Biotin-modified submicron latex particles for affinity precipitation of avidin

COLLOIDS B

AND SURFACES E LS EV I ER

Colloids and Surfaces B: Biointerfaces 7{1996) 55 64

Biotin-modified submicron latex particles for affinity precipitation of avidin C.S. Chern *, C.K. Lee, C.Y. Chert Department o(Chemical Engineering, National Taiwan Institute q(Technology 43 Keelung Rd., Sec. 4, Taipei, Taiwon Received 8 December 1995; accepted 19 March 1996

Abstract

The negatively-charged latex particles (latex COO ) is more effective in binding lysozyme via electrostatic interactions than avidin. The zeta potential data suggest that a portion of the carboxyl groups can be trapped inside the network of large flocs formed during the precipitation process. Thus the entrapped binding sites are no longer available for protein as the precipitation process proceeds. This action can reduce the maximum amount of protein adsorbed on the particle surface. The selectivity of the precipitation process using latex-COO as the polymeric support is in favor of lysozyme for the mixture of avidin and lysozyme. Latex COO- can be used to concentrate the targel protein avidin from a crude extract. The biotin-modified latex (latex-biotin) can then be used m a subsequent step to purify avidin via affinity interactions from an intermediate product containing avidin and lysozyme. The selectivity of the affinity precipitation process using latex-biotin as the support is satisfactory with respect to the protein mixture containing avidin and lysozyme. Keyw,,rds: Affinity precipitation: Avidin; Biotin: pH sensitivity; Protein puriiication: Submicron latex particles

1. Introduction It has been proposed to use submicron polymer particles to isolate and recover proteins from a crude biological mixture. Such a technique involves binding of the target protein onto the particle surface, sedimentation of the particles, removal of the supernatant, and dissociation and recovery of the protein product from the sediment. Binding of proteins to the particle surface can be achieved by either electrostatic interactions [ 1 - 8 ] or specific ligand-protein interactions [ 9 - 1 1 ] . Sedimentation of the particles can be carried out by centrifugation. Recently, the authors have prepared and characterized a pH-sensitive latex product ( l a t e x - C O O , * Corresponding author. 0927-7765/96/$15.00 c~) 1996 Elsevier Science B.V. All rights reserved PII S 0 9 2 7 - 7 7 6 5 ( 9 6 ) 0 1 2 8 1 - 7

0.5 ~m in diametert for selective precipitation of proteins [8]. The submicron particles carry negative charges on their particle surfaces (:~ 276/amol carboxyl groups g ~ polymerj at neutral pH and they can effectively precipitate positivelycharged proteins such as avidin and lysozyme. Precipitation of the particles can be greatly' enhanced by changes in pH and/or the ionic strength of the solution. Furthermore, the precipitated protein can be completely recovered from the particles by using 1 M NaC1 solution. Nevertheless, the selectivity of the precipitation process using the electrostatic interaction mechanism is expected to be poor with respect to the mixture of avidin and lysozyme. To overcome this problem, the ligand biotin can be chemically incorporated onto the particle surface and thereby the biotin-containing particles can specifically bind the

56

C.S. Chern et al./Colloids Surfaces B." Biointerfaces 7 (1996) 55 64

target protein avidin. Thus, the objective of this work was to demonstrate the feasibility of utilizing such biotin-containing submicron latex particles to obtain avidin from the crude extract (e.g. chicken egg white) that contains both avidin and lysozyme. The separation process proposed in this work is shown schematically in Fig. 1. The process involves addition of latex-COO- to the crude extract to bind both avidin and lysozyme via electrostatic interactions, followed by addition of the biotincontaining particles to the mixture of avidin and lysozyme in which affinity interactions between biotin and avidin take place. In this manner, the target protein, avidin, can be separated from the protein mixture. Subsequently, avidin can be recovered from the biotin-containing particles by using 8 M pH 1.5 guanidine-HC1 solution or by autoclaving [ 12].

2. Experimental 2.1. Materials

The pH-sensitive latex was prepared by a semibatch surfactant-free emulsion polymerization process. Detailed information concerning the polymeric support has been reported previously [8]. Other chemicals used in this work include 1,6-diaminohexane (99.5%; Hayashi Pure Chemical Industries), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Sigma), 2,4,6-trinitrobenzenesulfonic acid (TNBSA) (95%; Sigma), sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LC-biotin) (ImmunoPure; Pierce), avidin (ImmunoPure; Pierce), lysozyme (EC 3.2.1.17) from chicken egg white (Sigma), Micrococcus lysodeikticus cell (Sigma), and sodium

Crude Extract

Avidin, Lysozyme, Negatively Charged Proteins

Electrostatic Interactions

I|

! [

Latex-COO"

Negatively Charged ) Proteins

i

Avidin, L y s o z y m e

Affinity Interactions

<

l <

• Latex-Biotin

-~ Lysozyme

Avidin

Fig. 1. Schematicillustration of the bioseparation process for separating avidin from the crude extract containing both avidin and lysozyme.

C.S. Chern et al./Colloids SurJhces B: Biointerjaces 7 (1996) 55 64

57

phosphate (J.T. Baker). Deionized water (Barnsted, Nanopure Ultrapure Water System, specific conductance < 0.057 ItS c m - 1) was used throughout this work. i

2.2. Preparation of biotin-containing latex particles The amino groups were incorporated into the latex particle surface by reacting the carboxyl groups on the latex particle surface with EDC in the presence of 1,6-diaminohexane according to the method reported previously [8]. The amino content was determined by the TNBSA method [ 13,14]. Biotin was coupled to the NHz-modified latex by using the activated biotin, NHS-LC-biotin. Various amounts of NHS-LC-biotin were reacted with 4 ml NHz-modified latex (1% solids content) in 0.1 M pH 8.0 phosphate buffer at room temperature overnight. The biotin-containing latex was washed twice with phosphate buffer solution to remove the unreacted NHS-LC-biotin and recovered by centrifugation at 3000 rev min -1 for 20 min. These particles were then redispersed in a phosphate buffer solution and the latex product (latex-biotin) has a total solids content of 2%. The amount of biotin covalently coupled on the particle surface was determined according to the method proposed by Guzman et al. [15]. This method involves the measurement of the avidin concentration difference before and after reaction of latex-biotin with avidin at 25 ° C. It was assumed that the coupled biotin can only bind one avidin molecule, due to both the electrostatic repulsion and steric hindrance effects caused by two approaching particles, as illustrated schematically in Fig. 2. The avidin concentration was determined by UV absorbance (Shimadzu, UV-160A) at 233 nm (extinction coefficient= 1.36 x 105 M - l ) . Thus, the maximum amount of avidin adsorbed on the latex-biotin particle surface (q*) was taken as the amount of biotin covalently bound to the latex particles.

2.3. Precipitation of lysozyrne and avidin via electrostatic interactions L a t e x - C O O - with a total solids content of 0.5% was employed for precipitation of lysozyme and

Pamcles a b o t l t 4 nrll

.oul~uce

not feasible Fig. 2. Schematic illustration of the steric hindrance effect caused by two approaching particles.

avidin. Various amounts of protein prepared in 4 ml of 0.1 M pH 8.0 phosphate buffer solution were added to 1 ml latex to initiate the precipitation process. The reaction solution was stirred overnight at 25°C to ensure that the system was at equilibrium. To determine the zeta potential of the latex C O 0 - particles containing adsorbed lysozyme, 2 ml of the reaction solution was diluted with 13 ml of 0.1 M pH 8.0 phosphate buffer solution. The zeta potential of latex particles was determined by Zetamaster instrument (Malvern). The precipitate phase of the reaction solution was collected by centrifugation at 3000 rev rain-~ for 20 rain. The clear supernatant was filtered through a 0.2 Itm membrane to remove any residual particles. The protein concentration in the supernatant was determined by UV absorbance (Shimadzu, UV-160A) at 233nm for avidin or 280nm for lysozyme. Similarly, the effects of the amount of l a t e x - C O O particles and the pH on the lysozyme recovery yield at 25°C were investigated by adding 0.5 ml of 0.296 mg ml 1 lysozyme solution to 4 ml of l a t e x - C O O - sample with various solids contents. The pH of the solution was adjusted by using 0.1 M phosphate buffer solution. The effect of ionic strength on the lysozyme elution yield, defined as the weight percentage of the adsorbed protein that can be recovered from the precipitate, was investigated according to the following procedure. First, 0.5 ml of 0.296 mg ml ~

58

C,S. Chern et al./Colloids Surfaces B: Biointerfaces 7 (1996) 55-64

lysozyme solution was added to 4 ml of latexC O 0 - sample (pH 6 adjusted by a phosphate buffer solution, total solids content =0.5%) to initiate the precipitation process. The reaction solution was stirred overnight at 25'~C to ensure that the system was at equilibrium. The precipitate phase of the reaction solution was collected by centrifugation at 3000 rev rain -1 for 20min. The clear supernatant was filtered through a 0.2 ~tm membrane and the lysozyme concentration in the supernatant was determined by the UV absorbance method. The precipitate phase was redispersed in 4 ml of 0-1.5 M NaC1 solution. The redispersed sample was then allowed to stand for 20 rain to cause lysozyme to desorb out of the latex C O O particle surface. After centrifugation at 3000 rev rain -~ for 20 rain, the resultant supernatant was filtered through a 0.2 pm membrane. This step was followed by determination of the concentration of the eluted lysozyme in the supernatant. 2.4. Affinity precipitation of avidin

A protein mixture containing 0.17 mg of avidin and 0.30 mg of lysozyme was prepared in 7 ml of 0.1 M pH 8.0 phosphate buffer solution. The mixture was then mixed with 1 ml of latex-biotin (total solids content = 0.1%) to initiate the affinity precipitation of avidin. The reaction mixture was stirred overnight at 25 °C. The precipitate phase was collected by centrifugation at 3000 rev min -~ for 20 rain. Since the UV absorbance method cannot be applied to determine the lysozyme concentration in the avidin and lysozyme mixture due to the mutual interference, the lysozyme activity in the mixture was assayed according to the Micrococcus lysodeikticus cell lysis method [ 16].

3. Results and discussion 3.1. Precipitation o f lysozyme and avidin via electrostatic interactions

Both the avidin (pI 10.0 10.5) and lysozyme (pI 10.7) are positively-charged proteins in a neutral pH environment because of their high isoelectric points [ 17]. Therefore, these proteins can be reco-

vered by the selective precipitation technique using the negatively-charged latex-COO -. The Langmuir isotherm model [18] was used to describe adsorption of protein on the particle surface when the system was at equilibrium: (1)

q* = qmaxC*/(Kd + C*)

where q* is the amount of protein adsorbed per gram polymer particles, qmax is the maximum amount of protein that can be adsorbed on the particle surface, C* is the protein concentration in the aqueous solution, and g d is the dissociation constant for the protein-binding site pair. Rearrangement of Eq. (2) leads to the Scatchard equation [ 19]: q* = qmax - - Kdq*/C*

(2)

Thus the parameters K a and qmax can be obtained from the slope and intercept respectively, of the q* vs. q*/C* curve. Fig. 3 shows the Langmuir isotherm curves for lysozyme adsorbed on the l a t e x - C O O - particle surface at 25°C. The Scatchard plot is shown in the inset. The parameters K d and qmax estimated fi'om the Scatchard plot are listed in Table 1. The numerical values in the parentheses correspond to qmax (rag g i polymer). The magnitudes of qmax and Ka are approximately 4.06 g mole g a polymer (or 58.37mg g-~ polymer) and 2 . 8 8 × 1 0 - 6 M respectively. The zeta potential as a function of the amount of adsorbed lysozyme is shown in Fig. 4. The zeta potential first decreases rapidly and then levels off with increasing amount of adsorbed lysozyme. This observation suggests that the colloidal stability provided by electrostatic repulsion forces can be greatly reduced as the particle surface charges are partially neutralized by the adsorbed protein. A Table 1 Langmuir parameters for the precipitation of proteins using latex-COO- via electrostaticinteractions at 25°C Lysozyme

Avidin

qmax(gmole g 1) K a (M)

qmax(~tmoleg-1)

K a (M)

4.06 2.88 × 10 6 0.27 6.12 × 10-" (58.37 mg g-i polymer) (17.82 mg g 1 polymer)

59

C,S. Chern et al./'Colloids Sur/aces B: Biointer[iK'es 7 1996) 55 64

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50

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portion of the carboxyl groups can be trapped inside the network of large flocs formed during the precipitation process. As a result, the entrapped binding sites are no longer available for protein as the precipitation process proceeds. This action can reduce the maximum amount of protein adsorbed on the particle surface and thereby the parameter qmax obtained from the isothermal adsorption experiments should be regarded as an apparent value. Furthermore, the minimum zeta potential shown in Fig. 4 ( ~ - 2 0 mV when the adsorbed lysozyme is greater than 0.25 mg) implies that the latex C O 0 particle surface is saturated with lysozyme. Fig. 5 shows the effects of latex CO() solids content and pH on the lysozyme recovery yield. As expected, the protein recovery increases monotonically with the amount of polymeric support (i.e, with the amount of carboxyl groups) when the pH is kept constant. At a fixed quantity of latex,

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C.S. Chern et al./Colloids Surfaces B: Biointerfaces 7 (1996) 55-64 100

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Fig. 5. Weight percentage of lysozyme recovered as a function of total solids content of l a t e x - C O O - at various values of pH: (©) 6; (A) 7; ( ~ ) 8.

the lysozyme recovery yield decreases as the pH is increased from 6 to 8. This trend can be attributed to the decreased net charge on the lysozyme surface as pH increases. The Langmuir isotherm curves for avidin adsorbed on the latex-COO- particle surface at 25°C are shown in Fig. 6 and the estimated Ka and qmax values are listed in Table 1. The magnitudes of qmax and Kd are approximately 0.27 ~t mole g - i polymer (or 17.82 mg g-1 polymer) and 6.12 x 1 0 - 7 M respectively. It can be concluded that the negatively-charged latex particles are more effective in binding lysozyme than avidin. It is therefore postulated that lysozyme should have carried more positive charges under the precipitation condition studied. Another possible explanation is that the molecular weight of avidin is about four times greater than that of lysozyme and thereby the adsorbed avidin can exhibit a stronger steric hindrance effect towards approaching avidin molecules. However, the dissociation constant Ka for lysozyme is about one order of magnitude greater than that for avidin. This result implies that the electrostatic interaction between avidin and latex-COO- is stronger than that between lysozyme and latex-COO-.

--~

i

2

I

6 c * X104( ~ m o l e l m L )

Fig. 6. Langmuir isotherm curves for avidin adsorbed on the particle surface at 2 5 C .

Fig. 7 shows the effect of sodium chloride concentration ([NaC1]) on the lysozyme elution yield for the system using latex C O 0 as the polymeric support. Such elution yield data can provide information on the degree of electrostatic interaction between the adsorbed protein molecule and the binding site. The elution yield first increases rapidly 100

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Fig. 7. Elution yield as a function of sodium chloride concentration for the lysozyme/latex-COO system at 25°C.

C.S. Chern et al./'Colloids Sur[ctces B." Biointer/ctces 7 ~ 1996) 55 64

and then levels off when [NaC1] is increased from 0 to 1.5 M. This trend suggests that the electrostatic interaction between lysozyme and latex-COO is strongly dependent on the ionic strength of the aqueous solution. The dissociation constant K d for the lysozyme-binding site pair should increase with increasing [NaC1], as suggested by the elution yield data in Fig. 7. Note that the maximum elution yield (~80%) can be achieved when [NaCI] is greater than 0.4 M. The ionic-strength-dependent elution yield data for the avidin/latex COO system are not available. However, similar behavior is expected for the avidin/latex-COO system towards the added salt because avidin has a comparable pl and follows a similar adsorption mechanism (based on the electrostatic interaction) to lysozyme. For the mixture of avidin and lysozyme, the selectivity of the precipitation process using latexC O ( ) - as the polymeric support is in favor of the undesired product lysozyme (see Table 1). Nevertheless, it seems to be feasible to concentrate a target protein such as avidin from a crude extract ifsut'ficient latex COO is used in the precipitation process. The biotin-modified submicron particles (latex biotin) can then be used in a subsequent step to purify the target protein (avidin) from an intermediate product containing both the avidin and lysozyme.

3.2. Preparation o(biotin-mod![ied latex partich, s First, the latex particle surface was modified with amino groups by reacting the surface carboxyl groups with EDC in the presence of 1,6-diaminohexane. A series of activation experiments were conducted to study the effect of the molar ratio of 1,6-diaminohexane to EDC on the percentage of surface carboxyl groups that can be converted to amino groups. The molar ratio of EDC to the surface carboxyl groups was kept constant at 0.4: 1. The molar ratio of 1,6-diaminohexane to EDC was varied from 1:1 to 10:l. As shown in Fig. 8, the percentage of surface carboxyl groups converted to amino groups 1~ 8%) is relatively independent of the molar ratio of 1,6-diaminohexane to EDC. When the molar ratio of 1,6-diaminohexane to

61

12

.i. N

z "O

O O

0 0

1

2

3

5

8

10

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diaminohexanelEDC (molar ratio) Fig. 8. Percentage of the surface carboxyl groups convertcd to amino groups as a function of the molar ratio of 1,6-diaminohexane to EDC. The molar ratio of EDC to the surface carboxyl groups was kept constant at 0.4:1.

EDC was kept constant at 8:1, the percentage of surface carboxyl groups converted to amino groups increased from 0% to ~ 18% with an increase in the molar ratio of EDC to surface carboxyl groups from 0.2:l to 2:1, as illustrated in Fig. 9. It can be concluded that the percentage of surface carboxyl groups that can be converted to amino groups is determined by the EDC concentration. An experiment with COOH:EDC:NH2 (CH2)~, N H 2 = 1:0.62:1.86 (molar ratiosl was then carried out twice to prepare the amino-modified latex particles (latex-NH_,). The goal was to convert 10% of the surface carboxyl groups to amino groups. Indeed, about 10.3% of the surface carboxyl groups were converted to amino groups in the resultant latex-NH 2. Furthermore, the remaining 90% of carboxyl groups can impart the desired pH sensitivity to the polymeric support. Biotin was covalently coupled to the amino groups on the latex-NHz particle surface by reacting activated biotin (NHS-LC-biotin) with latex-NH2. Three biotin-modified lattices, designated latex-B4, latex-B5, and latex-B6) were prepared and their characteristics are shown in Table 2. The grafting efficiency, defined as the ratio of the coupled biotin

C.S. Chern et al./Colloids" Surfaces B." Biointerfaces 7 (1996) 55-64

62 2o

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2

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~

4

6

~

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8

10

e* X104( I~ molelmL)

Fig. 9. Percentage of the surface carboxyl groups converted to amino groups as a function of the molar ratio of E D C to the surface carboxyl groups. The molar ratio of 1,6-diaminohexane to E D C was kept constant at 8 : 1.

Fig. 10. Langmuir isotherm curves for avidin adsorbed on the particle surface at 25°C: ([]) latex-B4; ( ± ) latex-B5; (©) latex-B6.

Table 2 Some characteristics of the biotin-containing lattices" Lattice

NHS-LC-biotin added (i.tmole g 1 polymer)

NHS-LC-biotin (mole %)

Biotin-COOH (mole %)

Biotin-NH 2 (mole %)

Latex-B4 Latex-B5 Latex-B6

114.88 289.75 579.50

4.33 1.81 1.10

1.80 1.90 2.30

20.60 21.70 26.33

The amino-modified latex particles contain 276 g m o l e - C O O H and 24/.t mole - N H 2 per gram polymer.

to the added NHS-LC-biotin, decreases with an increase in the added NHS-LC-biotin (see Table 2). As a result, the amount of bound biotin only increases gradually with a significant increase in the added NHS-LC-biotin. Table 2 shows that about 2 mole % of the surface carboxyl groups were modified by the ligand biotin.

3.3. Affinity precipitation of avidin by latex-biotin Fig. 10 shows the Langmuir isotherm curves for avidin adsorbed on the biotin-modified lattices at 25°C. The qm,x and Kd values are obtained from the Scatchard plot and are summarized in Table 3. For comparison, the qmax and Kd data for the

avidin/latex-NHz system are also included in Table 3 (the Langmuir isothermal adsorption data are not shown here). The maximum amount of avidin adsorbed on the latex-NH2 particle surface (q~n,x=0.19 la mole g - t polymer) is only about 3% Table 3 Calculated Langmuir parameters for affinity adsorption of avidin on the biotin-modified lattices Lattice

qmax (~mole g - ' polymer)

Kd (M)

Latex-NH 2 Latex-B4 Latex-B5 Latex-B6

0.19 4.97 5.24 6.35

2.77 6.36 4.42 5.66

x × × x

10 v 10 7 10 7 10 -7

63

C S . Chern et al./Colloids Surfaces B: Biointer[aces 7 i 1996) 55-64

of that on the biotin-modified latex particle surface. Nevertheless, the dissociation constant Ka for the avid)n-surface carboxyl group pair is comparable to those for the avid)n/latex-biotin system. This result indicates that the strength of electrostatic interactions between avidin and latex-COO is quite high. The physically adsorbed protein molecules can be eluted easily by washing the precipitated latex particles with 1 M NaC1 solution [8]. However, the specific avid)n/latex-biotin bond is expected to remain stable in such a mild environment. This makes the bioseparation process illustrated in Fig. 1 feasible, The values of qmax and Ka for the avid)n/latexbiotin systems are about 5.5 g mole g ~ polymer and 5.5 × 10 -7 M respectively. The binding constant K (= I/Kd) is then equal to 1.8 x 106 M ~, which is much smaller than that for the free avid)nbiotin pair ( K = 1 0 ~s M ~) [20]. Reduction in K for the avid)n/latex-biotin system can be attributed to the steric hindrance effect caused by immobilization of the relatively small biotin molecule on the gigantic latex particles. This result is consistent with the work of Powers et al. [21]. They used a biotin-modified liposome ( ~ 74 nm in diameter) to bind avidin via affinity interactions. A value of 6 × 107 M ~ was obtained for the apparent association constant K which is approximately 30 times greater than that for the avidinqatex-B systems. Such a difference is reasonable because the latex particle size is ~ 500 nm, much greater than the diameter of liposome. Thus, a higher degree of steric hindrance is experienced in the avid)n/latexbiotin systems. Finally, latex C O 0 and latex-B5 were used separately in a series of experiments designed to demonstrate the feasibility of the bioseparation process shown in Fig. 1. It was found that each individual protein concentration in the supernatant containing avidin and lysozyme cannot be determined directly by the UV absorption method due to the mutual interference. Thus, the activity of unbound lysozyme in the supernatant was monitored and the lysozyme activity data were used to qualitatively assess the feasibility of the proposed process. Table 4 shows the lysozyme activity in the supernatant during each separation step. Before precipitation, the pure lysozyme solution has an

Table 4 Experiments designed to demonstrate the teasibility of purification of the target avidin from a biological solution containing both the avidin and lysozyme Solution

Lysozyme activity ill supernatanl (U ml ~) Without latex particles

Avidin 0 Lysozyme 1210 Avidin + lysozyme I 125

Latex COO

Latex-B5

0 611 911

0 1093 1093

activity of 1210 U m1-1 and the lysozyme avidin mixture has an activity of 1125 U ml-1. Using latex-COO- as the precipitation agent, the activity decreases from 1210 to 611 U ml 1 in the case of pure lysozyme solution. However, the activity merely decreases from 1125 to 911 U ml ~ in the presence of both avidin and lysozyme. This is duc to the fact that avidin and lysozyme compete with each other for the surface carboxyl groups and thereby less lysozyme can be adsorbed on the particle surface. When latex-B5 was employed as the precipitation agent, only a few lysozyme molecules could be adsorbed on the particle surface as the activity of lysozyme just drops from 1125 to 1093 U ml - 1. This small change in the activity of lysozyme is not as important as the change ( 1 1 2 5 - 9 1 1 = 2 1 4 U m l 1) for the protein latex C O O system. The reason for this observation is that latex-B5 carries less negative charges than latex COO does. In addition, the tendency for lysozyme to adsorb on the latex-B5 particle surface is greatly reduced when the particle surface is already saturated with the target avidin. Based on the above experimental data, the selectivity o[" the affinity precipitation process using latex-B5 as the support is satisfactory with respect to the avidin lysozyme mixture. More research work is reqnired to quantitatively understand the process performance.

4. Conclusions

Latex COO carries negative charges at neutral pH and it can effectively precipitate positively-

64

CS. Chern et al./Colloids Surfaces B: Biointerfaces 7 (1996) 55-64

charged proteins such as avidin and lysozyme. The Langmuir isotherm data show that latex-COOis more effective in binding lysozyme than avidin. In addition, the dissociation constant between latex-COO- and lysozyme is about one order of magnitude greater than that for avidin. The zeta potential of the latex-COO- particles first decreases rapidly and then levels off with increasing amount of adsorbed lysozyme. This observation implies that the colloidal stability can be greatly reduced as the particle surface charges were partially neutralized by the adsorbed protein. A portion of the carboxyl groups can be trapped inside the network of large flocs formed during the precipitation process. As a result, the entrapped binding sites are no longer available for protein as the precipitation process proceeds. This action can reduce the maximum amount of protein adsorbed on the particle surface. The selectivity of the precipitation process using latex-COO- as the polymeric support is in favor of lysozyme for the mixture of avidin and lysozyme. It is proposed to use latex-COO- to concentrate the target protein avidin from a crude extract. Latex-biotin can then be used in a subsequent step to purify avidin from an intermediate product containing avidin and lysozyme. Experimental data show that the selectivity of the affinity precipitation process using latex-biotin as the support is satisfactory with respect to the avidin-lysozyme mixture.

References 1-1] W. Norde and J. Lyklema, J. Colloid Interface Sci., 66 ( 1978} 277.

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