Adsorption and selectivity characteristics of several human serum proteins with immobilised hard Lewis metal ion-chelate adsorbents.

In this investigation, human serum has been used as an example of a crude protein mixture to define the protein binding characteristics and selectivity of several immobilised hard Lewis metal ion affinity chromatographic (IMAC) adsorbents. Specifically, the binding properties of immobilised O-phosphoserine (im-OPS) and 8-hydroxyquinoline (im-8-HQ), with immobilised iminodiacetic acid as a control system, have been investigated in combination with the hard Lewis metal ions, Al3+, Ca2+, Fe3+, Yb3+, and the borderline metal ion, Cu2+, over the pH range pH 5.5 to pH 8.0 with buffers of 0.5 M ionic strength. The same IMAC adsorbents were also investigated for their protein binding capabilities with buffers of an ionic strength of 0.06 M at pH 5.5 and pH 8.0. The binding behaviour of four "marker" proteins, namely transferrin, alpha2-macroglobulin, gammaglobulin and human serum albumin have furthermore been employed to monitor the differences in protein selectivity exhibited by these IMAC systems. The experimental findings confirm that these hard Lewis metal ion IMAC adsorbents function in a "mixed" binding mode with both coordination and electrostatic characteristics evident, depending on the ionic strength and pH of the equilibration or elution buffers. Based on a screening protocol, several members of the im-Mn+-8-HQ and im-Mn+-OPS adsorbent series have been identified with high selectivity for transferrin and alpha2-macroglobulin. These hard Lewis metal ion IMAC adsorbents thus provide attractive alternatives for selective fractionation of human serum proteins.

Characterization by potentiometric procedures of the Acid-base and metal binding properties of two new classes of immobilized metal ion affinity adsorbents developed for protein purification.

The acid-base protonation constants of two recently introduced chelating ligands for protein purification, O-phosphoserine and 8-hydroxyquinoline immobilized onto Sepharose CL-4B, and the stability constants of their derived immobilized metal ion chelate complexes have been determined by potentiometric methods. The data confirm that immobilization thermodynamically constrains the ligands, with the electron-withdrawing characteristics of the group linking the ligand to the support material affecting the magnitude of the stability constant of the immobilized metal ion complex vis-à-vis the free ligand-metal ion complex in solution. The influence of buffer composition, ionic strength, and pH on the stability constant of the immobilized hard metal ion chelate complexes has also been examined. Collectively, the results have confirmed that coordination complexes with stoichiometries other than the simply 1:1 ML-type exist with these systems, with hard metal ions exhibiting a preference for hydrolytic M(OH)(m)L(n) complexes where m or n > 1. These findings on the participation of coordination complexes of different stoichiometry depending on the characteristics of the chelating ligand and metal ion have fundamental implications for the interpretation of immobilized metal ion affinity chromatographic separation of proteins.

Application of immobilized metal ion chelate complexes as pseudocation exchange adsorbents for protein separation.

The interactions of horse muscle myoglobin (MYO), tuna heart cytochrome c (CYT), and hen egg white lysozyme (LYS) with three different immobilized metal ion affinity (IMAC) adsorbents involving the chelated complexes of the hard Lewis metal ions Al3+, Ca2+, Fe3+, and Yb3+ and the borderline Lewis metal ion Cu2+ have been investigated in the presence of low- and high-ionic strength buffers and at two different pH values. In contrast to the selectivity behavior noted with buffers of high ionic strength, with low-ionic strength buffers, these three proteins interact with the hard metal ion IMAC adsorbents in a manner more characteristic of cation exchange behavior, although in contrast to the cation exchange chromatography of these proteins, as the pH value of the elution buffer was increased, the retention also increased. The selectivity differences observed under these conditions appear to be due to the formation of hydrolytic complexes of these immobilized metal ion chelate systems involving a change in the coordination geometry of the im-M(n+)-chelate at higher pH values. The experimental observations have been evaluated in terms of the effective charge on the immobilized metal ion chelate complex and the charge characteristics of the specific proteins.

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues.

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al3+, Ca2+, Fe3+, and Yb3+ and the borderline metal ion Cu2+, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the range pH 5.5-8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized Mn+-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe3+-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe3+-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metal-ion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surface-exposed aspartic and glutamic acid residues on the protein with the immobilized Mn+-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu2+-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number of clustering of the surface-accessible aspartic and glutamic residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.

High-performance liquid chromatography of amino acids, peptides and proteins. CXXXI. O-phosphoserine as a new chelating ligand for use with hard Lewis metal ions in the immobilized-metal affinity chromatography of proteins.

Conditions for the immobilization of O-phosphoserine (OPS) to epoxy-activated Sepharose CL-4B are described. The binding behaviour of OPS and iminodiacetic acid (IDA) immobilized onto Sepharose CL-4B, toward the hard Lewis metal ions Al3+, Fe3+, Ca2+ and Yb3+, and Cu2+ ion as a borderline metal ion control, over the pH range pH 4.0 to pH 8.0, was examined. Immobilized OPS shows a stronger affinity for Fe3+ and Al3+ ions but a lower affinity for Cu2+ and Yb3+ ions, compared to immobilized iminodiacetic acid (IDA), over the equilibrating range examined. Immobilized OPS-Mn+ was screened for protein binding using as model proteins tuna heart cytochrome c (THCC), horse myoglobin (HMYO) and hen egg while lysozyme (HEWL) over the pH range 5.5 to 8.0. Immobilized OPS-Fe3+ bound THCC under all the examined equilibrating conditions, bound HMYO between pH 5.5 and pH 7.0 and did not bind HEWL under any condition examined. Immobilized OPS thus presents an additional mode of metal ion and protein selectivity in immobilized-metal affinity chromatography.

Development of an enzyme immunoassay for the detection of Clostridium novyi type B alpha toxin.

Clostridium novyi type B alpha toxin was purified to homogeneity and shown to have a molecular weight of 200 kD by SDS polyacrylamide gel electrophoresis. The toxin was toxoided and used to produce a pair of non-interfering monoclonal antibodies. Their specificity was confirmed by immunoblotting and bioassay. The monoclonal antibodies were used to develop an enzyme immunoassay which was more sensitive than bioassay, and permitted less than 1 ng/ml toxin to be detected in a rapid 10 min assay format. Use of the assay can eliminate the requirement for in vivo testing of novyi toxin and toxoid, provided measurements of biological activity are not required. Because of its speed and sensitivity, the assay can be used to monitor toxin production during fermentation and as an alternative to bioassay to measure antigen content during toxoiding and vaccine formulation.

Glucose-fructose oxidoreductase, a new enzyme isolated from Zymomonas mobilis that is responsible for sorbitol production.

The enzymes responsible for sorbitol formation in Zymomonas mobilis were investigated. A previously undescribed enzyme catalyzes the intermolecular oxidation-reduction of glucose and fructose to form gluconolactone and sorbitol. This enzyme has been purified; it had a subunit size of 40,000 daltons and is probably tetrameric at low pH. It contained tightly bound NADP as the hydrogen carrier and did not require any added cofactor for activity. In addition, a gluconolactonase has been isolated, although not completely purified. Together these two enzymes were capable of completely converting a 54% (wt/vol) equimolar mixture of glucose and fructose to sorbitol and sodium gluconate at the optimum pH of close to 6.2. The oxidoreductase had low affinities for its substrates, but natural environmental conditions would expose it to high concentrations of sugars. The amount of the enzyme in Z. mobilis cells was sufficient to account for the rate of sorbitol formation in vivo. However, the enzyme was present in the highest amounts when the cells were grown on glucose alone, and it was repressed by the presence of fructose; this was not the case with the gluconolactonase.

Gluconate kinase from Zymomonas mobilis: isolation and characteristics.

The enzyme gluconate kinase EC 2.7.1.12 has been found at high levels in glucose-grown Zymomonas mobilis cells. A simple procedure, based on differential dye-ligand chromatography, has been used to isolate the enzyme, purifying it some 600-fold. The purified enzyme is a monomer of molecular weight 18,000 Da, which is much smaller than other gluconate kinases reported. It has a relatively low affinity for ATP. (Km = 1.5 mM), but high for gluconate (Km = 0.33 mM), and has little activity with any other potential substrates.