1 Refractive Index Matching to Develop Transparent Polyaphrons:

2 Characterization of Immobilized Proteins.

3
4 Keeran Ward, David C Stuckey*
5
6 Department of Chemical Engineering, Imperial College London, SW7 2AZ London, UK.
7 Telephone: +44 20 7594 5591; Fax: +44 20 7594 5629. Email:keeran.ward10@ic.ac.uk,
8 d.stuckey@ic.ac.uk*
9
10
1
2 Short Statistical Summary
3
4 This article looks at the significance of Refractive index matching to characterize the
5 structure of proteins adsorbed onto the aphron surface. The paper consists of 4841 words
6 including the title page and references, 3 Figures and 1Table.
7

8
9
10
 1 Abstract
2
3 Refractive index matching was used to create optically transparent polyaphrons to enable

4 proteins adsorbed to the aphron surface to be characterized. Due to the significant light

5 scattering created by polyaphrons, refractive index matching allowed for representative

6 circular dichroism (CD) spectra and acceptable structural characterization. The method

7 utilized n-hexane as the solvent phase, a mixture of glycerol and phosphate buffer (30%

8 [w/v]) as the aqueous phase, and the non-ionic surfactants, Laureth-4 and Kolliphor P-

9 188. Deconvolution of CD spectra revealed that the immobilized protein adapted its

10 native conformation, showing that the adsorbed protein interacted only with the bound

11 water layer (soapy shell) of the aphron. Isothermal calorimetry further demonstrated

12 that non-ionic surfactant interactions were virtually non-existent, even at the high

13 concentrations used (5% [w/v]), proving that non-ionic surfactants can preserve protein

14 conformation.

15

16

17 Keywords: Protein; Conformation; Colloidal Liquid Aphrons (CLAs); Refractive Index

18 Matching; Adsorption, Immobilization.

19

20

21

22
 1
2 Introduction
3
4 Characterization of protein structures at oil-water interfaces has been of interest for some

5 years due to their inherent interfacial properties leading to their application in emulsion

6 stability (1, 2). A variety of spectroscopic techniques have been used to assess

7 conformational changes at the oil-water interface, such as Fourier transform infrared

8 spectroscopy (FTIR) and Tryptophan fluorescence spectroscopy, which have been used

9 to investigate changes occurring on solid surfaces and stabilized emulsions (3, 4), and

10 these techniques have found that proteins undergo some conformational change at the oil-

11 water interface. However, the quantification of protein secondary structure using FTIR is

12 not a trivial process since it requires careful deconvolution (using curve fitting software),

13 and solvent subtraction (5) .

14 Circular dichroism (CD) has been used extensively to assess protein structure (6-8), and

15 relies on the interaction between chromophores within the secondary structure of proteins

16 and polarized light (9). As this occurs, the chiral compounds of the protein backbone

17 absorb light, allowing for excitation. Different structural contributions, such as helical

18 and pleated sheets, relay different transitions during excitation leading to characteristic

19 spectra (10). However, the use of CD to assess protein conformation at the oil-water

20 interface has been difficult due to the high absorbance/light scattering of these systems,

21 which hinders reliable CD spectra from being obtained. Studies by Husband and

22 coworkers used glycerol and polyethylene glycol in the aqueous phase in an effort to

23 create refractive index (RI) matched emulsions (11). This modification created a fairly

24 transparent emulsion, which was also capable of enabling high quality CD spectra to be

25 obtained to determine protein conformation. Some of the conditions necessary for

 1 selecting an acceptable RI additive are as follows; a high RI, good water solubility, low

2 absorbance over the CD range used, and be nonhalogenic, non-chiral and non-denaturing

3 in nature. Although both glycerol and polyethylene glycol had all of the characteristics

4 desirable for RI matching, the high levels of glycerol needed (58% (v/v)) can affect the

5 conformation of proteins upon adsorption, and in solution (7, 11).

6 Colloidal Liquid Aphrons (CLAs) differ from conventional emulsions in that their

7 structural arrangement (micron sized oil droplets stabilized by surfactant bilayers)

8 decreases the likelihood of phase separation. Also, unlike emulsions, CLAs do not need

9 large volumes of additives for RI matching since only a small volume of aqueous phase is

10 necessary for formulation. Past studies utilizing anionic surfactants has attributed

11 surfactant interactions to superactivity as well as denaturation among CLA immobilized

12 enzymes, with very little known about immobilized enzyme conformation (12-15). The

13 novelty in this research lies in the creation of RI matched polyaphrons that are very

14 similar supports for protein immobilization as conventional polyaphrons. Furthermore,

15 the ability to characterize protein structure at these interfaces has not been attempted

16 before in past research, and hence can provide valuable information about the interactions

17 occurring during immobilization.

18 In this study we present a novel method for formulating RI matched non-ionic

19 polyaphrons for the characterization of four (4) proteins, Bovine Serum Albumin (BSA),

20 Ovalbumin, Lysozyme and -Chymotrypsin, using CD.

21

22 Materials and Methods

23 Materials
24
 1 The enzymes examined were; lysozyme from chicken egg white (EC 3.2.1.17,

2 Mucopeptide N-acetylmuramylhydrolase, 90% pure, Sigma), Bovine Serum Albumin

3 (BSA, 96% pure, Sigma Aldrich), lyophilized Albumin from chicken egg white

4 (Ovalbumin, 98% pure, Sigma Aldrich,), and -chymotrypsin from bovine pancreas

5 (Sigma Aldrich, E.C. 3.4.21.1). Polyaphrons were manufactured using Mineral Oil

6 (Sigma Aldrich), Tween 20 (Polyoxyethylene sorbitan monolaurate, 99% pure, Sigma

7 Aldrich), Tween 80 (Polyoxyethylene sorbitan monooleate, 99% pure, Sigma Aldrich),

8 Kolliphor P-188 (Poly (ethylene glycol)-block-poly (propylene glycol)-block-

9 poly(ethylene glycol), Sigma), Kolliphor-EL (Polyoxyl-35 castor oil, Sigma) and Triton

10 X-100 (Polyethylene glycol tert-octylphenyl ether, Sigma). n-Hexane (97% pure, VWR

11 International), Laureth-4 (tetraethylene glycol monododecyl ether) and glycerol (99.5%

12 pure, VWR International) were used for refractive index matching. Potassium phosphate

13 monobasic (KH2PO4, 99% Sigma) was used for buffering. The high-performance liquid

14 chromatography grade organic solvent, acetonitrile (99.9%, VWR), and reagent grade

15 trifluoroacetic acid (99.5%, Sigma) were used for assaying concentration. The water used

16 throughout the experiment was distilled and deionised.

17

18 Instruments
19
20 UV-VIS spectrometry/UV-VIS scanning spectrophotometer (UV-1800 Shimadzu, UK)

21 and High Performance Liquid Chromatography (HPLC Shimadzu, UK) were used for

22 analyzing enzyme samples using a silica-based C4 HPLC column (Phenomenex, UK).

23 An overhead stirrer (Heidolph RZR 2020) was used for polyaphron preparation and a

24 vortex (Fisher Scientific) used for mixing, while a Biofuge Stratos centrifuge (Heraeus
 1 Instruments) was used for CLA separation. Disposable syringes (B.Braun Melsungen

2 AG) and 0.22 m syringe filters (Millipore Co.) were used to remove particulates prior to

3 sample analysis. Circular dichroism (CD) spectral measurements were performed on a

4 Jasco J715 Spectropolarimeter using a 1-mm quartz cell, while Isothermal calorimetry

5 was performed using a Micro-CAL titration calorimeter (Micro Cal Inc. USA). Particle

6 Size measurements were performed using the Mastersizer 2000 (Malvern instruments).

8 Protein Preparation
9
10 BSA, Ovalbumin and Lysozyme protein samples were prepared in 20mM potassium

11 phosphate (monobasic) buffer, while -chymotrypsin samples were made up via stock

12 solutions using 1mM HCL solution consisting of 2mM CaCl2, and further diluted to

13 0.08mM. CaCl2 was necessary for protease stability.

14

15 Polyaphron Formulation.
16
17 Polyaphron phases were made by the dropwise addition of mineral oil/surfactant solution

18 (1% w/v Tween 80) from a burette into a stirred foaming aqueous phase (1% w/v Tween

19 20 in lysozyme solution) using overhead stirring until the required phase volume ratio

20 (PVR= Vorg.Vaq-1) was reached. The solvent was added at a flowrate of 0.3ml.min-1 at a

21 stirrer speed of 700 rpm; after addition of the total volume of oil, the formulation was

22 sheared using the same apparatus at 1000 rpm in order to achieve the desired droplet size

23 (~12m). The resulting formulation was very viscous with a creamy white appearance,

24 and showed no phase separation over a period of several weeks. Polyaphron formulations

25 were made up to a phase volume ratio (PVR) of 8 and diluted to PVR 4. PVR 8 was the
 1 optimum choice for the PVR taking into consideration viscosity limitations as well as

2 uniform particle sizes. However, PVR 8 formulations became difficult to measure

3 volumes in, and hence the formulation was consequently diluted for easier estimations,

4 while the droplet size and distribution was unaffected (16).

6 Refractive Index Matched Polyaphrons (RIMPs)

7
8 The aqueous phase consisted of 5% (w/v) Kolliphor P-188, 25% (w/v) glycerol, and 70%

9 (w/v) phosphate buffer. Protein concentrations investigated were 2-3 mg.mL-1, while the

10 solvent phase consisted of 1% (w/v) Laureth -4 in n-Hexane. Approximately 12 mL of

11 the solvent phase was carefully added to 3 mL of the aqueous phase using a pipette (at a

12 rate of 0.5 mL.min-1), at a stirrer speed of 260 rpm. Upon addition of all the solvent

13 phase, 4-5 drops of glycerol were then added to create a completely transparent

14 polyaphron formulation. A large volume of glycerol (~30% w/v) was needed to correct

15 for the changes in RI due to the uptake of water within the soapy shell of the aphrons.

16 Blank RIMPs (without immobilized protein), as well as samples of the aqueous phase,

17 were used as controls in the CD experiments.

18

19 CLA manufacture, Sample Preparation and Protein Assay

20 Polyaphron samples were dispersed into a bulk continuous buffer phase at a ratio of the

21 dispersed phase to the bulk of approximately 40% CLA: 60% water. Samples were vortex

22 mixed for 1-2 minutes for homogeneity and allowed to settle for 1 hour; after settling two

23 distinct layers were observed, and the samples were centrifuged at 8500 g for 25 minutes

24 for further separation without CLA deterioration. A 1-2ml sample of the supernatant was
 1 pipetted and filtered using 0.22 m syringe filters to ensure a CLA free sample for

2 protein assaying. Lysozyme samples (100L) were injected into a Phenomenex C4 HPLC

3 column equilibrated with 0.1% Trifluoroacetic acid (TFA) in water (mobile phase A).

4 The column was eluted using 0.1% TFA in acetonitrile (mobile phase B) using a gradient

5 from 2-30% B in 15 min, and then a ramp from 30-90% for another 15 min at a flowrate

6 of 1.5 mL.min-1. Retention time (tR) for lysozyme was around 13.8 minutes.

7 Chromatograms were analyzed using Origin Pro 9 software.

9 Error Analysis

10 Experimental errors for all results were measured by calculating the Coefficient of

11 Variance:

12 COV = (1)

13 where is the standard deviation, and is the mean value. Samples (n=4) were

14 reproduced using 4 replicates giving a COV of <10%.

15 Particle size analysis

16 A Malvern Particle Size analyser (Mastersizer 2000) was used to quantify the radius of

17 the CLA. Polyaphron samples were diluted by adding 0.1ml of the formulation directly

18 into the dispersion unit (120 ml volume) filled with deionised water, and stirred at 2400

19 rpm. For each sample 6 readings were taken, with the average particle size quoted as D

20 [4,3], giving a COV of 6%.

21

22 Isothermal Calorimetry (ITC)

23 Measurements were performed using a VP-ITC (Micro Cal) titration calorimeter, and the
 1 method was adapted from the work of Al-Anber (2013) [20]. The unit consisted of a

2 reference and sample cell, roughly 1.4mL in volume, and was insulated by an adiabatic

3 shield allowing for careful feedback to maintain both cells at the same temperature. The

4 sample cell and syringe were washed with distilled water before each run, and then the

5 sample cell was loaded with lysozyme solution, while the syringe was loaded with

6 surfactant. The initial delay was 60 s, reference power 10 cal/s, and the filter was 2 s;

7 the ITC unit was calibrated at 25C. A total of 17-20 injections were run for each sample,

8 with an injection time of 20 s, and a time interval between injections of 210 s. The

9 solution in the sample cell was stirred at 315 rpm using a rotating paddle, and the heat of

10 dilution for both the protein and surfactant was measured and subtracted to obtain the

11 heat of adsorption. Samples were run in triplicate with a COV <5%.

12 Circular Dichroism (CD)

13 The CD spectra of native and immobilized proteins were recorded over the far-UV

14 wavelength range of 200-240 nm with a scan at 20C in a thermostatic cell holder. The

15 path length was 1 mm, the step resolution 0.5 nm and the bandwidth 1 nm; the scan speed

16 was 10 nm.min-1.

17 1mL of the RIMP formulation was pipetted into a syringe fitted with a needle, and then

18 the sample was added to the CD cuvette; sample cuvettes were then centrifuged at 2000 g

19 for 5 minutes to expel air bubbles. Dissolved protein samples consisted of native protein

20 solutions formulated using the same aqueous phase as that used for the RIMP

21 formulation. Samples of the aqueous phase as well as blank RIMPs were measured and

22 subtracted from both the immobilized and native protein spectra, with data presented as

23 ellipticities ( millidegree). The observed ellipticities were converted into molar

 1 ellipticities based on the mean molecular mass per residue of each protein. Average

2 spectra of the replicate scans were analysed using deconvolution software (CDNN

3 program version 2.1), which calculates the secondary structure of the peptide by

4 comparison with a base set of 13 known protein structures. The COV was <5%.

6 Results and Discussion

7 Surfactant Lysozyme Interactions and CLA-Lysozyme Adsorption

8 In an effort to understand the effects of non-ionic surfactants on adsorbed protein

9 conformation, a study into the adsorption of lysozyme using different non-ionic

10 surfactants was carried out. An estimate of the amount of protein adsorbed was carried

11 out using a mass balance, as illustrated in Equation 1:

12 = (1)

13 where, Mi is the mass immobilized [mg], CL is the enzyme loading (concentration)

14 examined [mg.mL-1], VL is the volume of enzyme solution used [mL], CS is the enzyme

15 concentration in the supernatant [mg.ml-1], and Vs is the volume of the supernatant [mL].

16 The amount immobilized was calculated as the mass immobilized per CLA surface, as

17 described in Equation 2 (17):

18 = (2)
3

19 where, r is the radius of the CLA [m], and V is the volume of oil used for manufacture

20 [m3].

21 Percentage immobilization was approximated by comparing the immobilized mass to the

22 initial mass of protein used:


1 Percent Immobilization (w/w) = (3)

2 where, M is the mass of protein initially used during formulation .

3 The bar chart (Figure 1) shows that lysozyme immobilization was relatively unchanged

4 using different surfactants; the maximum amount of lysozyme adsorbed was 0.27 mg.m2,

5 accounting for 27% (w/w) immobilization (Equation 3). Several studies have been carried

6 out using non-ionic surfactants for protein solublization and extraction due to their non-

7 denautring nature (18-20). Furthermore, several reports have indicated high activity

8 retention amongst enzymes interacting with non-ionic reverse micelles, showing that

9 proteins can maintain activity at concentrations at or higher than the critical micelle

10 concentration (21, 22). Some studies suggest that specific groups of non-ionic surfactants,

11 in particular Tweens and Sorbitans, can possess a negative charge able to induce

12 electrostatic interactions (18, 23). Based on the results in Figure 1, there is no change in

13 the adsorption profile of lysoyzme (postively charged, pI=11) onto non-ionic CLAs, and

14 hence surfactant interactions are most likely too weak to support immobilization. For

15 non-ionic CLAs, the main forces contributing to adsorption are hydrophobic interactions

16 due to surfactant interactions, as well as those of the non-polar solvent phase (15, 24).

17 Due to the charge interactions of lysozyme, it is possible that repulsion between protein

18 molecules could decrease immobilization, however, for the adsorption of proteins onto

19 hydrophobic interfaces, hydrophobic interactions can overcome electrostatic interactions,

20 allowing for immobilization (25).

21 Figure 2 presents the calorimetric traces for lysozyme and surfactants Triton X-100 (TX-

22 100) and Kolliphor P-188. These results show that the heat generated from surfactant

23 dilution was much greater than that from lysozyme-surfactant interactions, and changing
 1 the concentration of lysozyme and surfactant by a factor of 10 had no influence on the

2 interaction. The integral enthalpy change for an interaction using ITC was recorded by

3 estimating the difference between the heat of dilution and that of the interaction between

4 lysozyme and surfactant molecules. However, since the heat of dilution was almost

5 identical to that of the interaction, the enthalpy change was recorded as null. The results

6 of this experiment differ considerably from those researchers investigating the interaction

7 of lysozyme with cationic surfactants where the enthalpy changes were much larger than

8 those of surfactant dilution (26). Hence, these results suggest that the interaction between

9 lysozyme and non-ionic surfactants was relatively small. By comparing the results from

10 Figures 1 and 2, it is clear that hydrophobic interactions due to the presence of a non-

11 polar solvent drives lysozyme adsorption rather than surfactant interactions.

12 Immobilized Protein Characterization

13 Figure 3 shows the spectra for both immobilized and dissolved proteins; each individual

14 spectrum has unique minimum points which indicate distinctive structural properties.

15 With -chymotrypsin, a negative shoulder is observed at 230nm corresponding to the

16 histidine-40-tryptophan-141 complex (27). For BSA, two negative peaks at 209 and

17 222nm indicate the transitions of amide bonds within the helical conformation of the

18 protein (28). Lysozyme gives similar peak contributions, while ovalbumin has a major

19 peak around 222nm.

20 For the immobilized enzyme, each spectrum shows the same unique characteristics of its

21 freely dissolved counterparts. Comparing the results in Table 1 shows that the

22 conformation of the immobilized protein is maintained upon adsorption to the CLA

23 surface. The spectra and deconvolution data is consistent with the findings of Banerjee
 1 and Pal (27) for -chymotrypsin, Huntington and coworkers (22) for ovalbumin, Reed

2 and coworkers (30) for BSA, and Greenfield (9) for lysozyme. Protein adsorption at

3 oil/water interfaces almost always results in conformational changes due to unfolding, as

4 non-polar residues become available for interaction; the adsorption kinetics for proteins

5 at an interface occurs in stages. Firstly, the protein is transported to the surface from the

6 bulk phase, allowing for surface interactions to occur. Secondly, spreading occurs due to

7 the extent of the interactions, whether hydrophobic or electrostatic. The spreading rate

8 depends both on the conformational changes arising from interactions, as well as protein-

9 protein interactions due to the proximity of neighbouring molecules (31, 32).

10 The effect of both hydrophobic and electrostatic interactions on conformation depends on

11 the nature and charge of the protein. For elctrostatic interactions, the charge of the protein

12 and the surface induces binding which allows for adsorption. However, as the adsorption

13 rate increases, replusive forces arising from like charges can limit the amount of protein

14 adsorbed (33). For hydrophobic surfaces, adsorption occurs regardless of the nature of

15 electrostatic forces since the driving force for adsorption is determined by the

16 hydrophobic exterior of the protein (34). In most cases, hydrophobic surfaces lead to a

17 greater adsorbed layer due to a large entropy gain arising from the interaction with the

18 surface; as this occurs, the likelihood for unfolding increases (33).

19

20 Lysozyme has been known to lose small amounts of -helix upon adsorption to interfaces

21 (35). However, for proteins such as BSA, -casein and myoglobin, drastic reductions in

22 secondary structure have been observed (7, 11, 28). The nature of the solvent phase has a

23 direct effect on the level of conformational change induced upon adsorption (36, 37), and
 1 interactions with highly non-polar solvents results in dehydration and a consequent

2 increase in unfolding. For the RIMPs generated with hexane, adsorption to the solvent

3 core directly results in conformational changes, and hence the results suggest that

4 immobilization occurs within the aqueous shell of the polyaphron. The structure of

5 aphrons has been debated in the literature over the past decades. Sebba (17) proposed that

6 polyaphrons are comprised of an inner solvent core encapsulated by a surfactant bilayer.

7 Studies by Princen (38) showed that these systems are similar to high internal phase ratio

8 emulsions (HIPREs), being both polyhedral in structure, but differing in the presence of a

9 second surfactant. The presence of this polyhedral structure is the direct result of the

10 closed packing arrangement of the aphrons. According to Sebba (17), the presence of a

11 thin aqueous film or soapy shell was directly responsible for the inherent stability of

12 these systems. Lye and Stuckey (39) investigated the structure of aphron dispersions and

13 found that these systems do in fact possess a soapy shell, with a thickness close to what

14 was first postulated by Sebba. The results suggest that upon adsorption, protein

15 conformation is preserved due to the hydration effect induced by the soapy shell of the

16 aphron.

17 Studies have also shown that in the presence of small amounts of glycerol [35 % (v/v)],

18 proteins exhibit very little change in conformation (40). This is due to the preferential

19 hydration of glycerol since it does not significantly disrupt the hydrophobic interactions

20 necessary for a stable conformation (41). The results also suggest that non-ionic

21 surfactant- interactions do not induce large conformational changes in the immobilized

22 protein; this was evident by comparing ITC data in Figure 2 with that of CD data in Table

23 1. Non-ionic surfactants are known to preserve protein conformation since they do not
 1 cooperatively bind to protein molecules (42). The presence of a water layer within the

2 CLA structure is similar to the water pool in reverse micelle systems. In many cases,

3 protein conformation has been preserved due to a hydration shell maintained by the water

4 pool (43). A study by Yan and coworkers (44) has revealed that the microstructure of

5 CLAs possess reverse micelles (microemulsions) in both the solvent and aqueous phases.

6 It is possible that the presence of these micelles can contribute to a level of hydration

7 necessary to preserve conformation upon adsorption to the CLA surface.

8
9 Conclusions
10
11 In this study we present a novel method of formulating refractive index matched

12 polyaphrons to enable us to characterise immobilized proteins. The results showed that

13 protein adsorption was mainly attributed to hydrophobic interactions resulting from the

14 non-polar solvent core of the aphron. Furthermore, CD spectra suggested that proteins

15 mainly interacted with the soapy shell of the polyaphron allowing the protein to adopt a

16 hydrated conformation, with very few changes in structure being observed between the

17 immobilized and dissolved states. The hydration required could be attributed to both the

18 bound water of the soapy shell, as well as microemulsions present in the aqueous phase

19 of the polyaphron. Finally, insight into surfactant interactions indicates that non-ionic

20 surfactants do not bind to proteins, even at high concentrations, allowing for the

21 preservation of protein conformation upon immobilization.

22

23 Acknowledgments
 1 The authors are grateful for the partial funding received from MC2 Biotek, Derek

2 Wheeler, Stephen Lenon and Fraser Steele from Drug Delivery Solutions for their

3 assistance in developing the methodology, and Hanna Barriga for assisting in carrying out

4 ITC experiments.

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10

11
1 Figures and Tables

2
3 Table 1: Deconvoluted CD spectra for Dissolved and Immobilized Proteins.
4
5
6 Protein Deconvoluted Data (%)
7 -Helix -Sheet -Turn Random
8 Coil
9 Immobilized Protein
10 -Chymotrypsin 9 34 22 35
11 BSA 53 10 14 23
12 Ovalbumin 28 21 18 33
13 Lysozyme 30 19 25 26
14 Dissolved Protein
-Chymotrypsin 10 32 23 35
15
BSA 54 9 14 23
16 Ovalbumin 29 22 17 32
17 Lysozyme 32 18 23 27
18
19
20
21
22 Figure Captions
23
24 Figure 1: Lysozyme adsorption as a function of changing non-ionic surfactant. Enzyme
25 concentration 4 mg.mL-1 at pH 6.2. Error bars report SD, n=4.
26
27 Figure 2: ITC isotherms of a) Kolliphor P-188 Dilution, b) TX-100 Dilution, c)
28 Lysozyme-Kolliphor P-188 and, d) Lysozyme-TX-100. Enzyme concentration 10
29 mg.mL-1, surfactant concentration 5% (w/v) at pH 6.2.
30
31 Figure 3: Far-UV Circular Dichroism spectra of Immobilized and Dissolved -
32 Chymotrypsin (a), BSA (b), Lysozyme (c), and Ovalbumin (d).
33
1 Figure 1
2

3
4
5
6
1
2 Figure 2
3
4
5

6
7
8
9
10
11
1 Figure 3
2
3