactancy of β-casein and chymosin at the air-water interface and that enzymatic reaction in both an oriented film and in protein-enzyme mixtures can be observed by X-ray reflectivity. In the reflectivity measurements on the separate proteins at different concentrations, the form of the SLD profiles changes at high protein concentrations, allowing best filling of the monomolecular layer to be estimated. A weak third sublayer is needed to give the best fit to the reflected X-ray intensity, consistent with a (13) Creamer, L. K.; Mills, O. E.; Richards, E. L. J. Dairy Res. 1971, 38, 269. (14) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105.
Langmuir 2010, 26(24), 18985–18991
mols chymosin/casein 1/10 beginning 18.9 (1.7) 11.2 (0.1) 2.8 (0.1) 20.0 (2.4) 10.1 (0.1) 6 60.0 (4.1) 9.7 (0.05) 6
equilibrium 16.2 (3.7) 11.2 (0.2) 3.1 (0.2) 18.3 (2.4) 10.4 (0.1) 5 46.5 (5.2) 10.0 (0.05) 6 41.2 (4.9) 9.8 (0.05) 6
mols chymosin/casein 1/1 beginning 22.0 (1.0) 10.8 (0.08) 2.9 (0.08) 23.4 (1.4) 10.1 (0.07) 4
equilibrium 22.4 (1.0) 10.6 (0.05) 2.7 (0.07) 23.9 (1.7) 9.9 (0.06) 4
train and loop model15 for the interface. The surface excesses; taking into account two layers only;are in accordance with the expected surface packing densities for proteins as shown in the tables. The different behavior of the chymosin-β-casein surface structure observed for the two styles of presentation of one molecule over the other (β-casein-established film with chymosin presented underneath and vice versa) is attributed simply to a difference in the surfactancy of the two molecules. When the order is chymosin on top, β-casein under, there are large changes in the interfacial structure attributed to the β-casein from the subphase solution displacing parts of the chymosin interfacial film to produce a new subsurface layer, mainly of chymosin. To a first approximation, the two proteins are behaving as independent surfactants. The apparently small extent of proteolysis in this configuration could not be estimated. At high chymosin subphase concentrations (0.15 mg/mL), cleavage reactions give the responses observed. The uppermost sublayer has a loss of only about 9% in surface excess; with the other two sublayers, at least 25% total loss of the surface excess is found. This is the extent of the enzyme effect on this film. The 9% result is consistent with the effects of orientation of this strong surfactant, making the 190-192 cleavage site less accessible to the chymosin and lateral binding of the β-casein molecules in the film. Surface retention of any hydrophobic proteolytic fragment of β-casein would obviously contribute. The premixed β-casein-chymosin solution experiment provides a “control”. The changes in the scattering function with time are not marked for low concentrations of chymosin, but, for the highest concentration, the SLD profile resembles that of β-casein itself (without the long tail and with a lower SLD at the top layer). The disappearance of the deep sublayers indicates that, at these high chymosin concentrations, extensive cutting in the β-casein chain leaves some of the more hydrophilic fragments away from the surface. No obvious clue is given by the scattering pattern or the SLD profile, but experiments in the future with fully deuterated recombinant β-casein should be able to resolve this matter. Acknowledgment. The authors thank the Australian Research Council and Dairy Ingredients Australia (DIGA) for the collaborative grant LP0774909 and acknowledge the valuable discussions held with other colleagues on the grant and members of DIGA. (15) Graham, D. E.; Philips, M. C. J. Colloid Interface Sci. 1979, 70, 403.
DOI: 10.1021/la103943b
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