Jerker Porath

Metal chelate affinity chromatography, a new approach to protein fractionation.

CONVENTIONAL nonspecific precipitation methods sometimes depend on affinities which can be used in a more selective fashion by modern chromatographic techniques. The affinity of proteins for heavy metal ions, for example, may provide a basis for their purification and analysis. A highly flexible method based on such affinities is described here.

Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography.

Phosphoproteins and phosphoamino acids bind to ferric ions immobilized on iminodiacetate-agarose gel and can be eluted by increasing pH or by introducing phosphate ions to the eluant. Some other metals were found to resemble iron with regard to the interaction with protein-bound phosphate and phosphoamino acids. These observations were utilized to develop purification procedures for phosphoproteins. Hen egg albumin (ovalbumin) was fractionated into three components of varying phosphate contents. Porcine pepsin was purified in a similar manner.

Preparation of adsorbents for biospecific affinity chromatography : I. Attachment of group-containing ligands to insoluble polymers by means of bifunctional oxiranes

Abstract A new method for preparing adsorbents for biospecific affinity chromatography is described. Bisoxiranes (e.g. 1,4-butanediol diglycidyl ether) have been used for the introduction of reactive oxirane groups into agarose, and for simultaneous stabilization of the gel by cross-linking. Optimal conditions for the activation and subsequent coupling of proteins, peptides and aliphatic and aromatic amines have been evaluated. Fractionation of different forms of trypsin on soya bean trypsin inhibitor agarose is described in order to illustrate the use of oxirane-agarose in biospecific affinity chromatography.

Gel filtration of proteins, peptides and amino acids

Abstract 1. 1. Mixtures of proteins, peptides and amino acids can be fractionated by filtration through beds of dextran gel containing only small amounts of carboxylic groups. 2. 2. Group separations are readily achieved. In highly cross-linked dextran proteins and large peptides move together ahead of amino acids. In dextran gels of low degree of cross-linking peptides and even proteins may be retained on the columns, so that a fractionation of substances within these groups may be obtained. 3. 3. Basic peptides and amino acids move slowly through the gels in certain basic solvents such as 1 M pyridine and faster in acidic solvents such as 1 M acetic acid. For acidic peptides and amino acids the influence of the solvents mentioned appears to be the reverse of that for the basic compounds. 4. 4. Aromatic substitution has a marked effect on the migration through the gels. The relative speed of dinitrophenylated amino acids is highly dependent on the buffer used. Such influence of the buffer was not noticed for phenylalanine, tyrosine and tryptophan, although these compounds are retarded to a different extent. 5. 5. When the columns are properly prepared, symmetrical distribution of each compound is always obtained. 6. 6. The column capacity is very high compared to other similar column methods (chromatography and zone electrophoresis). 7. 7. The reproducibility is very good. 8. 8. The gels are easily regenerated in the columns and may be used daily over a period of months without detectable deterioration.

Preparation of cyanogen bromide-activated agarose gels

Abstract By performing the cyanogen bromide activation of hydroxylic gels (e.g., agarose) in alkaline phosphate solutions of very high buffer capacity, the pH control that has hitherto been necessary can be omitted. Strongly, moderately and weakly activated gels can easily be prepared in a simple and reproducible manner.

Agar derivatives for chromatography, electrophoresis and gel-bound enzymes: I. Desulphated and reduced cross-linked agar and agarose in spherical bead form

Abstract A method for producing alkali- and thermo-stable, insoluble spherical agar particles with very low adsorption capacity (tested with cytochrome C) is described. Sulphate ester groups present in the original agar are removed by alkaline hydrolysis of cross-linked agar, in bead form, at an elevated temperature. The reaction is performed in the presence of sodium borohydride to prevent simultaneous oxidation. Further reduction of the adsorption capacity may be accomplished by treatment of the gel with lithium aluminium hydride in dioxane. Desulphated and reduced agar and agarose gels can be packed in beds with excellent flow and molecular sieve properties.

Characterization studies on a new lectin found in seeds of Vicia ervilia

As part of a screening program for the occurrence of lectins in leguminous plants [ 1,2] we have found a lectin in seeds of viciu ervilia, which we have purified and partially characterized. The serological specificity of extracts from seeds of this species was reported earlier [3-51. The compound was isolated directly from a raw extract of I/. ervilia seeds by specific adsorption to a D-mannose-substituted Sepharose gel and subsequently desorbed with a glucose solution. The material recovered from a second adsorption-desorption treatment was found to be homogeneous on ultracentrifugation and free zone electrophoresis although with a pH-dependent tendency for aggregation. Its mol. wt of 60 000 may be accounted for by four subunits which are identical pairwise with molecular weights of 21 000 and 4700, respectively. The lectin molecule lacks carbohydrate and furthermore lacks sulphur containing amino acids and is rich in acidic and hydroxylic amino acids, which are usual features of this class of molecules [6-91. The sugar specificity appears similar to that of Concanavalin A. The purified lectin has been submitted to other investigators for examination of its possible utility as a biospecific adsorbent, especially for virus purification.

Methodological studies of zone-electrophoresis in vertical columns. I. Fractionation in cellulose powder columns of substances of low molecular weight exemplified by amino acids and related compounds.

Abstract 1. 1. Apparatus for zone electrophoresis in columns has been described. 2. 2. Directions for packing and operation of the columns have been given. 3. 3. The behavior of some compounds of low molecular weight on cellulose columns under different conditions has been studied. Thus adsorption and zone-spreading as well as temperature rise within columns of different diameters as a function of current have been investigated. 4. 4. the usefulness of zone electrophoresis in cellulose columns for fractionating mixtures of amino acids and related compounds, pea root exudate and crude pituitary extract has been demonstrated.

Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography

High-performance immobilized metal ion affinity chromatography was utilized to evaluate the adsorption properties of 67 synthetic, biologically active, peptides ranging in size from 5 to 42 residues. The metal ions, Cu(II), Ni(II) and Zn(II), were immobilized by iminodiacetic acid (IDA) coupled to TSK gel 5PW (10 microns). Two types of gradient elution (imidazole and pH) were used to evaluate peptide retention by the metal ions. A decreasing pH gradient and an increasing imidazole gradient eluted the peptides in similar order. IDA-Cu(II) and IDA-Zn(II) showed very similar selectivities for the peptides analyzed; however, IDA-Zn(II) displayed a weaker affinity for the peptides. IDA-Ni(II) showed a slightly different pattern of selectivity. Peptide adsorption effects contributed by the metal-free gel matrix were found to be relatively minor. The concentration and type of salt included in the mobile phase could affect the relative affinities of the peptides for the immobilized metal ions. Retention coefficients were assigned to individual amino acid residues by multiple linear regression analysis. Histidine showed the largest positive correlation with retention, followed by aromatic amino acid residues. Modified N-terminal residues resulted in negative contributions to retention. Analyses of peptide amino acid composition alone allowed prediction of peptide retention behavior on immobilized metal ion affinity columns.

Radioimmunosorbent assay for proteins. Chemical couplings of antibodies to insoluble dextran

Abstract The use of immunosorbents for radioimmunoassays of proteins and polypeptides has considerably simplified these methods. The immunosorbents were prepared by chemical coupling of antibodies to derivatives of insoluble dextran (Sephadex). Four coupling methods were compared: three cyanogen halide methods and one isothiocyanato-method. It was concluded that immunosorbents prepared by CNBr-activation of Sephadex are stable for at least three months, have a high antigen binding capacity, have a very small tendency for nonspecific adsorption, and are easiest to prepare in comparison with the other immunosorbents investigated. This method is recommended for the preparation of immunosorbents to be used in the radioimmunosorbent assay system.