Biomedical subjects

J Porath

Publications and source records attributed to J Porath.

At least 19 recordsLinked to original sources

PubMed1998

Comparison of the protein adsorption selectivity of salt-promoted agarose-based adsorbents. Hydrophobic, thiophilic and electron donor-acceptor adsorbents.

Protein adsorption of human serum onto six different agarose-based chromatographic gels that were representative of the salt-promoted adsorbent family [octyl- and phenyl-Sepharose, mercaptoethanol-divinyl sulfone agarose (T gel), mercaptomethylene pyridine-derivatized agarose gel (MP gel), tricyanoaminopropene-divinyl sulfone agarose (DVS-TCP gel), tricyanoamino-propene-bisoxirane agarose (bisoxirane-TCP gel)] was studied in the presence of moderate or high concentrations of the water structuring salt, sodium sulfate. Study of the protein adsorption selectivity by two-dimensional gel electrophoresis revealed an opposed selectivity for hydrophobic interaction adsorbents and electron donor-acceptor adsorbents. The T gel, MP gel and TCP gels belonged to the electron donor-acceptor adsorbents, displaying a main selectivity for immunoglobulins, whereas octyl-Sepharose belonged to the hydrophobic adsorbents, displaying a main selectivity for 'hydrophobic' proteins. Phenyl-Sepharose for its part was described as an example of a composite selectivity of both families. The conclusion of this work is two-fold: (1) hydrophobic interaction chromatography (HIC) and electron donor-acceptor chromatography (EDAC) have opposed protein selectivities and are both salt-promoted. As a main consequence, it means that high concentrations of a water-structuring salt can promote different types of weak molecular interactions, resulting in different protein adsorption selectivities: (2) thiophilic adsorption chromatography (TAC) should be renamed EDAC as similar protein selectivity is demonstrated for electron donor-acceptor ligand devoid of sulfur atoms.

Adsorption↗

PubMed1997

Residue accessibility, hydrogen bonding, and molecular recognition: metal-chelate probing of active site histidines in chymotrypsins.

Subspecies defining the maturation pathway of bovine chymotrypsinogen to alpha-chymotrypsin have been separated in a single chromatographic run by affinity to iminodiacetic acid-Cu(II) [IDA-Cu(II)] immobilized onto Novarose. A major highlight of the elution pattern is that, as maturation proceeds, these subspecies exhibit a correlated increase in affinity toward IDA-Cu(II). This behavior is analyzed by a combination of physicochemical and molecular modeling techniques to assess the contribution of the two histidines present in chymotrypsins, at positions 40 and 57 on the protein surface. Catalytic His-57 features adequate surface accessibility to serve as a ligand to IDA-Cu(II), but its participation is clearly ruled out by specific chemical modification. In contrast, His-40, whose side chain is buried in the crystal structures of both zymogen and mature enzyme, surprisingly proves the most plausible candidate as an electron donor to IDA-Cu(II). This apparent conflict between histidine accessibility and their implication in IDA-Cu(II) recognition has been rationalized on the basis of their flexibility and/or hydrogen-bonding status, with the following outcome. First, histidine constitutes a useful reporter group for subtle protein conformational fluctuations. Second, static accessibility computation alone provides no unequivocal guideline as to whether a protein residue can serve as a ligand. Third, this study is the first to document the occurrence of a screening effect due to hydrogen bonding of an otherwise "accessible" histidine. A significant corollary to this finding would be that the catalytic histidine is rigidly entrapped in a remarkably strong hydrogen-bonding network, a situation that may pertain to mechanistic aspects of catalysis.

Animals↗

PubMed1997

Salt-independent adsorption of human serum proteins on cyanocarbon gels.

Electron donor acceptor gels based on cyanocarbons have been tested for human serum protein adsorption in the absence of salt-promotion by water-structuring salt. This phenomenon was compared with a normal adsorption process in the presence of salt. The tricyanoaminopropene-divinyl sulfone-agarose displayed unusual protein adsorption properties as binding could occur both independently or dependently of the salt-promotion. The absence of hydrophobic or ionic character of the salt-independent interaction suggests an electron donor acceptor adsorption mechanism which is shown, for the first time, to occur independently of salt-promotion in aqueous solution. Study of the protein adsorption specificity showed similar protein selectivity for the fractions adsorbed in both conditions.

Adsorption↗

PubMed1997

From gel filtration to adsorptive size exclusion.

Adsorption and size exclusion in starch and cross-linked dextran were phenomena discovered in Uppsala in the 1950s [Porath (1979), Biochem. Soc. Trans. 7, 1197; Porath (1981), Current Content 19, 21; Porath (1981), J. Chromatogr. 218, 241; Janson (1987), Chromatographia 23, 361; Laurent (1993), J. Chromatogr. 633, 1]. These discoveries were the background to the development of a variety of affinity chromatographic methods. At present attempts are being made to combine size exclusion chromatography (SEC) with adsorption into a single operation that we call adsorptive SEC (AdSEC).

Adsorption↗

PubMed1996

Cyanocarbons as ligands for electron donor acceptor chromatography of human serum proteins.

The adsorption of human serum proteins to mono-, di- and tri-cyanocarbon ligands was studied in the presence of the water-structuring salt Na2SO4. All of the ligands adsorbed proteins to varying degrees when coupled to agarose via a divinylsulfone-derived spacer arm (DVS), whereas an insignificant or very low protein adsorption capacity occurred when the ligands were coupled through the bisoxirane-derived spacer arm. Studies of the DVS-coupled cyanocarbons showed that adsorption capacity increases with the number of cyano substituents carried by the ligand. The selectivity toward human serum proteins appeared to be similar to that of other electron acceptor ligands, but different from that of hydrophobic ligands. Tricyanoaminopropene-DVS-agarose was found to be the most potent protein adsorbent.

Blood Proteins↗

PubMed1996

Purification of recombinant insect transferrin from large volumes of cell culture medium using high capacity Ni(2+)-dipicolylamine gel.

We report the purification of secreted recombinant Manduca sexta transferrin from Spodoptera frugiperda (Sf9) cell culture medium in a single step using high capacity Ni(2+)-dipicolylamine (DPA)-Novarose gel. Although the original sample was highly diluted (approximately 10 micrograms transferrin/ml medium) and the cell culture medium contained 10% surfactant (Pluronic F68) and a lipid emulsion, we were able to recover the recombinant transferrin (1 mg protein/100 ml) under gentle elution conditions with 70% yield at > 90% homogeneity. This work demonstrates the versatility of immobilized metal ion affinity chromatography using a high metal ion capacity gel to purify a recombinant protein and illustrates the potential of this affinity technique for protein separations from large volumes of cell culture media that contain surfactants.

Animals↗

PubMed1995

Rapid one-step purification of goat immunoglobulins by immobilized metal ion affinity chromatography.

A rapid, single step purification of immunoglobulins from goat serum was achieved using immobilized metal ion affinity chromatography (IMAC) on a new high capacity gel, Novarose, coupled to tris(2-aminoethyl)amine (TREN) chelated with copper. When goat serum was adsorbed to this gel in buffer pH 7 at 11 cm/h (8.6 ml/h), the immunoglobulin fraction was recovered in a decreasing linear pH gradient at about pH 5.5. When the adsorption buffer was adjusted to pH 6.0 and the linear velocity increased to 110 cm/h (221 ml/h), an immunoglobulin fraction of greater than 95% homogeneity was obtained. Protein purity was assessed by silver-stained native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The capacity of the gel for immunoglobulins was 17 mg immunoglobulin/ml at the low flow rate with adsorption at pH 7 and 15 mg immunoglobulin/ml at the high flow rate with adsorption at pH 6. No problems of back pressure or gel compression were observed at the higher linear velocity. The mild elution pH, high flow rate, and synthetic nature of the ligand support make this new metal-chelating gel a powerful alternative to the use of other currently available commercial gels commonly used for immunoglobulin purification.

Animals↗

PubMed1995

Amphiphilic agarose-based adsorbents for chromatography. Comparative study of adsorption capacities and desorption efficiencies.

A number of hydrophobic derivatives attached to cross-linked agarose were studied as protein adsorbents. Differences in the adsorption and desorption behaviour were determined as functions of type and concentration of selected salts. Whereas octyl- and phenyl-Sepharose adsorb serum albumin preferentially, pyridyl-S-agarose shows a much stronger preferential affinity for IgG in the presence of high concentrations of lyotropic salts, such as sulphates. In contrast to pyridyl-S-agarose, a large portion of proteins remained fixed to octyl- and phenyl-Sepharose after extensive washing with 1 M NaOH.

Adsorption↗

PubMed1995

Rapid and efficient isolation of transferrin and ferritin from Manduca sexta.

We report methods for the rapid purification of two iron-binding proteins from larval hemolymph of Manduca sexta. Ferritin was purified in two steps by density gradient ultracentrifugation. To accomplish this, we utilized the relatively high level of ferritin present in the hemolymph of this animal and augmented the density of the protein in vivo by injection of iron sulfate. Nitrocellulose blots analyzed by laser densitometry showed hemolymph from iron-injected insects contained about 0.4 mg of ferritin per ml (approximately 0.7% of total hemolymph protein); of this, 62% was found as pure ferritin in the pellet formed during ultracentrifugation. Following the density ultracentrifugation, we purified transferrin from the hemolymph subphase by immobilized metal ion affinity chromatography using a new gel, Novarose-SE1000/40 coupled to dipicolylamine (DPA) chelated with nickel. Higher capacity Ni2+DPA-gel permitted good resolution of transferrin in the first chromatography; a lower capacity of the same gel allowed purification of transferrin in a second step. Overall transferrin recovery was 52%. Larval hemolymph contained 0.770 mg transferrin/ml, representing about 1.3% of the total protein.

Animals↗

PubMed1994

Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes.

Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid residues, His84 and His85, that are suitably positioned for coordination to Ni(II)-IDA-agarose. This particular structural motif is lacking in GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineering afforded a mutant enzyme that displays affinity for Ni(II)-IDA-agarose, in contrast to the wild-type GST M1-1. The results identify a distinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins.

Amino Acid Sequence↗

PubMed1994

Immobilization of beta-galactosidase on metal-chelate-substituted gels.

The use of copper, zinc, iron, nickel and calcium in three different chelating gels was investigated for preparing immobilized beta-galactosidase. The chelated ligands [Cu(2+)-iminodiacetate (IDA), Cu(2+)-Tris(carboxymethyl)ethylenediamine (TED), Ni(2+)-IDA and Fe(3+)-IDA] absorbed the protein so strongly that it can be considered a true immobilization. The obtained enzyme derivatives were investigated with regard to activity and stability. Enzymic activity was highly preserved in general for the TED derivates (90% when compared with that for Cu(2+)-TED). The immobilized Ni2+ derivatives were more stable to high temperature and to storage than the Cu2+ derivatives. Temperature-stability of the immobilized enzyme was very much improved by adding a strong metal-chelating gel such as carboxymethylated tetraethylenepentamine-agarose. The gel could be re-used and reloaded after elution with chelator. beta-Galactosidase from Escherichia coli was purified using immobilized-metal-ion-chelate chromatography (i.m.a.c.). The potential use of beta-galactosidase immobilized on i.m.a.c. gels for technical purposes is discussed.

Chelating Agents↗

PubMed1992

Purification and determination of the binding site of lactate dehydrogenase from chicken breast muscle on immobilized ferric ions.

Lactate dehydrogenase from chicken breast muscle was purified to homogeneity in one step by immobilized metal ion affinity chromatography. The purified enzyme was used to localize the binding site to immobilized Fe(III) ions. After cyanogen bromide degradation and digestion with trypsin, small enzyme fragments capable of binding to immobilized Fe(III) ions were obtained. It is proposed that several histidyl groups are involved in the binding.

Amino Acid Sequence↗

PubMed1992

Selective adsorption of immunoglobulins and glucosylated proteins on phenylboronate-agarose.

Aminophenylboronate-substituted agarose in 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid, pH 8.5, selectively adsorbs immunoglobulins and complement factors C3 and C4 from human serum. The selectivity of binding is strongly influenced by the presence of magnesium chloride in the sample buffer. Adsorbed immunoglobulins are quantitatively eluted by sorbitol, but only partially by ethylene glycol or methylcellosolve. Aniline-agarose of a similar degree of substitution shows only weak adsorption of serum proteins under similar experimental conditions, thus indicating the important contribution of the boronate moiety to this interaction. Immunoglobulin adsorption seems not to be due to the cis-diol complexation used extensively for the chromatographic determination of non-enzymatically glucosylated proteins. Hydrophobic and pi-pi interactions with the aromatic structure of the ligand seem also to contribute to protein binding. The behaviour of aminophenylboronate-liganded agarose is, in some respects, rather similar to that of the so-called "thiophilic adsorbents".

Adsorption↗

PubMed1992

Separation of insect hemolymph proteins by cascade-mode multi-affinity chromatography.

The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multi-affinity chromatography (CASMAC) on a main-line tandem column chain containing Zn(2+)-TED, T-gel, Ni(2+)-DPA, and phenylsepharose and a side-line column containing Zn(2+)-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph.

Animals↗

PubMed1992

Purification of a novel heterodimer from shark (Carcharhinus plumbeus) serum by gel-immobilized metal chromatography.

1. Resolution of the fraction of sandbar shark (Carcharhinus plumbeus) serum that was soluble in 50% saturated ammonium sulfate by gel-immobilized metal-affinity chromatography allowed the isolation of a novel disulfide-bonded heterodimer of intact mass 70 kDa. 2. Following reduction, the molecule could be resolved into two chains of apparent mass 36 and 24 kDa. 3. The molecules were glycoproteins as determined by an observed reduction in molecular weight following enzymatic glycosylation. 4. The two separate chains were related to one another on the basis of amino-acid composition analysis and by comparison of the N-terminal amino acids (seven out of 10 identities). 5. The exact relationship of this molecule to characterized heterodimers of higher vertebrates is unknown. 6. Cross-linked agarose-acetate was synthesized and proved to be an efficient concentrating agent and also a hydrophobic interaction adsorbant.

Amino Acid Sequence↗

PubMed1992

Immobilized metal ion affinity chromatography.

The introduction of immobilized metal ion affinity chromatography, directed toward specific protein side chains, has opened a new dimension in protein purification. This review covers the principles and practice of IMAC that can be performed under very mild, nondenaturing conditions. IMAC is particularly suitable for preparative group fractionation of complex extracts and biofluids, but can also be used in high-performance mode: "HP-IMAC." Single-step purifications of 1000-fold or more may allow isolation of a particular protein from crude extracts on a milligram or gram scale. With respect to separation efficiency, IMAC compares well with biospecific affinity chromatography, and the immobilized metal ion ligand complexes are more likely to withstand wear and tear than are antibodies or enzymes. The enormous potential of IMAC and related metal affinity techniques is only in the initial stages of being explored and exploited. Synthesis of IMA adsorbents, and various modes of performing IMAC are discussed and exemplified with selected applications. Advantages and disadvantages are listed. Effective means of counteracting the few undesirable effects that can occur are suggested.

Chromatography, Affinity↗

PubMed1991

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.

Animals↗

PubMed1991

Thiophilic adsorbents for RIA and ELISA procedures.

Three types of agarose derivatives have been prepared and investigated as adsorbents for radioimmunoassay and ELISA analysis. The analytical systems were evaluated using beta 2 microglobulin as a model. After a competitive reaction between the immunocomponents in solution, the formed immune complexes were adsorbed onto the adsorbent in the presence of 0.5 M potassium sulfate in 0.1 M Tris, pH 7.5. The binding constant between the interaction site on human IgG and the adsorbent 3-(2-pyridylthio)-2-hydroxypropylagarose (Py-S-gel) was determined to be 1.5 x 10(7) M-1 and the binding capacity was 20 mg/ml gel. The immune complex was desorbed by deleting potassium sulfate from the buffer, and only 0.5% of the total applied protein remained after washing the adsorbent with 0.5 M NaOH. The same adsorbent can be used repetitively with different systems.

Animals↗