Affinity-based reversed micellar protein extraction: II. Effect of cosurfactant tail length

Aff inity-Based Reversed Micellar Protein Extraction: 1. Principles and Protein-Ligand Systems

Brian D. Kelley, Daniel I.C. Wang, and T. Alan Hatton' Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139

Received September 11, 1992/Accepted July 14, 1993

Affinity cosurfactants, consisting of hydrophilic ligands derivatized with hydrophobic tails, increase the efficiency of selective protein recoveries using reversed micelles by extending the operating range of pH and salt concentration over which an extraction can be performed. Three different affinity cosurfactant-protein pairs have been used to demonstrate the principles of this extrac- tive technique: (i) concanavalin A, a lectin, was extracted with the addition of octyl glucoside; (ii) natural amphiphiles, such as lecithin, were used to extract myelin basic protein, a membrane-associated protein known to recognize and bind the phosphatidylcholine headgroup; and (iii) alkyl boronic acids were used to extract chymotrypsin. The enhancement in protein transfer correlated with the binding strength of the free ligand and protein in aqueous solution. Several control studies confirmed the biospecificity of the interactions of protein and affinity cosurfactant. 0 1993 John Wiley & Sons, Inc. Key words: hydrophilic ligands reversed micelles concanavalin A myelin basic protein chymotrypsin protein extraction affinity partitioning

INTRODUCTION

Reversed micelles are water-in-oil microemulsion droplets stabilized by surfactants in apolar solvent^.^^^^^ Hydrophilic species, such as amino acids and proteins, may be solubilized within these water pools, suggesting that reversed micelles may be useful for liquid-liquid extraction separation processes. Reversed micelles have been employed for the extraction and purification of several industrially rele- vant proteins, including lipases,* amylases," proteases,2° and food proteins. They have shown robust responses to complex feed mixtures, including whole and distributed cells,14 fermentation broths,35 and dried solids.28 While the results of these purifications are encouraging, there are several issues which require attention before reversed micellar extraction will compete with established technolo- gies. Recovery is usually below 80%, reuse of the reversed micellar phase is not addressed, and the overall purification factor is often not very high. The latter problem results from the selection mechanism, which is based on electrostatic interactions between the protein and the charged surfactant headgroups lining the interior of the reversed micellar wall.

Each of these purifications has exploited the unique charge characteristics of the protein of interest to partition

* To whom all correspondence should be addressed.

Biotechnology and Bioengineering, Vol. 42, Pp. 1199-1208 (1993) 0 1993 John Wiley & Sons, Inc.

it selectively into the reversed micellar phase. Adjustment of solution pH controls the protein charge, and protein extraction is closely tied to the protein's isoelectric point.16 In addition, the water content of the reversed micellar phase can be controlled by the salt concentration of the supporting aqueous phase.27 As the salt concentration increases, the reversed micelles shrink and effectively exclude proteins by size, although this apparent size exclusion effect has thermodynamic origins, and does not reflect a mechani- cal constraint on the protein uptake. These manipulations of pH and salt concentration are very similar to those used in purification by ion-exchange adsorption, because proteins are generally extracted under conditions where the pH results in a charge opposite that of the surfactant headgroup and the ionic strength is low. In many cases, recovery of the protein can be obtained by altering the pH of the back extraction buffer, and increasing the salt c~ncentration, '~,~' as in ion-exchange processes. Also, the addition of disrupting agents such as ethyl acetate44 or ethanol2 can be used to achieve dewatering of the reversed micelles, with subsequent protein rejection. Temperature increases, too, have been exploited to reduce the water content of a cationiclnonionic reversed micellar phase, with excellent concentration of the product, amylase.'

Two different approaches to increasing the selectivity of the extraction can be adopted. First, the protein charge can be modified to strengthen the electrostatic or hydrophobic attraction to the reversed micellar phase. Sassenfeld and Brewe?' used genetic engineering to modify argi- nine residues to urogastrone, increasing the protein charge at acidic pH. Such proteins would be expected to have increased transfer into the reversed micellar phase both because of the increased charge and because of the charge asymmetry.43 This approach would work well for proteins whose genes have been cloned, and can be altered and expressed in easily manipulated organisms, but for native, nonrecombinant proteins, a different strategy will need to be implemented.

Kabanov et aI.'l showed that covalent attachment of an alkyl group to the protein resulted in stronger protein- reversed micelle interactions. This approach is of limited appeal for separation processes because of the difficulty in detaching the alkyl tail once the separation has been achieved; a reversible complexation of the anchoring group

CCC 0006-3592/93/01001199-010

with the protein would be desirable. Thus, it has been suggested that a small concentration of a biospecific ligand which resides in the reversed micellar phase will increase the efficiency and extend the operating range of the extraction. Indeed, a mixed-micellar system consisting of a primary surfactant and an affinity cosurfactant should improve both protein uptake and selectivity. The primary surfactant should be capable of forming large reversed micelles, and should tolerate the presence of other amphiphiles without gross disturbance of the reversed micellar structure. The affinity cosurfactant should have a hydrophilic ligand which the protein is able to recognize and bind, and an attached hydrophobic tail, which will impart surface activity to the resulting protein-ligand complex, as shown in Figure 1. The ligand could be a substrate analog or product inhibitor, and the interaction must be reversible to allow the dissociation of product and cosurfactant after the back extraction.

In previous investigations, two protein-affinity cosurfactant systems have been examined, and some of the basic tenets of this extraction system have been highlighted. Woll et al.45 used octyl glucoside to extract concanavalin A, and demonstrated increased protein loading in the extracting phase. Control studies confirmed that the interaction within the reversed micellar phase was biospecific. Coughlin and Baclaski used an alkylated derivative of biotin to purify avidin from an aqueous solution.’ Excellent protein transfer resulted from the extremely strong interaction of avidin and biotin. Glycoproteins have also been extracted by their association with concanavalin A in the reversed micellar phase, although this was attributed to the altered charge properties of the lectin-glycoprotein complex and not to an increased surface activity.34

The purpose of this study was to establish further the fundamentals of protein extraction using affinity cosurfactants; to extend the methodology to different proteins; and to design, synthesize, and test affinity cosurfactants for a variety of applications. Several affinity cosurfactants were examined, including nonionic and zwitterionic species. In this article, we report the results of affinity partitioning

Figure 1. versed micellar extraction of proteins.

Schematic representation of affinity partitioning in the re-

studies, with an emphasis on showing that the enhancement in partitioning is due to biospecific interactions, and that the strength of binding of the ligand to the protein is an important consideration in selecting systems for affinity partitioning studies and processes. To this end, the extraction of the target protein was conducted in the presence and absence of affinity cosurfactant, for a variety of pHs and salt concentrations, the extent of protein transfer into the reversed micelle being the principal measure of performance. The back transfer of protein from the extract phase was not addressed in these studies. In a companion article,23 we develop a clearer understanding of the role played by the hydrophobic tail of the affinity cosurfactant in determining the efficiency of the extraction process.

MATERIALS AND METHODS

Materials

Except where noted below, all proteins and chemicals, including the surfactant Aerosol OT (AOT), were purchased from Sigma Chemical Co. Catecholborane and alkenes were obtained from Aldrich Chemical Co., and isooctane from Mallinckrodt. Tetradecane boronic acid was provided by the Alfred Bader Library of Rare Chemicals, Aldrich Chemical Co. All materials were used as received. The water used for all solutions was from the Milli-Q Water System (Millipore Corp.), and had a resistance of at least 10 M R * cm.

Protein Partitioning

The proteins were transferred into the reversed micelles under conditions of phase transfer equilibrium, in which the final reversed micellar phase consisted of solubilized water, protein, and salts, while the supporting aqueous phase contained equilibrium concentrations of AOT, cosurfactant, and isooctane. Protein solutions were made in buffer stocks to concentrations of either 0.5 or 1.0 mg/mL. Precise control of the reversed micellar phase water content was achieved through manipulation of the cation concentration in the supporting aqueous phase. All buffers contained only sodium as the cationic species; with other cations, ion exchange between the aqueous and reversed micellar phases can have a significant effect on the overall properties of the reversed micellar p h a ~ e , ’ ~ . ~ ~ complicating the interpretation of the experimental results. Mixtures of the three stock buffers, sodium lactate, phosphate, and carbonate, allowed exploration of the pH range 4 to 11, simply by mixing buffers with the same sodium ion concentration. The salt concentration listed in the figure captions is the concentration of the sodium ions derived solely from the aqueous buffer, and does not include the counterions contributed by the surfactant. This mixing of buffers of identical sodium concentrations yields reproducible water transfers independent of the pH of the aqueous phase, and allows the two effects of pH and counterion concentration to be addressed separately.22

1200 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 10, NOVEMBER 20, 1993

Equal volumes of a dry reversed micelle solution and an aqueous protein solution were mixed in a test tube using a Vortex mixer for 1 to 5 s. For low salt samples, the biphasic mixture was centrifuged at 2000g for 3 min to effect a preliminary phase separation. All samples were capped and equilibrated in a water bath at 25°C for 16 to 20 h. This period of incubation resulted in perfectly transparent reversed micellar and aqueous phases, with no precipitation at the interface. Samples of both the aqueous and organic phases were subjected to UV/VIS analysis (Hewlett-Packard 8452A diode array spectrophotometer) to determine protein concentration, using 280 nm as the primary wavelength and 410 nm as the internal reference. Calibration curves were constructed for each protein. Mass balance closures using separate aqueous and organic phase measurements were usually better than 5%, and there was very good agreement between the organic protein concentrations obtained directly and those obtained by difference. Karl Fisher titration was used to determine the water content of the reversed micellar phase (Mettler DL18).

For the AOT concentration range of 50 to 200 mM used here, the AOT present in the aqueous phase was usually less than 2 mM, with over 99% of the surfactant present in the reversed micellar phase.4 These low aqueous phase concentrations are unlikely to cause protein d e n a t ~ r a t i o n . ~ ~

Protease Activity

Protease activity was determined by a method similar to that of Estell et a1.l' Protease samples were diluted and mixed with a 0.1 mM solution of the substrate succinyl- Ala-Ala-Pro-Phe-p-nitroanilide in 100 mM sodium phosphate buffer, pH 8.0. The samples (0.1 mL) were mixed with substrate (1.0 mL) and placed in a temperature- controlled cuvette holder at 25°C. The increase in absorbance at 415 nm was measured as a function of time, with correction for nonenzymatic hydrolysis under the same conditions.

Alkyl Boronic Acid Synthesis

The hexyl and octyl boronic acids were synthesized by the reaction of catecholborane with terminal alkenes.26 Catecholborane was mixed with the corresponding primary alkene (1.1 : 1) under a nitrogen atmosphere. The mixture was immersed in a boiling water bath and reacted for 1 to 2 h. The reaction mixture was hydrolyzed with water overnight. The aqueous phase was cooled, and the resulting waxy solid filtered and recrystallized from both hot water and benzene. Analysis by Galbraith Laboratories confirmed the expected mass percentages of carbon, oxygen, and boron.

Chymotrypsin Alkylation

Chyrnotrypsin was alkylated by the method of Brown and Wold,' in which 10 mg/mL of the protein in 50 mM

phosphate buffer (pH 6.7) was titrated by the addition of the alkyl isocyanates in ethyl alcohol. Total alcohol concentration in the final aqueous mixture was kept below 10% to avoid denaturation. Residual activity was measured by hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide under standard conditions.

PMSF Inactivation of Chymotrypsin

Chymotrypsin was inactivated by the addition of a 10% mole excess of phenylmethylsulfonyl fluoride (PMSF). The reaction was carried out at a protein concentration of 10 mg/mL in 50 mM phosphate buffer (pH 6.7). Residual activity was determined by hydrolysis of succinyl-Ma-Ala-Pro-Phe-p-nitroanilide under standard conditions. Residual activity was less than 2%.

RESULTS

Extraction As a Function of Salt, pH, and AOT Concentration

Protein transfer from the aqueous to the organic phase has been examined as a function of salt concentration, pH, and AOT concentration. It is reported as the percentage of protein in the reversed micellar phase compared to the total protein initially present in the aqueous phase, taking into account the phase volume of the swollen reversed micellar phase as determined by Karl Fisher titration.

The principal effect of changing the salt concentration in the aqueous phase is shown in Figure 2. For the control study of concanavalin A transfer, in the presence of a pure AOT reversed micellar phase, the extraction was quite poor. Less than 50% transfer was achieved even at the lowest salt concentration used (lower concentrations are impossible to achieve under these conditions due to the formation of a single phase). This value rapidly decreased to zero as the

100 1 I

a t

0 0.2 0.4 0.6 0.8 1 1.2

Salt Concentration (M)

Figure 2. Protein transfer into the reversed micellar phase decreases as sodium ion concentration increases. The addition of 10 mM octyl glucoside as an affinity cosurfactant to a 100 mM AOT phase increases concanavalin A uptake and extends the operating range over which an effective extraction can be conducted.

KELLEY, WANG, AND HATTON: AFFINITY-BASED REVERSED MICELLAR PROTEIN EXTRACTION 1201

sodium ion concentration increased past 0.5 M. When 10% of the total surfactant in the extracting phase was octyl glucoside, a dramatic increase in protein uptake was seen. Fully 90% of the concanavalin A was taken up at the lower ionic strengths, and the transfer decayed to zero only above 1.0 M salt. This represents a significant increase in the operating range of the extraction, as most other proteins will be excluded at these high concentrations of salt.

The addition of cosurfactants not only affects protein transfer, but alters the subtle balance of forces which determine the equilibrium water uptake and the size of the reversed micelles. It is common to report water transfer as the value of W,, the molar ratio of water in the reversed micellar phase to the primary surfactant, AOT. Both geometric arguments3’ and neutron scattering data39 confirm that W, is proportional to the reversed micellar radius. Because the bulky nonionic headgroup of the glucoside is much larger than the sulfosuccinate of the AOT, it is expected that the larger increase in interfacial area on addition of the glucoside will increase water transfer; this is seen to be the case in Figure 3. High protein transfer has been correlated in the past with increases in water content, independent of the surfactant concentration.26

This correlation is explained in Figure 4a. Ribonuclease was solubilized in a pure AOT reversed micellar phase, and the protein uptake plotted as a function of W, (this plot is called a “transfer curve”). Similar results were obtained for chymotrypsin. The AOT concentration was varied eightfold, from 50 to 400 mM, and all the data collapsed to a single sigmoidal curve. At low W, values, where the salt concentration was high, very little protein was transferred. As W , increased, a critical micelle size was reached, beyond which significant protein transfer occurred, which increased with further increases in W,.

The protein transfer at the high W, plateau is determined solely by the pH of the aqueous phase, as suggested by Figure 4b. Here, at a single surfactant concentration, the protein transfer curves are shown as a function of pH. The transitional W , values shift up as the pH is increased and the

a 60 % m a 3 C 0

- 0 40

g r 20 8

0 ’

40 -

30 -

20 -

o 50mM

o 200mM - 0 300mM o 400mM

A 100mM - so

d 0 .

- I I

‘1 AOT + Octyl glucoside 1-

u 0 0.2 0.4 0.6 0.8 1 1.2

Salt Concentration (M)

Figure 3. The addition of octyl glucoside increases the transfer of water to the reversed micellar phase.

100

(a)

100 I

0 10 20 30 40

Water Uptake Number, W,

Figure 4. Transfer curves for ribonuclease A. (a) The effect of surfactant concentration is normalized, as, at constant salt concentration, the number of the reversed micelles changes with surfactant concentration, but not their size. Here, ribonuclease has identical transfer curves for an eightfold range in AOT concentration. (b) , For a given surfactant concentration (100 mM AOT), the transfer curves are dependent on pH, demonstrating the importance of electrostatic interactions in the protein solubilization process.

protein is more negatively charged. Thus, a larger reversed micelle is required to overcome the reduced electrostatic interaction between protein and the surfactant m ~ n o l a y e r . ~ ~ At the highest pH examined, above the isoelectric point of the protein, only poor transfer is achieved with even the largest reversed micelles. Thus, the strength of interaction of the reversed micellar phase with the protein is evident from the position and high W, plateau of the transfer curve. Proteins that have the strongest transfer driving force are shifted up and to the left compared to conditions of weaker interactions. This effect should be mirrored in the transfer curve derived from the affinity cosurfactant data presented in their raw form in Figures 2 and 3. Further control studies confirming the biospecificity of the interaction will be presented in a separate section.


Transfer Curves for Affinity Interactions

The addition of an affinity cosurfactant should increase the transfer of a targeted protein through its association with the protein binding site and subsequent imparting of surface activity to the protein. Concanavalin A has a strong affinity for carbohydrate moieties, binding to both monosaccharides as well as longer sugars or derivatized mon~saccharides.'~ The nonionic surfactant, octyl glucoside, has been used to increase concanavalin A partitioning in reversed micelles? and will be used in this study to illustrate typical characteristics of protein transfer with affinity cosurfactants.

The transfer curve of the concanavalin A-octyl glucoside data is shown in Figure 5, with protein transfer plotted as a function of W,. The transfer curve is shifted to the left, which, by interpretation of Figure 4b, is indicative of a stronger (or additional) driving force for protein uptake into the reversed micelle than that observed in the absence of the glucoside. The transitional W, is lower, and the high W , plateau indicates better protein transfer. These results emphasize improvements in extraction performance in two important regimes. If protein recovery is the most important goal, the extraction should be conducted at low salt and high W, values, where the maximum protein is solubilized. This region also has the highest protein concentration in the reversed micellar phase. If selectivity is to be maximized, however, the region around a W , value of 25 is likely to give the largest increase in specific activity. Here, other proteins generally show very poor transfer, especially at higher pH levels (see Fig. 4b for comparison). The modest concanavalin A transfer results in poor recovery for a single-stage operation, but the use of hollow-fiber liquid-liquid contractors or other multistage configurations would increase product recovery to an acceptable level.

Other protein-affinity cosurfactant interactions have been characterized. The use of naturally occurring, commod- ity amphiphiles as affinity cosurfactants demonstrates an

100 1

80 -

60 -

40 -

0

AOT + Octyl glucoside f l v-

0 10 20 30 40 50 60

Water Uptake Number, Wo

Figure 5. The transfer curve for concanavalin A with octyl glucoside is shifted up and to the left in comparison with the control containing AOT only. This indicates a stronger interaction between the protein and the extracting phase, which is the result of an additional driving force for concanavalin A transfer.

extension of this work to less specialized systems. The largest group of biologically derived amphiphiles is the phospholipids, whose twin-tailed long-chain hydrophobic group should provide a very strong anchor for the protein complex into the surfactant monolayer. The interaction of myelin basic protein and lecithin has been studied by NMR spectroscopic methods, and shown to be the result of protein recognition and binding to the phospholipid h e a d g r ~ u p . ~ ~ Only phospholipid monomers bind to the myelin basic complex, as found from binding studies with immobilized protein.33

Phosphatidylcholine, or lecithin, has been used to extract myelin basic protein with high efficiency, as shown in Figure 6. The protein transfer is much higher at all values of W,, with nearly complete transfer over the range investigated. This is an improvement over the concanavalin A-octyl glucoside system, and is due to the increased interaction strength between the protein and the affinity cosurfactant. Measured values for the dissociation constant for concanavalin A and P-methyl glucoside are in the range of 6 mM>* whereas for the myelin basic protein and lecithin they are much lower, around 0.1 mM.37 This stronger interaction is assurance that with an excess of lecithin in the reversed micellar phase, essentially all of the myelin basic protein will be bound to the phospholipid headgroup.

A control study was conducted by replacing lecithin with lysolecithin, the single-tailed phospholipid produced when lecithin is subjected to partial digestion with phospholipase A2. Myelin basic protein has a greatly weakened interaction with lysolecithin, with measured binding only when the concentration of lysolecithin in the aqueous phase is at or above its critical micelle concentration (about 0.5 mM)." Because very little of the lysolecithin will be present in the aqueous phase to promote aqueous micellar binding, and some component of the full

100 AOT + Lecithin

75 -

50

AOT + Lysolecithin

8 . , , , I , , , , , , , ~ , , , , , , , , , , , , , , 0

20 25 30 35 40 45 50

Water Uptake Number, Wo

Figure 6. Natural cosurfactants can increase protein uptake. Myelin basic protein is much more concentrated in the extracting phase when 4% lecithin is added. The addition of lysolecithin drops transfer back to the AOT-control curve, as the binding of protein and affinity cosurfactant is greatly diminished by the removal of one of the fatty acids from the affinity cosurfactant.

KELLEY, WANG, AND HAlTON: AFFINITY-BASED REVERSED MICELLAR PROTEIN EXTRACTION 1203

phospholipid is required for association with protein, it is not surprising that the addition of lysolecithin instead of lecithin results in a return of protein transfer to the control transfer curve of the single surfactant system, as seen in Figure 6.

The synthesis of affinity cosurfactants will be necessary for cases in which the ligand is not available in an am- phiphilic form. A class of such surfactants is discussed here. Boronic acid and its derivatives inhibit some serine proteases by the formation of a reversible, covalent acyl- enzyme c ~ m p l e x . ~ ~ , ~ ~ Chymotrypsin has been shown to form such an intermediate with alkyl boronic acids, and the active site cleft has been mapped with a homologous alkyl series of boronic acids.3 Boronic acids also inhibit many other serine hydrolases, including lipase,I3 lactamase: and cholesterol e ~ t e r a s e . ~ ~ Only butyl and tetradecyl boronic acids are available commercially, however, and intermediate alkyl tail lengths must be synthesized. A synthesis based on the reaction of catecholborane and terminal alkenes was used to produce hexyl and octyl boronic acids.6 High yields were obtained, and analysis of the material for carbon, oxygen, and boron content were consistent with the expected compositions. Because of their high pK, of the alkyl boronic acids will be neutral for all the investigated pHs, and thus available to form the acyl-enzyme intermediate. The addition of hexyl boronic acid is shown to increase the transfer of chymotrypsin, seen in Figure 7. The higher protein transfer plateau at high W , is about 15% above the pure AOT monolayer value.

A control experiment is included for the chymotrypsin- boronic acid system using an irreversibly alkylated chymotrypsin. Chymotrypsin reacts with isocyanate in aqueous solution to yield an inactivated enzyme which is alkylated at the reactive site serine by the formation of a carbamate ester. The reaction is quite specific, with nearly complete

100

80

60

40

AOT + Hexyl boronic acid

0 10 20 30 40 50 60 Water Uptake Number, W,

Figure 7. The addition of 10% hexyl boronic acid to the reversed micellar phase increases chymotrypsin transfer above that of the pure AOT reversed micellar phase. The relatively small increase reflects the weak interaction between the protein and the affinity cosurfactant. The alkylated chymotrypsin shows a higher transfer curve plateau; this is the result that would be expected if the native chymotrypsin were saturated with hexyl boronic acid.

1204

selectivity for the reactive serine and no other nucleophilic groups on the surface of the protein.34 These alkylated chymotrypsins act as an important control for studying transfer into the reversed micelle, as they represent an irreversible analog to the reversible protein-affinity cosurfactant complex. As expected, the transfer of hexylated chymotrypsin in Figure 7 shows the same shift up and to the left seen for the addition of hexyl boronic acid. The earlier transition W , and higher plateau of the alkylated chymotrypsin suggest that there was not a complete satu- ration of the chymotrypsin by the hexyl boronic acid. This is a reflection of the relatively weak interaction between alkyl boronic acids and chymotrypsin, with a dissociation constant of only 1.5 mM.32 This investigation of both reversible and irreversible inhibitor complexes with chymotrypsin emphasizes the importance of having a strong binding between the protein and the ligand, to increase the quantity of complexed protein.

The increases in protein transfer for the three protein-affinity cosurfactant pairs are summarized in Table I. The partition coefficients between the two phases are listed as ratios to the pure AOT system, all taken at the high W,, plateau. As expected, the increases are ordered with the strongest protein-ligand interaction providing the largest increase in protein transfer. Although the concanavalin A-glucoside system has a higher dissociation constant, the enhancements in its transfer are greater than for the a-chymotrypsin-boronic acid systems because there are two binding sites for the glucoside in the concanavalin A dimer which is formed.33 These increases in partition coefficients could be considered representative for each system, as they were not optimized for pH or affinity cosurfactant loading. Clearly, larger increases could be obtained by the synthesis of affinity cosurfactants having smaller dissociation with the targeted protein.

Control Experiments

The use of transfer curves to analyze a protein-affinity cosurfactant pair for biospecific interaction is convenient because of the simple measurements required. It is important to examine other control experiments performed with the above proteins and affinity cosurfactants to confirm that the association of protein and cosurfactant is required for this increased protein transfer, and that the surface activity of such a complex is indeed the additional driving force for protein uptake. Two controls have been included in the above plots for simplicity. These include the modification of the affinity cosurfactant to abolish its binding properties, as for the case of lysolecithin and myelin basic protein, and the similar transfer curve shift of irreversibly alkylated chymotrypsin compared with the addition of hexyl boronic acid. More controls are needed, however, and are summarized below.

Cosurfactant addition to the reversed micellar phase has been shown to increase the transfer of the desired proteins, but we have not yet discussed how they affect the

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 10, NOVEMBER 20, 1993

Table I. maximum partition coefficient in the absence of affinity cosurfactants.

Summary of partition coefficient increases for three systems, normalized with the

Cosurfactant Structure Protein extracted K/Ko K d

Boronic acids

Phospholipids

?kc-pc

Concanavalin A 8 6 mM

a-Chymotrypsin 3 3 mM

Myelin basic protein 20 0.08 pM

interphase partitioning of other proteins. For contaminating proteins, the transfer should be independent of the added cosurfactant, if the results of Figure 4a are universal. To examine this question, the cosurfactants with the largest change in water transfer, the glucoside family, were added to AOT and used to extract ribonuclease, a protein not expected to have any recognition of and binding to the cosurfactant. Figure 8 shows the resulting transfer curve, and it is obvious that the addition of hexyl glucoside decreases the driving force for ribonuclease transfer as the curve shifts down and to the right. It is hypothesized that this is the result of adding a nonionic surfactant to a pure AOT monolayer comprised of anionic headgroups. Nonionic surfactant headgroups are present in the surface monolayer, and the average surface charge density will decrease. A decrease in the surface charge density should weaken the interaction of proteins with the reversed micellar interface, for proteins which are transferred strictly due to electrostatic effects. This hypothesis was tested by performing an extraction with another nonionic surfactant, Span 20, at both 5% and 10% concentrations. This cosurfactant is a monolaurate

100

5 60

m a, v 40 3 c 0 0

2 -

a 20 8

10% Hexyl glucoside A 5% Span 20 A 10% Span 20

0 ~ ~ " " " ' ~ ' ' ~ ~ ~ ' " ' ~ ' ~ ~ ~ ' ~ -

10 20 30 40 50 60 Water Uptake Number, W,

Figure 8. The addition of hexyl glucoside during the extraction of ribonuclease A shifts its transfer curve down and to the right. This is consistent with a dilution in the surface charge density of the reversed micellar interface, decreasing the attractive interaction between the positively charge protein and the reversed micelle. This hypothesis is confirmed by the addition of Span 20, another nonionic cosurfactant, to the reversed micellar phase.

anhydrous sorbitol, and depresses the ribonuclease transfer in the same fashion as the glucoside. The higher concentration of Span 20 results in lower transfer, suggesting that the additional decrease in surface charge density decreases the electrostatic attraction of the protein with the reversed micelle. It is important to note that, while the addition of a nonionic cosurfactant increases the driving force for the targeted protein uptake, it decreases the driving force for contaminating proteins. This results in a divergence of the two transfer curves, exaggerating their differences, which should improve selectivity for protein mixtures when compared with the AOT control transfer curves.

The association of protein and ligand is a competitive process, with ligands of higher affinity displacing those with weaker interactions. The addition of a hydrophilic, non-surface-active ligand to the aqueous phase will result in some protein binding the free ligand, reducing the number of protein-affinity cosurfactant complexes. This should reduce the protein transfer into the reversed micellar phase. Such an experiment was conducted with the concanavalin A-glucoside system, with the monosaccharides galactose, glucose, and mannose added to the aqueous phase before contacting with the reversed micellar phase containing octyl glucoside. Figure 9 shows

100

Galactose

0 2 4 6 8 10

Carbohydrate Concentration (mM)

Figure 9. Water-soluble inhibitors compete with the octyl glucoside for the binding site of the concanavalin A. The level of transfer without carbohydrate decreases as the sugar concentration increases, in accordance with the known affinities of the protein for the individual hexoses.

KELLEY, WANG, AND HATON: AFFINITY-BASED REVERSED MICELLAR PROTEIN EXTRACTION 1205

the resulting equilibrium values of protein transfer. The control transfer in the presence of octyl glucoside is given by the value at zero carbohydrate concentration, approx- imately 60% transfer under these conditions of salt and pH. The monosaccharides have different effects on protein transfer; galactose does not change the protein transfer, glucose results in a slight decrease, and mannose rapidly competes for the binding site of the concanavalin A and displaces the octyl glucoside headgroup. The lowest protein transfer reached by the addition of 10 mM mannose is the same as the control study of concanavalin A transfer under the same conditions of salt and pH, but without the affinity cosurfactant present. The dissociation constants for these monosaccharides and concanavalin A have been reported, and fall in the order expected from the above competition e~periment.~' Concanavalin A has virtually no affinity for galactose ( K d > 100 mM), moderate affinity for glucose ( K d = 2.0 mM), and high affinity for mannose ( K d = 0.2 mM). This altered affinity is due to the C-2, C-3, and C-4 hydroxyl positions on the pyranosyl ring. These results suggest that one means of back extracting the protein would be to employ soluble inhibitors to reduce the number of protein-affinity cosurfactant complexes formed, decreasing the protein partition coefficient.

The binding strength of the affinity cosurfactant headgroup and the protein should mediate the degree of protein transfer. Concanavalin A binds anomers of glucosides with different affinities, showing a 30-fold larger dissociation constant for p-methyl glucoside as compared with a- methyl g l u ~ o s i d e . ~ ~ The transfer curves for these two anomers are compared in Figure 10, at the same concentration of cosurfactant. The transfer is higher for the a-octyl glucoside, with a sharper transition at smaller values of W,. This result again emphasizes the importance of having the

40

20

0 10 15 20 25 30 35 40


Figure 10. Alpha or beta anomers of octyl glucoside have different effects on concanavalin A transfer. The alpha anomer increases the driving force for protein solubilization, shifting the transfer curve to the left. The aqueous dissociation constant is lower for this species, indicating that the stronger the interaction of the protein and ligand, the better the extraction performance.

100 .

AOT + Hexyl boronic acid

0 0 10 20 30 40 50


Figure 11. Chymotrypsin inactivated by phenylmethylsulfonyl fluoride (PMSF) does not interact with hexyl boronic acid, and its transfer curve collapses to that of the AOT control. Thus, the use of affinity cosurfactants is specific to active protein capable of recognizing and binding active protein.

strongest binding possible between the headgroup of the cosurfactant and the protein to be extracted.

The extraction of protein by the addition of affinity cosurfactant should be selective for active enzymes, and not result in additional transfer of inactivated species. For the chymotrypsin study, it was informative to look at the transfer of the protease inactivated irreversibly with phenylmethylsulfonyl fluoride (PMSF); this chemical reacts with the catalytic serine residue with which the boronic acid forms the acyl-enzyme intermediate.12 Transfer curves of this PMSF-chymotrypsin with and without hexylboronic acid are given in Figure 11. The transfer curves are nearly identical, confirming the need for binding of the affinity cosurfactant to the protein to increase the driving force for protein uptake into the reversed micelles.

CONCLUSIONS

Reversed micelles containing only pure surfactant solubilize proteins on the basis of electrostatic interactions with the protein, such that control of both extraction and back extraction can be achieved through manipulation of pH and salt concentration. The addition of a small concentration of affinity cosurfactants to the reversed micellar phase increases the amount of protein extracted and extends the operating range of pH and salt concentrations over which proteins can be extracted. The shift in the transfer curves, when affinity cosurfactant is added, indicates a stronger interaction between the extracted protein and the reversed micellar phase in the presence than in the absence of the affinity cosurfactant. Higher protein concentrations can be achieved at the highest water transfers (lowest salt concentrations), while greater selectivity is possible at higher salt concentrations. The binding strength of the protein and the ligand headgroup of the cosurfactant determines the increase in protein transfer, the most dramatic increases in


protein partition coefficients being observed for dissociation constants on the order of 1 X lo4 M.

Control experiments confirmed the need for the interaction between the protein and the ligand headgroup of the affinity cosurfactant for enhanced transfer to occur. Nonionic cosurfactants decreased the transfer of ribonuclease, as the surface charge density of the reversed micellar wall decreased. Free ligands competed with the affinity cosurfactant for the protein binding site, reducing the protein uptake into the reversed micelle. The transfer of an inactivated protein incapable of binding ligands was not affected by the addition of affinity cosurfactant. Finally, the irreversible alkylation of proteins shifted their transfer curves in the same manner as the addition of affinity cosurfactants.

The optimization of affinity-based reversed micellar protein extraction will require a better knowledge of the interactions between the cosurfactant, the target protein, and the reversed micellar interface. As the cosurfactant will be present in both the aqueous and reversed micellar phases, analysis of the optimal phase partitioning is needed if we are to better predict what properties of an affinity cosurfactant determine the extent of protein uptake into the reversed micellar phase. This is the subject of other studies.

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Affinity-based reversed micellar protein extraction: II. Effect of cosurfactant tail length

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