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Biomedical subjects

J Porath

Publications and source records attributed to J Porath.

At least 37 records · Page 2Linked to original sources
PubMed1991

Cascade-mode multiaffinity chromatography. Fractionation of human serum proteins.

The group-resolving power of cascade-mode multiaffinity column chromatography (CASMAC), was demonstrated with human serum as a model mixture. More than 99% of the serum proteins were adsorbed in the same high salt-containing buffer on a tandem column consisting of (1) immobilized Zn2+ on triscarboxymethyl diamine gel followed by (2) thiophilic (T) gel, (3) Zn2+ bound to the new tridentate chelating adsorbent dipicolylamine (DPA) agarose, (4) hexyl-thioether C6-S agarose and (5) Ni(2+)-DPA agarose. After the adsorption step the immobilized metal ion affinity gels were attached to the top of tandem columns of other adsorbents (T gel, Sephadex G-25 for desalting and Mono-Q) and the elution conditions were selected such that further group separation was achieved. High resolution, high recovery, easy manipulation and high capacity are characteristic features of the cascade process with these adsorbents. The advantage of CASMAC is particularly striking when, with a given number of adsorbents, the overall number of operations involving adsorption, desorption, washing, buffer change and substance concentration can be effectively minimized.

Blood Protein Electrophoresis↗
PubMed1991

Purification of insect vitellogenin and vitellin by gel-immobilized ferric chelate.

Vitellogenin and vitellin of Manduca sexta and some other insect species were purified by immobilized metal ion affinity chromatography. Ferric ion was chosen as the immobilized metal ion. Agarose-bound carboxymethylpicolylamine was used as the chelating adsorbent for the ferric ion. Vitellogenin and vitellin, both phosphorylated lipoproteins, were shown to bind specifically to the iron. The general applicability of immobilized ferric ion affinity chromatography for the purification of insect vitellogenin and vitellin is suggested.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Further characterization of carboxymethylated aspartic acid agarose. Purification of human alpha 2-macroglobulin and hemopexin.

alpha 2-Macroglobulin and hemopexin were purified by affinity chromatography on a recently introduced chelating matrix, i.e., carboxymethylated aspartic acid agarose, coupled with cobalt(II). Adsorption was performed at neutral pH and the proteins were eluted by lowering the pH to 5.0. An alternative method for desorption as well as comparison with iminodiacetic acid agarose coupled with cobalt(II) is also described.

Animals↗
PubMed1991

Purification of factor VIII:c coagulant activity from rat liver nonparenchymal cell culture medium by immobilized metal ion affinity chromatography.

The purification of factor VIII:c coagulant activity on the basis of its affinity for calcium is described. For this purpose, use was made of a recently introduced chelating matrix, i.e., carboxymethylated aspartic acid agarose, coupled with calcium--thereby creating a gel with specificity comparable with biospecific affinity chromatography. In a single step factor VIII:c activity was purified from rat liver nonparenchymal cell culture medium with a purification factor of 85-fold. The material exhibits a single band on polyacrylamide gel electrophoresis.

Animals↗
PubMed1990

Immobilized metal ion affinity chromatography. Effect of solute structure, ligand density and salt concentration on the retention of peptides.

The adsorption characteristics of a variety of synthetic peptide hormones and di-, tri- and tetrapeptides on Cu(II) immobilized on two commercially available high-performance chelating gels run under various experimental conditions are described. Methods for determining the concentration of immobilized Cu(II) in situ are also described. The Cu(II)-charged columns exhibit a net negative charge as judged from the significantly higher retention of some basic peptides in the absence of NaCl in the equilibration and elution buffers. At higher NaCl concentrations (2-4 M), aromatic interactions seem to be superimposed on the metal ion affinity characteristics of the peptides. The relationship between resolution of peptides and the concentration of immobilized Cu(II) ions has also been established for the Chelating Superose gel where 40 mumol Cu(II) ml-1 gel apparently gives the optimum resolution. The nature of the gel matrix also plays a role in the resolution of some peptides, the extent of which is difficult to predict. The results obtained also suggest that peptides containing aromatic and hydroxy amino acids are retarded more than those which lack them. Moreover, these same amino acids apparently strengthen the existing strong binding of peptides containing His, Trp or Cys to a Chelating Superose-Cu(II) column. Dipeptides with C-terminal His (i.e., X-His) are neither bound nor retarded on a column of Chelating Superose-Cu(II) whereas those having the structure His-X are strongly bound. Some tri- and tetrapeptides containing His were also found not to bind to the column. The underlying cause of this anomalous adsorption behaviour is discussed and is ascribed to "metal ion transfer" arising from the relatively higher affinity of such peptides towards immobilized Cu(II) ions than the chelator groups (iminodiacetate) which are covalently bound to the gel matrix.

Amino Acids↗
PubMed1990

Protein chromatography with pyridine- and alkyl-thioether-based agarose adsorbents.

In an attempt to identify the part of the ligand of 3-(2-pyridylthiol)-2-hydroxypropylagarose that is responsible for the specific adsorption of immunoglobulins and alpha 2-macroglobulin from serum, nine agarose derivatives were prepared: (I) 3-(N-2-iminopyridyl)-2-hydroxypropyl-, (II) 3-(4-pyridylthio)-2-hydroxypropyl-, (III) 3-(2-pyridylthio-N-oxide)-2-hydroxypropyl-, (IV) 3-(2-pyridylthio)-2-hydroxypropyl-, (V) 3-(ethylthio)-2-hydroxypropyl-, (VI) 3-(n-butylthio)-2-hydroxypropyl-, (VII) 3-(2-aminoethylthio)-2-hydroxypropyl-, (VIII) 3-(2-hydroxyethylthiol)-2-hydroxypropyl- and (IX) 3-(N-2-pyridyl-2-one)-2-hydroxypropylagarose. The selective adsorption of the above serum proteins to these derivatives was analysed by chromatography. The electron distributions were calculated for three of the investigated pyridine derivatives in order to establish whether there is any relationship between the electron distribution in the molecule and the absorption properties of the pyridine derivatives. By optimizing the preparation methods for the different derivatives, the possible side-reactions were minimized and checked. The adsorbed serum proteins were determined by the Ouchterlony technique and electrophoresis. The concentration of human serum albumin in the different fractions was determined by conventional radioimmunological methods. These data make it possible to classify the adsorbents according to their selectivity and hydrophobic thiophilic behaviour.

Chromatography, Agarose↗
PubMed1990

Differential salt-promoted chromatography for protein purification.

A range of hydrophobic-type adsorbents for protein chromatography has been screened for the binding, at high salt concentrations, of 10 enzymes from a bacterial extract. Adsorbents were chosen for tandem chromatography, in which the first adsorbent removed much of the protein, and the second and subsequent columns bound the desired enzymes. Simple schemes for isolating Zymomonas mobilis and yeast alcohol dehydrogenases are described, in which the enzymes are affinity eluted by NAD+.

Alcohol Dehydrogenase↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

Covalent chromatography and salt-promoted thiophilic adsorption.

Covalent chromatography on 3-(2-pyridyl disulfido)-2-hydroxypropyl agarose, abbreviated PyS2, turns out to involve more complex interactions than has been supposed heretofore. Unexpectedly, the sorption is highly salt dependent. The relative affinities for serum proteins have therefore been determined in the absence and presence of different types of salts at different salt concentrations and with different degrees of ligand substitution on the adsorbent. In the presence of water-structuring salts the PyS2-gel shows an adsorption pattern for serum proteins resembling that of the "thiophilic" T-gel (J. Porath; F. Maisano, and M. Belew (1985) FEBS Lett. 185, 306-310). Superimposed on thiophilic adsorption we have found, as expected, covalent attachment of thiol-containing proteins. Also the thiol-disulfide exchange increases from 4-5% in the absence of potassium sulfate or sodium chloride up to about 40% of the applied serum proteins when such a water-structuring salt is present. We have thus shown that the interaction of a protein with the ligand is greatly facilitated by a water-structuring salt--and in this case the product is a covalently as well as a thiophilically immobilized protein. A cautious interpretation of protein interaction phenomena is justified whenever ligands containing sulfide, disulfide, or pi-electron-rich structures such as aromatic moieties are involved.

Adsorption↗
PubMed1989

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).

Calmodulin↗
PubMed1989

Purification of human serum amyloid P component (SAP) by calcium affinity chromatography.

Serum amyloid P component (SAP) has been purified from human serum by means of immobilized metal ion affinity chromatography (IMAC). It was selectively concentrated on carboxymethylated aspartic acid agarose (CM-Asp-agarose) loaded with calcium and, employing very mild conditions, purified to electrophoretical and immunological homogeneity in a single step amounting to about 1900-fold purification. As a purification method our procedure thus compares well with bio-specific affinity chromatography.

Amino Acids↗
PubMed1989

Purification of two muscle enzymes by chromatography on immobilized ferric ions.

Two enzymes, glycogen phosphorylase and lactate dehydrogenase, were purified simultaneously in a single step. Ferric ions immobilized on a chelating gel were used as the adsorbent. Adsorption and desorption steps were accomplished by changes in buffer composition. The recoveries were better than 80% and the capacities were about 5 mg of protein per milliliter of adsorbent. The procedure worked well both on a small and on a preparative scale. The homogeneity of the purified enzymes was checked by FPLC.

Animals↗
PubMed1988

Purification and characterization of CS2, a sialic acid-specific haemagglutinin of enterotoxigenic Escherichia coli.

CS2 fimbriae of enterotoxigenic Escherichia coli were purified and characterized. The surface haemagglutinins (fimbriae) were detached by sonication from a strain producing only the CS2 fimbriae. Isolation was carried out by gel filtration on a Sepharose 4B column. After depolymerization, the fimbriae subunits were purified on a Sephacryl S-300 column in 8.0 M-guanidinium chloride. From 1 litre of medium, 4-6 mg of purified fimbriae was obtained. We found that CS2 fimbriae were completely dissociated by saturated guanidinium chloride into subunits with a molecular mass of 16.5 kDa. CS2 fimbriae was sialic acid-specific, since sialic acids were the most potent inhibitors, and neuraminidase treatment of erythrocytes abolished haemagglutination. Both fimbriae and fimbrial subunits were found to bind to bovine erythrocytes. The binding of subunits to erythrocytes could be inhibited with low concentrations of sialyl-lactose.

Amino Acids↗
PubMed1988

High-performance immobilized-metal-ion affinity chromatography of peptides and proteins.

The present status of the undeveloped branch of high-performance immobilized-metal-ion affinity chromatography (HPIMAC) is reviewed. As demonstrated, high resolution of peptides and protein mixtures can be obtained. Under specified conditions, more than 1000 runs can be made repeatedly on a single column without much change in pattern or recovery. Occasionally, 10(3)- to 10(4)-fold purification of a protein can be obtained in a single chromatographic run.

Chromatography, Affinity↗
PubMed1988

High-performance immobilized metal ion affinity chromatography of peptides: analytical separation of biologically active synthetic peptides.

The separation of more than 30 biologically active synthetic peptides and their analogs on a high-performance immobilized metal ion affinity chromatography column is described. The metal chelating gel (TSK gel chelate-5PW) contains iminodiacetic acid (IDA) covalently coupled to a hydrophilic, resin-based matrix with a bead diameter of 10 micron. The retention of the peptides on Cu(II), Ni(II), and Zn(II) ions immobilized on the chelating gel showed that some of them can be separated by isocratic elution while the majority of them are retained and are separated into distinct fractions by elution with a linear imidazole gradient or with a continuously decreasing pH gradient. Of the three immobilized metal ions investigated here, the IDA-Cu(II) chelate column gave the best resolution irrespective of the type of gradient used. This is amply illustrated by the resolution of angiotensins I and II and their seven synthetic analogs. The results obtained here serve as guidelines for the future exploitation of this separation method for the efficient fractionation of a wide variety of peptides on an analytical or preparative scale.

Buffers↗
PubMed1988

Protein interaction with immobilized ligands: quantitative analyses of equilibrium partition data and comparison with analytical chromatographic approaches using immobilized metal affinity adsorbents.

Quantitative or analytical affinity chromatography has been successful primarily for the analysis of biologically determined macromolecular affinity relationships. Quantitative approaches are also needed to better characterize simpler, chemically defined immobilized ligands with potential for selective interaction with specific, predetermined protein surface groups. Protein interaction with immobilized metal is a rather selective and versatile, high-affinity adsorption technique for which there is little quantitative information. Using model protein interactions with immobilized Cu2+ ions, we have compared analytical frontal affinity chromatographic methods to a simple, nonchromatographic protocol for the rapid determination of quantitative affinity relationships. Values obtained for the equilibrium dissociation constant (Kd) and binding capacity (Lt) characterizing the interaction of lysozyme with immobilized Cu2+ were quite similar by frontal analysis (Kd = 37-42 X 10(-6) M; Lt = 6.8-7.4 X 10(-6) mol protein/ml gel) and by equilibrium binding analyses (Kd = 33 +/- 4.7 X 10(-6) M; Lt = 5.8-6.1 X 10(-6) mol protein/ml gel; 14 determinations). The interaction of ovalbumin with immobilized Cu2+ was characterized by an affinity (Kd = 4.2-4.8 X 10(-6) M) and capacity (Lt = 1.5-2.1 X 10(-6) mol protein/ml gel) which were also the same regardless of the method for affinity analysis. These values indicate that the total protein bound at saturation corresponds to as much as 17% of the total immobilized Cu2+ ions (approximately 40 X 10(-6) mol/ml gel). Thus, depending on the fraction of total immobilized Cu2+ available for interaction with a given protein (e.g., lysozyme), the number of individual immobilized ligands actively participating as well as those rendered unavailable upon individual protein binding events may be greater than 1. Linear Scatchard plots obtained for both lysozyme and ovalbumin (purified) suggest the presence of only a single type of immobilized Cu2+-protein interaction operative under the experimental conditions employed. However, Scatchard analyses of data obtained by the nonchromatographic equilibrium binding method also demonstrated the ability to simultaneously resolve the contribution of two components whose presence was predicted by frontal chromatography. Our results support the validity and utility of equilibrium binding data analyzed according to the equations outlined by Scatchard and others as an alternative to analytical chromatographic methods.

Chromatography, Affinity↗