BIOTECHNOLOGY TECHNIQUES Volume 7 No.4 (April 1993) pp.277-282 Received 22nd January

TOSYL CHLORIDE ACTIVATION OF A RAYON/POLYESTER CLOTH FOR

PROTEIN IMMOBILIZATION

Scott Boyd and Biroshi Yamazaki* Department of Biology and Institute of Biochemistry,

Carleton University Ottawa, Canada KlS 5B6

SUMMARY Rayon/polyester cloth was treated with NaOH, washed and

dehydrated with acetone, and without drying activated with p- toluenesulfonylchloride in acetone. The activated cloth was used to immobilize bovine serum albumin and anti-horseradish peroxidase antibody. Tosyl activated cloth offers several advantages including low cost, high capacity for protein immobilization, rapidity, and high linkage stability.

INTRODUCTION Immunoaffinity chromatography is an industrially

attractive procedure for the purification of antigens (e.g. proteins) or antibodies as it provides a high degree of purification in a simple procedure of adsorption and elution. It has been recently demonstrated that rayon/polyester cloth may be used in immunoaffinity chromatography as a support for protein immobilization (Howlett et al ., 1991). When compared to currently available immunoaffinity supports, such as cross-linked agarose or dextran beads, rayon/polyester cloth offers the advantages of lower expense and greater physical stability. Its macroporous and compressed structure also allows for easier washing, the capacity for higher flow rates with larger sample volumes, faster immunoreaction rates, and the ability to concentrate dilute samples. The hydroxyl groups of rayon are easy to activate after treatment with strong alkali which presumably disrupts H-bonding involving the hydroxyl groups. Recently, carbonyldiimidazole (CDI) has been used in the activation of alkali-treated rayon/polyester cloth (Howlett et al., 1991). While CD1 activation is simpler to perform on cloth than cyanogen bromide (CNBr) activation (because it does not require the vigorous mixing that is necessary for CNBr activation), both types of activation produce similarproteinimmobilizationcapacities. Because the linkage formed between CDI- or CNBr-activated support and proteins are unstable at pH greater than 10, alkaline elution conditions cannot be used for these supports. Furthermore, CNBr and the solvents used in CDI activation are unsafe to use for industrial scale operations. This paper demonstrates that p-toluenesulfonyl chloride activation overcomes these disadvantages of CD1 and CNBr activation while allowing for more rapid immobilization of greater amounts of protein.

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MATERIALS AND METHODS

Materials The following materials were purchased from Sigma

Chemical Co.: bovine serum albumin (BSA) (A-7030), horseradish peroxidase (HRP) (P-6782), anti-horseradish peroxidase antibody (anti-HRP) (P-5774), p-toluenesulfonyl chloride (tosyl chloride) (T-5384) and 3,3',5,5'- tetramethybenzidine (TMB) (T-2885). A micro bicinchoninic acid (BCA) protein assay (No. 23235) was obtained from Pierce. The non-woven rayon/polyester blend (70%/30%) cloth (Sontara 8423) was acquired from DuPont.

Tosvl chloride activation of ravon/polvester cloth Segments (6 mm square) of the rayon/polyester cloth were

soaked in 10% (w/v) NaOH (0.5 ml/segment) for 10 min at room temperature (ca 25OC). The cloths were then washed three times with water, air-dried under suction followed by five more washes with acetone (1 min soak between washes). The dehydrated cloths weretransferred immediately without drying to a solution of 10% (w/v) tosyl chloride in acetone (0.5 ml/segment) for 20 min at room temperature. The tosyl chloride activated cloths (tosyl cloths) were washed three times with acetone, followed by three times with water under suction, and then thoroughly blotted.

Assay of immobilized BSA Fifty ~1 of 1% (w/v) bovine serum albumin (BSA) in 0.5

M sodium phosphate buffer (pH 7.5) was added to each tosyl cloth for an overnight incubation at room temperature. To remove any loosely bound BSA, each cloth segment was heated in 1% (w/v) sodium dodecyl sulphate (SDS) at 100°C for 10 min. The segments were then washed three times with 1% SDS followed by three washes with distilled water. A BCA protein assay was used to measure the immobilized BSA. Due to the lack of immobilized protein standards, the degree of protein immobilization was compared by absorbance at 562 nm of the developed colour following dilution to an absorbance range of 0.1 to 1.0.

Anti-HRP antibodv immobilization Ten microlitres of anti-HRP antibody (750 pg/ml in PBS)

was reacted with each tosyl cloth. To block any unreacted sites and any non-specific HRP binding sites, the cloths were soaked in 0.5% BSA in PBS overnight at room temperature. Each cloth segment was then reacted with 50 ~1 of 2 j.Lg/ml HRP in PBST (PBS plus 0.05% Tween 20) for 20 min. A TMB indicator system was used to indirectly measure the amount of antibody immobilized onto the cloth. The activity of HRP that reacted with immobilized anti-HRP antibody was measured by mixing each cloth separately in 1 ml of a TMB indicator system at room temperature. The absorbance of the resulting colour at 370 nm was determined after the reaction was stopped with 0.1 ml of 0.2 M NaF. The TMB indicator system was prepared by mixing 9.25 ml of distilled H20, 0.5 ml of 0.2 M sodium acetate buffer (pH 5.0), 0.25 ml of 0.8 mM TMB in ethanol, and 50 ~1 of 0.2 M H202 together immediately prior to use.

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RESULTS AND DISCUSSION

Tosvl chloride activation of rayon/polyester cloth Pretreatment of the rayon/polyester cloth with 10% NaOH

was found to be necessary for activation with tosyl chloride as with CDI. Since hydrogen bonding is destabilized by strong alkali, this pretreatment presumably breaks the hydrogen bonding involving the hydroxyl groups of rayon. Preliminary studies show that NaOH treatment increased BSA immobilization onto tosyl cloth by at least six-fold. Prior to tosyl chloride activation it is essential that the cloths be dehydrated, as the presence of even small amounts of water results in poor activation of the cloths. Dehydration of traditional supports (e.g. cross-linked agarose) is performed in a sequential washing with increasing ratios of acetone:water mixtures in order to maintain the structure of the support (Harlow and Lane, 1988). With cloth supports the dehydration step is performed with straight acetone which allows for easier and more rapid dehydration. The dehydrated cloths must be transferred without the complete removal of acetone to the tosyl chloride solution. Drying at this stage reduced BSA immobilization six-fold possibly due to the reformation of hydrogen bonds which reduces -:he activation by tosyl chloride. For the activation of the NaOH-treated cloth, pyridine (used for the activation of agarose beads) need not be added to the activation solution to neutralize the HCL generated. This is advantageous in industrial scale activation since pyridine is unsafe and has a very unpleasant odour. Acetone used in the activation is safe, inexpensive, and easy to remove.

Effect of tosvl chloride concentration and time of tosvl chloride treatment on BSA immobilization

Table 1 shows that BSA immobilization increased when higher concentrations of tosyl chloride were used during a 10 min activation reaction.

Table 1: Effects of concentration of tosvl chloride and length of tosyl chloride activation of on BSA adsorptiona.

Concentration Length Absorbanceb % (w/v) (min) (BSA Binding)

2.5 10 4.7 f 0.8 5.0 10 5.7 ?I 0.7

10.0 10 6.2 + 0.7

10.0 10 6.1 + 0.5 10.0 20 5.8 + 0.4 10.0 40 4.7 * 0.5 10.0 60 4.1 + 0.5

a Rayon/polyester cloths were activated with varyi

cloth

9 concentrations of tosyl chloride for varying lengths of time, and then assayed for BSA immobilization as described in Methods. The amount of BSA immobilized to each cloth segment was determined by measuring the absorbance at 562nm of the golour developed in the BCA protein assay.

Average absorbance + standard deviation (n=6)

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Instead of using concentrations higher than lo%, the time of activation in 10% tosyl chloride was lengthened. Table 1 indicates that as the time of activation increased, the immobilization capacity of the cloths actually decreased. The reason for this decrease is not clear. Here, the BSA immobilization is compared in the absorbance of the colour developed by the immobilized BSA because immobilized protein standards are not available. The immobilized BSA would be less efficient than free BSA in developing the colour of the protein assay because the protein-assay reaction would be slower with immobilized proteins. However, the colour developed with immobilized BSA can provide a minimum BSA immobilization capacity of tosyl chloride activated cloth: one gram of the activated cloth was capable of immobilizing at least 25 mg of BSA. Consequently one major advantage of tosyl cloths is their very high immobilization capacity. Comparison of the colour developed shows that tosyl cloths have the capacity to immobilize three times more BSA than CD1 or CNBr activated cloths.

Effect of Molaritv and pH of the Sodium Phosphate Buffer 0.5 M phosphate buffer (pH 7.5) has been used in the

immobilization of protein onto tosyl agarose (Harlow and Lane, 1988). Table 2 shows the effects of concentration and pH of the buffer on the binding capacity of the cloths. The amount of BSA immobilized increased as the molarity of the phosphate buffer increased from 0.05 to 0.5. The pH of the buffer within the range of 7.5 to 10.5 had little effect on the immobilized capacity of the cloths.

Table 2: The effect of changing the molarity and pH of the sodium phosphate buffer on BSA immobilization to the tosyl chloride activated clothsa.

Molarity (Ml

PH Absorbanceb (BSA Binding)

0.05 7.5 4.9 F 0.4 0.1 7.5 5.5 * 0.5 0.5 7.5 6.0 + 0.7

0.5 7.5 6.0 T!I 0.7 0.5 8.5 5.8 f 0.5 0.5 9.5 6.1 + 0.6 0.5 10.5 6.0 f 0.6

a Rayon/polyester cloth segments were activated with 10% (w/v) tosyl chloride for 10 min and incubated in 1% (w/v) BSA in phosphate buffer of varying molarity and pH overnight at room temperature as described in Methods. The amount of BSA immobilized to each cloth segment was determined by measuring the absorbance at 562 nm of the colour developed in the BCA

B rotein assay. Average absorbance * standard deviation (n=6)

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Linkaqe Stability Following immunoaffinity chromatography, antigen-antibody

dissociation is often achieved by adjusting the pH of the system to high alkalinity (e.g. pH 11) (Harlow and Lane, 1988). The linkage between the affinity support and proteins that is formed on CD1 activated supports is unstable at high pH. To test the stability of the linkages produced through tosyl chloride activation, BSA immobilized on tosyl cloth was incubated at room temperature for 20 hours in 0.5 M sodium phosphate buffer (pH 7.5 or 11). After incubation, the cloths were thoroughly washed with buffer and the amount of BSA still immobilized onto the cloths was measured. There was no significant difference between the amount of BSA still immobilized onto both sets of cloths (data not shown).

Kinetics of Immobilization of BSA and anti-HRP antibody Rayon/polyester cloth segments were activated with tosyl

chloride and then incubated with 1% BSA in 0.5 M sodium phosphate buffer (pH 7.5) at room temperature for various lengths of time including 1, 2, 4, 8 and 20 hours. The amount of BSA immobilized did not change significantly after 1 h. In immunoaffinity purification of proteins, generally small amounts of the antibodies are immobilized. It has been advertised that tresyl chloride activated support is ideally suited for the efficient immobilization of very small amounts of proteins in a short period of time (e.g. one hour) (Anonymous, 1991). To examine the kinetics for the immobilization of very small amounts of antibodies, tosy1 cloths were treated with anti-HRP (about 7.5 ,ug/cloth segment) for various time periods. After blocking non-specific binding sites on the cloth with BSA, HRP was allowed to react with the anti-HRP cloth. The captured HRP was assayed using the TMB indicator system. The amount of immobilized anti-HRP was measured indirectly by determining the colour formed.

Table 3: Rate of anti-HRP immobilization to tosyl- chloride activated rayon/polyester clotha'

Immobilization time Absorbanceb (min) (HRP activity)

control 0.22 I!I 0.06

15 2.2 It 0.1

30 2.3 k 0.2

60 2.1 + 0.1

120 2.0 ?I 0.1 ' Rayon/polyester cloths were activated with 10% (w/v) tosyf chloride for 10 min. Following the reaction of the tosyl cloths with anti-HRP for the various time periods indicated, the cloths were incubated with BSA overnight and then reacted with HRP as described in Methods. The amount of anti-HRP antibody immobilized to each cloth segment was measured indirectly by determining the absorbance at 370 nm of the colour developed in the TMB indicator system. The control cloths were not reacted with anti-HRP. b Average absorbance t standard deviation (n=6)

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Table 3 indicates that even a very low concentration of anti- HRP antibody was maximally immobilized within 15 min. Most of the HRP bound to the cloth represents HRP that has reacted with immobilized anti-HRP. This is indicated by the observation that tosyl cloth segments lacking anti-HRP (control) have only 10 percent of the amount of HRP present on tosyl cloth segments that have immobilized anti-HRP. The results also indicate that antibodies immobilized on tosy1 cloth retain their immunological activity.

This paper demonstrates that tosyl chloride activated cloth is an attractive method for industrial scale protein immobilization. Three times as much BSA can be immobilized onto tosyl chloride activated cloths than can be acheived with CD1 or cyanogen bromide activated cloths. Tosyl chloride activation is about 50 times less expensive than CD1 activation and 1000 times less expensive than tresyl chloride. Unlike many activation methods such as CD1 and CNBr, the ligand- affinity support linkage produced through tosyl chloride activation is stable at high alkali conditions (e.g. pH 11). Finally, tosyl chloride activated supports are capable of immobilizing smaller amounts of ligand faster than is possible with other activation methods.

ACKNOWLEDGEMENTS

This work was supported by Natural Sciences and Engineering Research Council of Canada Grants to HY.

REFERENCES

Anonymous (1991). Affinica Affinity Media. Keene: Schleicher and Schuell, Inc.

Harlow, E. and D. Lane (1988). Antibodies - A Laboratory Manuaf. pp. 535-536, New York: Cold Spring Harbor Laboratory.

Howlett, J.R., Armstrong, D.W. and H. Yamazaki (1991). Biotechnology Techniques. 5, 395-400.

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