Thiophilic interaction chromatography of prostate-specific antigen.

Prostate-specific antigen (PSA) protein and complexes of PSA with alpha1-antichymotrypsin (PSA-ACT) or alpha2-macroglobulin (PSA-A2M) prepared in vitro, have strong affinity for different thiophilic gels (T-gel). Free PSA, and these PSA complexes can be isolated due to their affinity for T-gels. The average recovery of PSA from several of the T-gels, based upon ELISA measurements, was 84 to 94%. The data suggest that T-gel affinity can be explored for the purification of free and complexed PSA from various biologic fluids.

Alzheimer's beta-amyloid peptide: affinity for metal chelates.

Alzheimer's amyloid peptide, A beta(1-42) and its fragments, A beta(1-28) and A beta(1-16), were chromatographed on IDA-M(II) columns (M: Cu2+, Ni2+ and Zn2+). The retention of A beta(1-42) and its fragments on IDA-Cu(II) could not be reversed in decreasing a gradient of pH, from 7.0 to 4.0. All A beta peptides were recovered from IDA-Ni(II) columns in a decreasing pH gradient from 7.0 to 4.0, within the pH range from 5.6 to 5.1. A beta(1-42) peptide was strongly retained on IDA-Zn(II) at pH 4.0, but its A beta(1-28) and A beta(1-16) were only transiently retained on IDA-Zn(II) columns when applied at pH 6.1. We submit that histidine clusters, residing both in the Alzheimer's beta-amyloid peptide and in most of the APP/APLP superfamily of proteins, constitute high-affinity binding sites for immobilized metal chelates.

Immobilized metal-ion affinity chromatography: imidazole proton pump and chromatographic sequelae. I. Proton pump.

Complexation of imidazole (Im) with an iminodiacetate (IDA) metal chelate [IDA-M(II)] ligand of chelating gel results in an acidification of the mobile phase. The scope of the action of this IDA-M(II)Im 'proton pump' in IMAC is determined by: (a) IDA-M(II) density of the gel; (b) concentration of applied Im; and (c) the buffering capacity of the mobile phase. Application of Im onto a metal chelate column in a gradient rather than in a stepwise manner, mitigates the proton pump's action, as it does an increase of buffer concentration in the mobile phase. However, only an antecedent conversion of the metal chelate gel, IDA-M(II), to its Im derivative, IDA-M(II) Im, can effectively circumscribe the action of the proton pump. The same holds true, as anticipated, when another chelating ligand (nitrilotriacetate) is used.

Immobilized metal-ion affinity chromatography: imidazole proton pump and chromatographic sequelae. II. Chromatographic sequelae.

Isocratic elution with imidazole of some protein models from a chelating gel, CSFF-IDA-M(II), resulted in their desorption owing to the low pH(6-->4) of the mobile, phase rather than to the imidazole itself (imidazole-generated fall in pH; proton pump). Gradient elution with imidazole was best accomplished when the chelating gel was initially converted into its imidazole complex, CSFF-IDA-M(II)Im. The exploitation of the imidazole-generated proton pump in the IMAC of proteins may enhance the versatility of this type of chromatography.

Spontaneous conversion of PrPC to PrPSc.

Octa-repeats of prion proteins (PrP) contain histidine and tryptophan residues which are known to function as ligands for transition metals. It is proposed that the spontaneous conversion of the PrPC (cellular) isoform into PrPSc (scrapie) isoform may be triggered by the coordination of these metals.

Evaluation of the interaction of protein alpha-amino groups with M(II) by immobilized metal ion affinity chromatography.

The adsorption properties of various peptides and proteins, lacking histidyl groups, on immobilized Cu(II), Ni(II), Zn(II) and Co(II) ions are described; at pH 6 and below they were little retarded. At higher pH the retention became pronounced for iminodiacetate (IDA)-Cu(II) gel. This effect seems to be related to the presence of a terminal alpha-amino group; in the absence of this group the retention of the protein was largely eliminated. At pH 8.5 a terminal alpha-amino group is adsorbed as strongly as a histidyl group. IDA-Ni(II), IDA-Zn(II) and IDA-Co(II) gels display little or no attraction for the terminal alpha-amino group of a protein.

Surface topography of histidine residues in lysozymes.

Several avian and mammalian c-type lysozymes were chromatographed on chelated (to iminodiacetate) and immobilized transition metal ions (Co2+, Ni2+, Cu2+ and Zn2+) under a variety of experimental conditions. The varied affinity of evolutionary variants of the lysozyme family for chelated metal ions, IDA-M(II), can be rationalized primarily in terms of the presence, multiplicity and microenvironments of histidine residues. The chromatographic resolution of some of these closely related proteins attests to the analytical power of immobilized metal-ion affinity chromatography.

Immobilized metal ion affinity chromatography of serum albumins.

The interaction of several serum albumins with chelated (iminodiacetate, IDA) and immobilized (agarose-IDA) metal ions, Co2+, Ni2+, Cu2+ and Zn2+, was studied. There was no retention of human, bovine, porcine, murine and avian albumins on IDA-Zn(II) and IDA-Co(II) columns. However, all albumins studied, i.e., those of: man, cow, pig, dog, rabbit, rat, mouse, chicken and pigeon were retained on IDA-Cu(II) columns, and all except dog albumin were retained also on IDA-Ni(II). The recognition of albumins by chelated and immobilized transition metals seems to be related to an affinity for the imidazole side chains. It is postulated that one to three imidazoles is involved in this interaction, under the employed experimental conditions (pH 7.0; 1 M sodium chloride). There is no evidence for any significant contribution of tryptophan or cysteine (Cys 34) residues to the chromatographic event. The retention of defatted albumin and albumin oligomers (human), on IDA-Cu(II) columns was not significantly different from that of non-defatted albumin or albumin monomer, respectively.

Glass ampules and associated hazards.

Accessing drugs dispensed in glass ampules is always associated with glass fragment contamination of the ampule contents on opening. In this study, the glass fragment contamination from the external surface of the ampule occurred 60% of the time with 1-ml sized ampules (p less than .01). Glass contamination has been shown to increase with larger ampule size. The glass ampule poses a potential source of microbial infection to the patient, as well as other hazards to the user. Increased precautions and improved drug container design appear to be desirable.

The saga of IMAC and MIT.

Immobilized Metal-ion Affinity Chromatography, IMAC, has been gaining in popularity as the purification technique of choice for proteins and peptides. IMAC of proteins on transition metals (Co, Ni, Cu, Zn) can be rationalized in terms of the coordination of histidine residues. Brief accounts of the principles of IMAC, its anticipated development and plausible applications are presented. Metal Ion Transfer, MIT, may offer an efficient means to deplete a metal ion from a metalloprotein or, conversely, to charge its apo form with a metal.

Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography.

Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins--thioredoxin, ubiquitin, calmodulin, lysozyme, cytochrome c, and myoglobin on immobilized transition metal ions (Co2+, Ni2+, Cu2+, and Zn2+)--allows establishment of the following facets of the histidyl side chain distribution: (i) either interior or surface; (ii) when localized on the surface, accessible or unaccessible for coordination; (iii) single or multiple; (iv) when multiple, either distant or vicinal. Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution).

Facile resolution of alpha-fetoproteins and serum albumins by immobilized metal affinity chromatography.

We have explored immobilized metal affinity chromatography as a means of resolving alpha-fetoprotein from its homologous albumin, a problem perennially encountered in the purification of an alpha-fetoprotein or its detection. Human alpha-fetoprotein and human serum albumin were chromatographed on immobilized iminodiacetic acid charged with either Co2+, Ni2+, Cu2+, or Zn2+. Neither human alpha-fetoprotein nor human serum albumin displayed any affinity for Co2+ and Zn2+. However, both proteins were bound to Cu2+ and were partially resolved by affinity elution with imidazole. By contrast, human alpha-fetoprotein and human serum albumin were completely resolved on immobilized Ni2+. Similar results were obtained using bovine alpha-fetoprotein and bovine serum albumin. The resolution of an alpha-fetoprotein from serum albumin should aid the purification of alpha-fetoprotein from a biological fluid containing overwhelming quantities of albumin, for example, serum. Importantly, the separation of human alpha-fetoprotein from human serum albumin may improve and help maintain the accuracy of immunoassays for alpha-fetoprotein, making the chromatography on immobilized Ni2+ a valuable diagnostic tool.

Improved large scale purification procedure of natural human fibroblast interferon.

Human fibroblast interferon, produced in tissue culture by a superinduction procedure, was purified 10,000-fold by chromatography to ca 50% purity as judged by SDS-PAGE. The specific activity of the interferon preparation is 2 X 10(8) and the yield is 70%. The interferon preparation is suitable for clinical use.

Purification of human urinary erythropoietin on controlled-pore glass and silicic acid.

Erythropoietin (Epo) has been isolated from raw human urine of anemic patients by controlled-pore glass and silicic acid chromatography. A purification factor of not less than 100-fold and recovery of about 80% of activity was routinely achieved. The resulting Epo preparation is active both in vivo and in vitro and can be neutralized by an antibody to human Epo indicating the native state and identity of the isolated Epo molecule. The preparation is stable at near neutral pH and is amenable to further purification.

Adsorption of human alpha (leukocyte) interferon on glass: contributions of electrostatic and hydrophobic forces.

Human leukocyte-derived interferon (HuIFN-alpha) can be displaced from controlled-pore glass (CPG) with alkylamines at 0.1-0.3 M concentration. The eluting power of an alkylamine correlates positively with the extent of its alkylation: CH3NH3 + less than (CH3)2NH2 + less than (CH3)3NH+ less than (CH3)4N+. Interferon can also be recovered using an electrolyte and ethylene glycol as cosolvents. Apparently, both hydrophobic and electrostatic forces are involved in the adsorption of HuIFN-alpha to CPG.

Partial purification and characterization of Syrian hamster interferon.

Syrian golden hamster interferon was made by stimulating secondary or benzo(a)pyrene-transformed embryo cells with Newcastle disease virus. Titres of 1000 to 6000 units/ml of tissue culture fluid were obtained. The ionic properties of this interferon were characterized by chromatography on cation and anion exchangers and also by isoelectric focusing when two components were observed: a major component, pI 5.8 and a minor component, pI 6.6. Hamster interferon was partially purified on a tandem of sorbents of diverse chromatographic bias: anion exchanger lead to metal chelate lead to hydrophobic ligand. The purified preparation had a specific activity of about 1 X 10(6) units/mg protein; the recovery of activity was nearly complete. The apparent mol. wt. of hamster interferon, as estimated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions, is 17000. Crude Syrian hamster interferon preparations had some activity on mouse cells; this heterologous activity was entirely due to a small subpopulation of interferon molecules which could be isolated on phenylagarose.

Chromatography of human leukocyte interferon on controlled pore glass.

Human leukocyte interferon (HuIFN-alpha ) was adsorbed on controlled pore glass (CPG) beads in 0.01 M potassium phosphate, pH 7.2. Partial recovery fo HuIFN-alpha from CPG, ranging from 50% to 75%, was obtained with ethylene glycol (6.8 M), ammonium chloride (1.0 M) and Tris.HCl (1.0 M). A near complete recovery (90%) was obtained with potassium phosphate (1 M), but there was no selectivity in the elution vis á vis other proteins. An efficient elution of HuIFN-alpha was accomplished with tetramethyl-, and tetraethylammonium chlorides: the recovery of HuIFN-alpha was complete at low concentrations (0.25 M to 0.50 M) of these salts at pH 8.0 and the elution of interferon was more selective than with other eluants.