Affinity chromatographic purification of human immunoglobulin a from chinese hamster ovary cell culture supernatant

Hexamer peptide ligand HWRGWV, initially screened from a solid phase combinatorial peptide library for immunoglobulins G (IgG) purification, is shown to also have potential for immunoglobulin A (IgA) purification. The determined dissociation constants for hIgA on HWRGWV resins at three different peptide densities from 0.11 to 0.55 meq/g fall in the range of 10^?6–10^?7 M, which are somewhat lower than those for hIgG. Although relatively low dynamic binding capacity (DBC) in the range of 9.2–16.8 mg IgA/mL resin at linear flow rates from 173 to 35 cm/h were obtained for IgA compared to IgG, the DBC value of HWRGWV for IgA is much greater than current commercially available affinity ligands. Although relatively lower binding affinity to secretory IgA compared to monomeric IgA was observed, the peptide ligand resins exhibit great potential for large‐scale purification of both human IgA and secretory IgA. Recoveries of 96.0% and 94.3%, and purities of 90.3% and 91.7% were achieved for human IgA and secretory IgA purification, respectively, from spiked Chinese hamster ovary cell culture supernatants without an extra afterwash step. Over 95% in purities were achieved for IgA and secretory IgA with an extra afterwash step; however, the recoveries would decrease at least 15% and 40% for IgA and secretory IgA, respectively. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Purification of animal immunoglobulin G (IgG) using peptoid affinity ligands

The availability of highly pure animal antibodies is critical in the production of diagnostic tools and biosensors. The peptoid PL16, previously isolated from an ensemble of peptoid variants of the IgG-binding peptide HWRGWV, was utilized in this work as affinity ligand on WorkBeads resin for the purification of immunoglobulin G (IgG) from a variety of mammalian sources and chicken immunoglobulin Y (IgY). The chromatographic protocol initially optimized for murine serum and ascites was subsequently employed for processing rabbit, goat and sheep, donkey, llama, and chicken sera. The PL16-WorkBeads resin proved able to recover all antibody targets with values of yield between 50 and 90%, and purity consistently above 90%. Notably, PL16 not only binds a broader spectrum of animal immunoglobulins than the reference ligands Protein A and G, but it also binds equally well with all their subclasses. Unlike the protein ligands, in fact, PL16 afforded excellent values of yield and purity of mammalian polyclonal IgG, namely murine (47 and 94%), rabbit (66.5 and 91.7%), caprine IgG (63 and 91–95%), donkey, and llama (93 and 97%), as well as chicken IgY (42 and 92%). Of notice, it is also the ability of PL16 to target monomeric IgG without binding aggregated IgG; when challenged with a mixture of monomeric and aggregated murine IgG, PL16 eluted <3% of fed aggregates, against 11–13% eluted by Protein A and G. Collectively, these results prove the potential of the proposed peptoid ligand for large-scale purification of animal immunoglobulins.     

Affinity chromatographic purification of human immunoglobulin M from human B lymphocyte cell culture supernatant

Compared to immunoglobulin G purification with extensively studied affinity ligands such as protein A and protein G, little work has been done on affinity chromatographic purification of immunoglobulin M. Hexamer peptide ligand HWRGWV, previously shown to bind specifically to the Fc fragment of IgG, also demonstrated potential for IgM purification. This study presents further characterization and investigation of this ligand for its potential for purification of IgM. Different running conditions were employed in order to improve the recovery and purity of IgM. The final recovery and purity of the antibody is feedstock dependent, but can reach levels of both recovery and purity as high as 95%. The dependence of the recovery and purity on total loading amount and initial IgM concentration were investigated and discussed. Although relatively low dynamic binding capacities (DBC) in the range of 4.6–13.1 mg IgM/mL resin at linear flow rates from 173 to 35 cm/h were obtained for IgM compared to IgG because of the large molecular weight of IgM, the DBC value of HWRGWV for IgM is much greater than protein-based IgM affinity ligands found in the literature and is competitive with current commercially available affinity ligands, such as KAPTIVE-M, CaptureSelect IgM and Ultralink Immobilized Mannan Binding Protein.     

Three-Dimensional Structure of the Human Myeloma IgG2

Human immunoglobulin G, subclass 2 (hIgG2), plays an important role in immunity to bacterial pathogens and in numerous pathological conditions. However, there is a lack of information regarding the three-dimensional (3D) structure of the hIgG2 molecule. We used electron microscopy (EM), differential scanning microcalorimetry (DSC) and fluorescence for structural analysis of the hIgG2. DSC and fluorescence indicated two types of interaction between C_H1 domain of Fab (antigen-binding fragment/subunit) and C_H2 domain of Fc (complement fixation fragment/subunit) simultaneously present in the sample: close interaction, which increases the thermostability of both, C_H1 and C_H2 domains, and weak (or no) interaction, which is typical for most IgGs but not hIgG2. Thermodynamics could not determine if both types of interactions are present within a single molecule. To address this question, EM was used. We employed a single-particle reconstruction and negative staining approach to reveal the three-dimensional structure of the hIgG2. A three-dimensional model of hIgG2 was created at 1.78 nm resolution. The hIgG2 is asymmetrical: one Fab subunit is in close proximity to the upper portion of the Fc subunit (C_H2 domain) and the other Fab is distant from Fc. The plane of Fab subunits is nearly perpendicular to Fc. EM structure of the hIgG2 is in good agreement with thermodynamic data: a Fab distant from Fc should exhibit a lower melting temperature while a Fab interacting with Fc should exhibit a higher melting temperature. Both types of Fab subunits exist within one molecule resembling an A/B hIgG2 isoform introduced earlier on physicochemical level by Dillon et al. (2008). In such an arrangement, the access to the upper portion of Fc subunit is partially blocked by a Fab subunit. That might explain for instance why hIgG2 mildly activates complement and binds poorly to Fc receptors. Understanding of the three-dimensional structure of the hIgG2 should lead to better design of antibody-based therapeutics.     

The YvfTU Two-component System is involved in plcR expression in Bacillus cereus

Background

Protein A, protein G and protein L are three well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The single domains of protein A, protein G and protein L were all demonstrated to have function to bind to Ig. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding is interesting.

Results

We used a combinatorial phage library which displayed randomly-rearranged various-peptide-linked molecules of D and A domains of protein A, designated PA(D) and PA(A) respectively, B2 domain of protein G (PG) and B3 domain of protein L (PL) for affinity selection with human IgG (hIgG), human IgM (hIgM), human IgA (hIgA) and recombinant hIgG1-Fc as bait respectively. Two kinds of novel combinatorial molecules with characteristic structure of PA(A)-PG and PA(A)-PL were obtained in hIgG (hIgG1-Fc) and hIgM (hIgA) post-selection populations respectively. In addition, the linking peptides among all PA(A)-PG and PA(A)-PL structures was strongly selected, and showed interestingly divergent and convergent distribution. The phage binding assays and competitive inhibition experiments demonstrated that PA(A)-PG and PA(A)-PL combinations possess comparable binding advantages with hIgG/hIgG1-Fc and hIgM/hIgA respectively.

Conclusion

In this work, a combinatorial phage library displaying Ig-binding domains of protein A, protein G, or protein L joined by various random linking peptides was used to conducted evolutional selection in vitro with four kinds of Ig molecules. Two kinds of novel combinations of Ig-binding domains, PA(A)-PG and PA(A)-PL, were obtained, and demonstrate the novel Ig binding properties.     

Molecular Basis for the Dissociation Dynamics of Protein A-Immunoglobulin G1 Complex

Staphylococcus aureus protein A (SpA) is the most popular affinity ligand for immunoglobulin G1 (IgG1). However, the molecular basis for the dissociation dynamics of SpA-IgG1 complex is unclear. Herein, coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field were used to study the dissociation dynamics of the complex. The CG-MD simulations were first verified by the agreement in the structural and interactional properties of SpA and human IgG1 (hIgG1) in the association process between the CG-MD and all-atom MD at different NaCl concentrations. Then, the CG-MD simulation studies focused on the molecular insight into the dissociation dynamics of SpA-hIgG1 complex at pH 3.0. It is found that there are four steps in the dissociation process of the complex. First, there is a slight conformational adjustment of helix II in SpA. This is followed by the phenomena that the electrostatic interactions provided by the three hot spots (Glu143, Arg146 and Lys154) of helix II of SpA break up, leading to the dissociation of helix II from the binding site of hIgG1. Subsequently, breakup of the hydrophobic interactions between helix I (Phe132, Tyr133 and His137) in SpA and hIgG1 occurs, resulting in the disengagement of helix I from its binding site of hIgG1. Finally, the non-specific interactions between SpA and hIgG1 decrease slowly till disappearance, leading to the complete dissociation of the SpA-hIgG1 complex. This work has revealed that CG-MD coupled with the Martini force field is an effective method for studying the dissociation dynamics of protein-protein complex.     

Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold

We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies – histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG_1–4), and recognize hIgG_1–4 as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.     

Protein 1a: A major wheat germ agglutinin binding protein on the surface of human granulocytes associated with the cytoskeleton

Lectin-receptors on leukocyte and endothelial surfaces are becoming more important in the light of increasing evidence which implicates lectin-carbohydrate interactions in diverse physiological phenomena. This study reports the identification of a major 118 kDa granulocyte surface protein, (Protein 1a) which binds the lectin wheat germ agglutinin (WGA), and is distinctly different from reported WGA binding granulocyte membrane proteins. Protein 1a has been isolated from the Triton-soluble and Triton-insoluble lysates of normal individuals and patients with Chronic Myeloid Leukemia (CML) using a combination of differential solubilization, lectin affinity, ion exchange chromatography and HPLC. The protein from the detergent lysates of both normal and CML granulocytes has similar pI values, lectin affinities, and hydrophobicity. However, its solubility in Triton is different in the two cell types. In 71% of CML cases examined, Protein 1a exhibits decreased Triton solubility suggesting its increased association with the cytoskeleton (CSK). Stimulation of normal granulocytes with WGA leads to the translocation of the soluble form of Protein 1a to the Triton-insoluble fraction. This cytoskeletal recruitment of Protein 1a is sustained only under conditions of excess WGA and occupied receptor. The CSK disruptive agent dihydrocytochalasin B (H₂CB) releases the insoluble form of the receptor into the Triton-soluble fraction. Investigation of a CSK-involving process such as ligand internalization revealed that CML granulocytes exhibit slower kinetics of internalization of fluorescent WGA molecules. Since Protein 1a is a major WGA receptor on the granulocyte surface, its decreased Triton solubility in CML granulocytes suggests that this may be one of the factors contributing to the defective receptor-mediated endocytosis of WGA by CML cells, arising as a consequence of altered membrane-CSK interaction — a nodal point in the signal transduction cascade.     

Streptococcal protein G-gold complex: comparison with staphylococcal protein A-gold complex for spot blotting and immunolabeling

Protein G, a cell wall protein isolated from human group G streptococci strain G148, binds in a similar manner as protein A from Staphylococcus aureus to the Fc portion of IgG molecules. Indeed, protein G has been proposed as a superior Fc binding protein due to its broader species reactivity. Thus, we have prepared a complex of protein G with particles of colloidal gold and determined its applicability for spot-blot analysis and postembedding immunolabeling by comparing it with protein A-gold complex. By spot-blot analysis no difference in binding of protein G-gold or protein A-gold to IgG molecules from a whole spectrum of animal species was observed. Moreover, using rabbit, sheep, or goat anti-rat albumin antibodies to detect nitrocellulose-immobilized rat albumin or antigenic sites in paraffin and Lowicryl K4M thin sections from rat liver, no difference was found with protein G-gold or protein A-gold. Similarly, no difference in binding to protein G-gold or protein A-gold was observed with a battery of monoclonal antibodies. However, in contrast to expectations, protein A-gold reacted well with both sheep and goat IgG molecules; indeed, for the light and electron microscopic localization of albumin with sheep or goat antibodies it was as efficient as protein G-gold. These results demonstrate, therefore, that both protein G-gold and protein A-gold are useful second step reagents for immunolabeling and that protein G-gold was not a superior probe in the systems tested.     

Mechanical properties of BiP protein determined by nano‐rheology

Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N‐terminal Nucleotide‐Binding Domain (NBD), and the C‐terminal Substrate‐Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano‐rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides‐ ATP, ADP, and AMP‐PNP‐, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (K_D) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.     

Stability and Inter-domain Interactions Modulate Amyloid Binding Activity of a General Amyloid Interaction Motif

The M13 tip protein, g3p, binds the C-terminal domain of the bacterial membrane protein TolA via β-sheet augmentation, facilitating viral entry into Escherichia coli. G3p binding leads to rearrangement of the β strands and partial unfolding of TolA. G3p also binds multiple amyloid assemblies with high affinity, and it can remodel them into amorphous aggregates. We previously showed that amyloid binding activity is defined by the two g3p N-terminal domains, which we call the general amyloid interaction motif (GAIM). GAIM–hIgG1Fc fusions, which add immune effector function to amyloid targeting of GAIM, mediate reduction of two CNS amyloid deposits, Aβ plaques and tau tangles, in transgenic animal models of neurodegenerative disease. We carried out site-directed mutagenesis of GAIM to identify variants with altered amyloid binding and remodeling activity. A small set of residues along the inner strands of the two domains regulates both activities. The specificity of amyloid binding is governed by individual domain stability and inter-domain interactions. Our studies reveal several lines of similarity between GAIM binding to amyloids and g3p binding to its E. coli membrane target, TolA. Based on these studies, we designed new GAIM fusions that show enhanced binding potency towards multiple amyloid aggregates.     

Antibody/antigen affinity behavior in liquid environment with electrical impedance analysis of quartz crystal microbalances

Electrical impedance analysis has been used to study anti-human immunoglobulin G (anti-h IgG) adsorption and the subsequent human immunoglobulin G (hIgG) or rabbit immunoglobulin G (rIgG) affinity reaction in aqueous liquids on a polystyrene (PS)-modified quartz crystal microbalance (QCM) surface. Time-dependent adsorption data of both the frequency shift and the electrical equivalent parameters (motional resistance, shunt capacitance, quality factor, etc) are monitored. It was found that the motional resistance, R, increases while the resonance frequency, f, decreases during both the anti-h IgG immobilization and the subsequent affinity process. Decreasing f primarily arises from the increased mass loading. Increasing R indicates more power dissipation (increased losses) in the system. The change in motional resistance, delta R, in the affinity reaction is considerably larger than that in anti-h IgG immobilization adsorption process, although the resonant frequency shifts, delta f, are very close in these two processes. Specifically, for a saturated solution, the ratio of delta R/delta f is 9.45 x 10 (-3) Omega/Hz for anti-h IgG adsorption and 28.1 x 10 (-3) omega/Hz for anti-h IgG/hIgG binding respectively, indicating the increased power dissipation with the increasing binding molecules. The shunt capacitance changes little in the hIgG binding process ( approximately 0.01 pF).     

Evaluation of a novel methacrylate‐based protein a resin for the purification of immunoglobulins and Fc‐fusion proteins

Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc‐fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline‐tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab‐scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014     

Crystal Structures of the Human G3BP1 NTF2-Like Domain Visualize FxFG Nup Repeat Specificity

Ras GTPase Activating Protein SH3 Domain Binding Protein (G3BP) is a potential anti-cancer drug target implicated in several cellular functions. We have used protein crystallography to solve crystal structures of the human G3BP1 NTF2-like domain both alone and in complex with an FxFG Nup repeat peptide. Despite high structural similarity, the FxFG binding site is located between two alpha helices in the G3BP1 NTF2-like domain and not at the dimer interface as observed for nuclear transport factor 2. ITC studies showed specificity towards the FxFG motif but not FG and GLFG motifs. The unliganded form of the G3BP1 NTF2-like domain was solved in two crystal forms to resolutions of 1.6 and 3.3 Å in space groups P2₁2₁2₁ and P6₃22 based on two different constructs, residues 1–139 and 11–139, respectively. Crystal packing of the N-terminal residues against a symmetry related molecule in the P2₁2₁2₁ crystal form might indicate a novel ligand binding site that, however, remains to be validated. The crystal structures give insight into the nuclear transportation mechanisms of G3BP and provide a basis for future structure based drug design.     

Monitoring nonenzymatic glycation of human immunoglobulin G by methylglyoxal and glyoxal: A spectroscopic study

The accumulation of dicarbonyl compounds, methylglyoxal (MG) and glyoxal (G), has been observed in diabetic conditions. They are formed from nonoxidative mechanisms in anaerobic glycolysis and lipid peroxidation, and they act as advanced glycation endproduct (AGE) precursors. The objective of this study was to monitor and characterize the AGE formation of human immunoglobulin G (hIgG) by MG and G using ultraviolet (UV) and fluorescence spectroscopy, circular dichroism (CD), and matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS). hIgG was incubated over time with MG and G at different concentrations. Formation of AGE was monitored by UV and fluorescence spectroscopy. The effect of AGE formation on secondary structure of hIgG was studied by CD. Comparison of AGE profile for MG and G was performed by MALDI–MS. Both MG and G formed AGE, with MG being nearly twice as reactive as G. The combination of these techniques is a convenient method for evaluating and characterizing the AGE proteins.     

Inclusion of ionization states of ligands in affinity calculations

When estimating binding affinities of a ligand, which can exists in multiple forms, for a target molecule, one must consider all possible competing equilibria. Here, a method is presented that estimates the contribution of the protonation equilibria of a ligand in solution to the measured or calculated binding affinity. The method yields a correction to binding constants that are based on the total concentration of inhibitor (the sum of all ionized forms of the inhibitor in solution) to account for the complexed form of the inhibitor only. The method is applied to the calculation of the difference in binding affinity of two inhibitors, 2‐phosphoglycolate (PGA) and its phoshonate analog 3‐phosphonopropionate (3PP), for the glycolytic enzyme triosephosphate isomerase. Both inhibitors have three titrating sites and exist in solution as a mixture of different forms. In this case the form that actually binds to the enzyme is present at relative low concentrations. The contributions of the alternative forms to the difference in binding energies is estimated by means of molecular dynamics simulations and corrections. The inhibitors undergo a pK_a shift upon binding that is estimated by ab initio calculations. An interesting finding is that the affinity difference of the two inhibitors is not due to different interactions in the active site of the enzyme, but rather due to the difference in the solvation properties of the inhibitors. Protein 2009. © 2008 Wiley‐Liss, Inc.     

A multipurpose capacitive biosensor for assay and quality control of human immunoglobulin G

We report a flow‐injection biosensor system with a capacitive transducer for assay and quality control of human immunoglobulin G (hIgG). The sensing platform is based on self‐assembled monolayers (SAMs) of carboxylic acid terminated alkyl‐thiols with covalently attached concanavalin A. The electrochemical characteristics of the sensor surface were assessed by cyclic voltammetry using a permeable redox couple (potassium ferricyanide). The developed biosensor proved capable of performing a sensitive label‐free assay of hIgG with a detection limit of 1.0 µg mL^?1. The capacitance response depended linearly on hIgG concentration over the range from 5.0 to 100 µg mL^?1, in a logarithmic plot. Typical measurements were performed in 15 min and up to 18 successive assays were achieved without significant loss of sensitivity using a single electrode. In addition, the biosensor can detect hIgG aggregates with concentrations as low as 0.01% of the total hIgG content (5.0 µg mL^?1). Hence, it represents a potential post‐size‐exclusion chromatography–UV (post‐SEC–UV) binding assay for in‐process quality control of hIgG, which cannot be detected by SEC–UV singly at concentrations below 0.3% of the total hIgG content. Biotechnol. Bioeng. 2009; 104: 312–320 © 2009 Wiley Periodicals, Inc.     

Physico-Chemical Characterization of Hamster Interferon

The glycoprotein nature of Syrian hamster interferon was tested on several immobilized lectins. The specific retention of small portion (20%) of interferon activity was observed only on concanavalin A-agarose; Component I of the interferon (not retained) has an apparent molecular weight of 23,500 whereas Component II (retained) is larger, 31,500. The apparent hydrophobicity of Syrian hamster interferon was probed by its chromatography on: (a) straight chain hydrocarbons of varied length; (b) aromatic ligands (aminobenzene, benzylamine, β-phenylethylamine, γ-phenyl-propylamine); ligands listed in (a) and (b) were immobilized to cyanogen bromide-activated agarose (isoure linkage); and (c) phenyl-agarose (Phenyl-Sepharose CL-4B), an aromatic ligand immobilized vi 2-hydroxypropyl arm to the agarose (ether linkage).

For a hydrophobic interaction to occur under physiological solvent conditions, the hydrocarbon arm (isourea 1inkage) must be C₉ to C₁₀ carbon atoms long, whereas the aromatic ligand (isourea linkage) must be removed from the agarose matrix by a molecular arm of C₃ carbon atoms. There is no significant binding of hamster interferon to phenyl-agarose (ether linkage) near neutral pH.

The apparent hydrophobicity of Syrian hamster interferon can be profoundly influenced by the pH of the solvent. At lower pH values, interferon binds to both octyl-agarose and phenyl-agarose. The midpoint of transition between binding of interferon and its release from these ligands is about the same, pH 6.0, suggesting the identity of the binding site on the interferon molecule for both aliphatic and aromatic hydrocarbons.