Alkyl-bound silica was modified using chelating surfactants and the resulting adsorbent was used in immobilized metal affinity chromatography of proteins and peptides. Brij-76, a non-ionic amphiphilic surfactant with an alkyl moiety and an ethylene oxide chain, was reversible adsorbed to alkyl silica (C 18). The hydroxyl group at the end of the ethylene oxide chain was chemically modified previously with an iminodiacetate functionality as chelating agent of transitional metal ions. Cu(II) was studied as immobilized ion for the adsorption of peptides and proteins. Three chromatographic supports were prepared having different Cu(II) capacities. For a low Cu(II) capacity case, the generated adsorbent behaved as a controlled access media preventing the adsorption of large molecular weight proteins, such as BSA, while small peptides, such as Angiotensin III, or amino acids could be retained. For a medium and high Cu(II) capacity, the synthesized adsorbent no longer behaved as a controlling access media and all molecules in this study, either large or small, were retained by the immobilized ion. Nonetheless, most of the BSA was strongly retained by the system and a pH change did not remove any of the adsorbed BSA while the small molecules were removed by the same pH change.
Alkyl silica, a common adsorbent for reversed phase chromatography of biological macromolecules [
During the creation of alkyl silica saturated with a surfactant that inhibits the interaction of a protein with alkyl residues, a surfactant containing a polyethylene glycol residue is necessary. Polyethylene glycol (PEG), either covalently grafted or hydrophobically adsorbed, has shown to prevent protein adsorption onto surfaces [
The recovery of proteins by immobilized metal affinity chromatography (IMAC) is widely known after the work of Porath et al. [
Chelating surfactants have been used to immobilize proteins on chromatographic beads. Ho et al. [
a transitional metal ion and the resulting adsorbent was used for the immobilization of luciferase on the polystyrene beads.
Here we describe the synthesis of a chelate-derivatized surfactant using Brij derivatives and iminodiacetic acid (IDA) to form surfactant-IDA complexes and the reversible adsorption of these surfactant-chelates to octadecyl-bonded silica. Octadecyl-bound silica beads were coated with the surfactant-IDA derivatives and after loading with Cu(II) at different densities, used to study protein adsorption by IMAC interactions.
Vydac silica C18 (218 TP) was purchased from Grace (USA). TP silica is a polymeric bonded phase (end-capped) with a pore size of 30 nm. The surfactant Brij-76 was purchased from Sigma (USA). Thionyl chloride, ammonium hydroxide, absolute ethanol, bromoacetic acid, sodium hydroxide, hydrochloric acid, ethyl acetate, chloroform, sodium phosphate, sodium chloride, acetonitrile, bovine serum albumin (BSA), ribonuclease A (RNAse A), tryptophan (Trp) and histidine (His) were also acquired from Sigma (USA). The peptide Angiotensin III was obtained from Bachem (USA).
Chromatographic analyses were performed using a Gilson HPLC system (USA) equipped with two isocratic pumps, a mixer, a manual injection valve (with a 0.5 mL sample loop), a UV-Vis detector, and a fraction collector. The system was controlled by the Unipoint software from Gilson. The chromatographic column was a glass column from Amersham Biosciences (USA) having an internal diameter of 0.5 cm and a length of 5 cm. Synthesis of the chelating-surfactant derivative was performed using a Parr (USA) mini reactor with 4843 controller.
The synthesis of chelating surfactants was performed by a three-step reaction scheme. In step 1, as an example, 10 g of Brij-76 were melted at 55˚C under vacuum to remove water for 1 h. Afterwards 5.2 mL of thionyl chloride were added under a nitrogen atmosphere and the temperature was increased to 65˚C. Chlorination continued for 6 h. Then vacuum was applied to remove unreacted thionyl chloride for 2 h. The product was dried overnight at 50˚C in an oven. In step 2, 40 mL of absolute ethanol were used to dissolve the modified surfactant and the solution was added to a reactor containing 400 mL of ammonia-saturated ethanol. The reactor was closed and the temperature raised to 100˚C, the reaction was let to proceed for 4 h. The mixture was then cooled to room temperature (RT) and concentrated under vacuum. The aminated residue was finally dried overnight at 50˚C in an oven. In step 3, the resulting solid was dissolved in 40 mL of hot ethanol (70˚C) and 2.05 g of bromoacetic acid (in 60 mL of ethanol with 2.16 mL of 10 M NaOH) were added. The temperature was raised to 80˚C and the reaction proceeded for 14 h controlling the pH in the range 8 - 9 adding 10 M NaOH. The temperature was then lowered to RT and the solvent was eliminated under vacuum. The residue was suspended in 200 mL of water, acidifying the mixture to pH 1 - 2 by adding concentrated hydrochloric acid. The resulting aqueous suspension was first extracted with ethyl acetate and then with chloroform. The organic solvent was evaporated and the residue dried in an oven at 50˚C overnight. The involved reactions during the surfactant-chelating synthesis are depicted schematically in
Surfactant concentration in the native and chelating derivative was determined by the Bradford method [
Octadecyl-bound silica was packed in a glass column (0.5 cm ID) to a height of 2.7 cm using methanol at a flow rate of 2 mL/min. The column was then washed with DI water to remove the alcohol. The interaction and saturation of the silica matrix with the chelating surfactant was carried out by feeding the surfactant solution at a flow rate of 0.5 mL/min until saturation. This was followed with a washing step with DI water to remove unbound surfactant. Afterwards, the column was equilibrated with a 10 mM Cu(II) solution. Unbound Cu(II) ions were also removed with DI water.
The Cu(II) capacity in the prepared adsorbents was measured as follows. A glass column packed with surfactant modified adsorbent was fed with a 10 Mm Cu(II) solution at a flow rate of 0.5 mL/min until saturation. Afterwards, DI water was fed to the column to remove unbound copper ions from the tubing and from the void volume in the column. The entire process was followed by measuring the absorbance of the eluate at 825 nm. Once the breakthrough and washing curves were obtained, Cu(II) capacity was measured by the mass balance expressed in Equation (1).
where cm is the measured Cu(II) concentration (mM), cm0 is the fed Cu(II) concentration (mM), t is the time (min), tf is the Cu(II) solution feeding time (min), tw is the final time of experiment (min), F is the flow rate (mL/min), and V is the column volume (mL).
The surfactant capacity of the reversed-phase adsorbent was measured by the same method used for Cu(II) capacity, with the quantification of surfactant concentration on the eluate by the Bradford method as previously described. A similar equation to Equation (1) was then used to estimate the amount of surfactant retained by the alkyl silica.
Once the alkyl-bound silica was modified with the chelating surfactant and loaded with Cu(II) ions, the column was equilibrated with buffers B and A at a flow rate of 0.5 mL/min. Buffer A consisted of 20 mM PO4, 500 mM NaCl, pH 7.0 while buffer B was 100 mM PO4, 500 mM NaCl, pH 4.0.
Once the system was equilibrated with buffer A (adsorption buffer), a 0.5 mL sample was injected. Absorbance of the eluate was followed at 220 or 280 nm. After a washing step with the same buffer A, buffer B (desorption buffer) was fed to remove specifically retained molecules.
Brij-76 was successfully modified to bear an iminodiacetic moiety. First the efficiency of Brij-76 amination was quantified using the TNBS test. This test revealed that approximately 60% of the original Brij-76 molecule was aminated. The same TNBS was used to determine the absence of amino groups in the Brij-76 molecule after carboxymethylation. The chemical structures of the native and the chelating surfactants are shown in
The surfactant capacities on the reversed-phase columns for native and modified Brij-76 were determined. For the modified surfactants, the Cu(II) capacity was also determined and the results are shown in
The measured Cu(II) capacities for chelating surfactants were comparable with Cu(II) capacities from commercial IMAC systems reported in the literature [
The HLB value for the original Brij-76 is reported in
Once the C18 silica was saturated with Brij-76-IDA1 and equilibrated with the adsorption buffer, pulses of BSA (1 mg/mL), RNAse A (1 mg/mL), Angiotensin III (0.05 mg/mL), Trp (0.05 mg/mL) and His (0.03 mg/mL) were injected separately for analysis. The chromatograms are shown in
f
Surfactant | Surfactant capacity, mg/mL | Cu(II) capacity, µmol/mL | CMC, µM | HLB |
---|---|---|---|---|
Brij-76 | 117 | 0 | 3 | 12.7 |
Brij-76-IDA1 | 124 | 10 | - | - |
Brij-76-IDA2 | 105 | 27 | - | - |
Brij-76-IDA3 | 95 | 66 | - | - |
loaded with Cu(II) ions and thus, none of the test molecules were retained. The proteins or peptides left the column with the void volume. The molecules were not retained by ion exchange processes either since the adsorption buffer had a high ionic strength to inhibit electrostatic interactions. These results corroborate that the adsorbent has changed polarity due to the presence of the ethylene oxide chain, changing from a hydrophobic to a hydrophilic adsorbent. Moreover, the test molecules are being excluded from the hydrophobic surface by the widely known rejecting properties of polyethylene glycol [
Two theories have been considered towards the molecular description of the interaction between PEG and proteins for the creation of non-fouling surfaces. The first theory considers that the PEG layer induces a steric repulsion of proteins associated with an entropic repulsion originated from the compression of the PEG layer [
In this case the Brij-76-IDA1-saturated C18 silica was equilibrated with Cu(II) ions and with desorption and adsorption buffers. The modified adsorbent was equilibrated with the desorption buffer to remove weakly bound Cu(II) ions. Pulses of BSA and RNAse A were tested and the resulting chromatograms are shown in
The observed non-retention of proteins could be associated to several factors. First it is necessary to consider the structure of the adsorbent which is silica modified polymerically for the incorporation of the alkyl chains. Once the surfactant is adsorbed on these alkyl moieties, and with the fact that the number of surfactant molecules bearing an IDA functionality is less than the native surfactant, the probability that an IDA-modified surfactant is buried by the presence of native surfactant is high and thus, Cu(II) ions are not accessible for protein retention. Another possibility is the consideration of the hemi-micelle formation in which the IDA-modified surfactant is closer to the surface and it is being protected by the ethylene oxide chains of the native surfactants that complete the hemi-micelle. Finally, the reduction in pore size has to be considered since this effect will make difficult for the proteins to enter the adsorbent.
Another factor that could be considered is the expected distribution of molecular weight oligomers present with the surfactant Brij-76. This surfactant theoretically contains 10 ethylene oxide units but the reaction product will also contain, in large proportion, surfactant molecules with higher or lower ethylene oxide units according to a Poisson distribution [
By considering any of these possibilities, a small peptide or protein could be more easily retained since the native surfactant will only exert a minor influence on their adsorption. To test the latter concept, Angiotensin III, Trp, and His were injected to the system and the results are shown in
According to the previous results, it was considered to determine if mixtures of large molecules with small molecules could be separated to corroborate a potential restricted access situation. Thus, mixtures of BSA (0.5 mg/mL) with Angiotensin III (0.025 mg/mL) or His (0.015 mg/mL) were injected to the system to determine the retention properties of the modified adsorbents. The results for these experiments are shown in
Alkyl silica was saturated with Brij-76-IDA2 and after equilibration with the adsorption buffer pulses of BSA (1 mg/mL), RNAse A (1 mg/mL), Angiotensin III (0.05 mg/mL), Trp (0.05 mg/mL), and His (0.03 mg/mL) were injected and the chromatograms obtained were similar to the results presented with the alkyl silica saturated with Brij-76-IDA1 (data not shown). As expected, none of these test molecules were retained by the system. Now the system was loaded with Cu(II) ions and pulses of the test molecules were performed. Desorption of adsorbed molecules was accomplished by a pH change. Despite the presence on an unretained peak for the injection of BSA (
Also, mixtures of BSA with Angiotensin III or His were injected to determine if the small molecules continued to be recovered by a pH change while BSA remains adsorbed on the chromatographic system. Both the peptide and the amino acid were successfully separated from the large molecular weight protein (data not shown).
The chloroform fraction of the reaction mixture from the surfactant modification recovered a highly IDA-subs-
tituted Brij-76. A clean silica C18 was saturated with this surfactant and with a Cu(II) solution. After Cu(II) saturation, the original silica support changes from white to bright blue upon Cu(II) complexation. Pulse studies of BSA, RNAse A, Trp and His were performed on this system and the results are comparable to the system using the derivative Brij-76-IDA2-Cu(II). None of the proteins or the amino acids were retained with the system lacking Cu(II) ions (data not shown).
The surfactant Brij-76 was successfully modified to bear an iminodiacetate group at the end of the ethylene oxide chain. This modified surfactant was strongly adsorbed by its hydrophobic tail to silica C18. The leakage of surfactant from the silica C18 was minimal once the surfactant molecules were not present on the mobile phase. The adsorption of surfactant to the silica C18 clearly changed the polarity of the original support that originally presents adsorption of peptides or proteins by hydrophobic interactions. Once the surfactant was loaded with Cu(II) ions, the generated support served as a stationary phase for immobilized metal affinity chromatography and peptides and proteins were retained through interactions with the immobilized ions that were disrupted by a pH change allowing the recovery of some of the studied molecules.
Depending on the Cu(II) capacity of the attached surfactant (or in the number of surfactant molecules bearing an iminodiacetate group), the generated adsorbent could prevent the adsorption of large molecular weight proteins allowing the interaction of small peptides and amino acids with the immobilized ions creating thus, a restricted access media. Once the Cu(II) capacity was increased, all the studied molecules (having large and small molecular weights) were retained by the modified adsorbent. When BSA was retained by interaction with the immobilized ions, its recovery by a pH change was not possible revealing a high affinity likely derived from multipoint interactions between histidine residues from the surface of BSA and the Cu(II) ions.
O. G. O. acknowledges funding from PROMEP/103.5/12/3953 to complete the present work.