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Biomedical subjects

J Porath

Publications and source records attributed to J Porath.

At least 55 records · Page 3Linked to original sources
PubMed1987

Thiophilic adsorption: a comparison of model protein behavior.

A newly recognized type of protein-ligand interaction phenomenon has resulted in the preparation of simple, nonionic, and highly specific gel derivatives for selective adsorption chromatography. The essential structure of the immobilized ligand can be represented as agarose-CH2CH2SO2CH2CH2SCH2CH2OH, which was prepared by using mercaptoethanol to derivatize [0.9-1.0 mmol (g of dry gel)-1] divinyl sulfone activated agarose (thiophilic or T-gel). Proteins interacting with this ligand are provisionally termed "thiophilic" to recognize their affinity for the definitive sulfone-thioether constituents. To better understand the experimental variables affecting adsorption efficiency and selectivity, several well-characterized proteins with diverse physicochemical features have been evaluated for thiophilic properties. Thiophilic interaction chromatography was investigated as a function of pH as well as the type and concentration of water-structure-forming salts required to promote adsorption. The model proteins characterized varied distinctly in their individual thiophilic affinities. At acidic pH values, a salt-independent adsorption process was observed. Furthermore, a minimum in the salt-promoted thiophilic adsorption tendency at pH 5-6 was found, with varying magnitude, for each of the model proteins evaluated. Recovery of adsorbed proteins routinely varied from 90% to 100%. There does not appear as yet to be any easily recognized physicochemical property associated with either thiophilic or nonthiophilic behavior. These results suggest that thiophilic interaction chromatography is a process that utilizes a previously unrecognized protein-ligand interaction mechanism. We suggest that salt allows the protein into close proximity with the sulfone-thioether group where short-range forces are effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1987

A one-step purification method for monoclonal antibodies based on salt-promoted adsorption chromatography on a 'thiophilic' adsorbent.

A convenient and fast method for the purification of mouse monoclonal antibodies from the culture media of cloned cells or from ascites fluids by means of salt-promoted chromatography on a 'thiophilic' adsorbent is described. The adsorbent has a capacity to adsorb about 20 mg/ml of immunoglobulins and a broad specificity towards immunoglobulins derived from various animal species irrespective of the type or subclass to which they belong. The recovery of the purified IgG is better than 90% while that for IgM is considerably less, probably due to dissociation occurring during the adsorption-desorption process. This one-step procedure also leads to a considerable concentration of dilute solutions of immunoglobulins. Moreover, the purified Igs are eluted by an essentially salt-free buffer at near neutral pH thus obviating the need for post-treatment of the sample before storage or subsequent conjugation to enzymes for use in immunoassays. This purification method is also well suited for large-scale operations since sample preparation requires only the addition of 0.5 M K2SO4 to the ascited fluid or cell culture medium. The degree of purification obtained is, in certain instances, comparable to that obtained by biospecific affinity chromatography based on antigen-antibody interactions. In contrast to immunosorption and desorption methods, however, there is no risk of contamination of the immunoglobulins purified on the 'thiophilic' adsorbent by foreign proteins.

Animals↗
PubMed1987

Interaction of proteins with immobilized Cu2+. Quantitation of adsorption capacity, adsorption isotherms and equilibrium constants by frontal analysis.

The interaction of lysozyme, ovalbumin, bovine and pig serum albumins with Cu2+ immobilized on Chelating Sepharose Fast Flow or TSK gel chelate-5PW was studied by frontal analysis at various initial concentrations of these solutes. The chromatographic data so obtained served as a basis for evaluating some relevant affinity chromatography parameters by adapting previously reported equations to this system. The TSK-based adsorbent had lower adsorption capacity for all the model proteins compared to the agarose-based adsorbent, due primarily to its lower porosity which has a marked influence on the accessibility of the immobilized ligand to the proteins. On the other hand, the TSK-based adsorbent offers almost ideal conditions for studying adsorption equilibria under column chromatographic conditions. The adsorption capacity of these adsorbents for the model proteins ranges from about 0.6 to 7 mumol/ml, equivalent to 40-100 mg/ml, of adsorbent. The following equilibrium constants for the interaction of the proteins with immobilized Cu2+ were obtained: lysozyme, 1.8.10(4); ovalbumin, 1.5.0(5); BSA, 1.7.10(5); PSA, 3.7.10(5) and imidazole, 8.10(3) M-1. Despite the comparatively low affinity of imidazole for the adsorbent, it is an effective competing ligand, at comparatively high concentrations, for adsorbed proteins primarily because all adsorption sites are available to it. The results obtained suggest that about 1/3 to 1/2 of the potential adsorption sites on the model proteins are involved in forming coordination complexes with Cu2+ immobilized to covalently bound iminodiacetate groups on insoluble gel matrices.

Adsorption↗
PubMed1987

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.

Chromatography, Affinity↗
PubMed1986

Thiophilic adsorption of immunoglobulins--analysis of conditions optimal for selective immobilization and purification.

Immunoglobulins have been selected by their general affinity for adjacent sulfone-thioether sulfur groups as a useful model system for the characterization of thiophilic interaction chromatography. Mercaptoethanol coupled to divinylsulfone-activated agarose (thiophilic or T-gel) provided an affinity matrix for the efficient and reversible immobilization of the immunoglobulins. The adsorption/desorption process was investigated as a function of protein concentration, temperature, flow rate, and pH in different concentrations of ammonium sulfate. Immobilization of these proteins was (as a function of pH) found to be both dependent and independent of the adsorption-promoting effects of water-structure-forming salts. Buffer conditions are recommended for the selective adsorption of immunoglobulins from unfractionated human serum. These results indicate that thiophilic interaction chromatography provides a new and effective alternative for the immobilization and purification of immunoglobulins and other proteins under conditions known to preserve structure and biological activity.

Chromatography, Affinity↗
PubMed1986

Selective adsorption of phosphoproteins on gel-immobilized ferric chelate.

Ferric ions are very strongly adsorbed to iminodiacetic acid substituted agarose. This firmly immobilized complex acts as a selective immobilized metal affinity adsorbent for phosphoproteins. Chromatography based on this principle is illustrated by the adsorption-desorption behavior of egg yolk phosvitin before and after dephosphorylation as well as by the change in the chromatographic pattern before and after enzymic phosphorylation of selected histones. The strength of binding is dependent on the phosphate content. The difference in binding before and after phosphorylation of a single amino acid residue is demonstrated. Affinity elution can be accomplished by inclusion in the buffer of phosphoserine or a displacing metal ion such as Mg2+.

Amino Acids↗
PubMed1986

Salt-promoted adsorption: recent developments.

Efficient fractionation of human serum proteins is accomplished by use of a series of tandem-coupled beds of group-affinity adsorbents. The general fractionation strategy for group fractionation of a complex protein mixture is discussed.

Adsorption↗
PubMed1986

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.

Animals↗
PubMed1986

Epidermal growth factor from deer (Cervus elaphus) submaxillary gland and velvet antler.

Epidermal growth factor (EGF)-like activity was isolated for the first time from the submaxillary gland (SMG) and the velvet antler of red deer (Cervus elaphus) by a combination of Sephadex gel or DEAE-Sephacel and IMAC columns in succession. The semipurified cervine EGF-like activity (cEGF), with specific activity of 4.7 ng/micrograms protein from the velvet tissues, can generate a completely parallel competitive binding curve against mouse EGF in both radioreceptor assay (RRA) and radioimmunoassay (RIA). Mitogenic activity of EGF from both tissues was demonstrated by stimulating the incorporation of [3H]thymidine in two different cell lines of fibroblast culture in a dose-dependent manner. The velvet layer may be the site of EGF synthesis outside the SMG.

Animals↗
PubMed1985

Isolation of the epidermal growth factor from the shrew submaxillary gland.

The epidermal growth factor (EGF) from the submaxillary glands of the Chinese Shrew (Suncus murinus) is purified to apparent homogeneity by using a sequence of four chromatographic steps, viz. gel filtration on Sephacryl S-200, affinity chromatography on immobilized Ni, hydrophobic interaction on phenyl-Sepharose CL-4B and reverse-phase HPLC. An 800-fold increase in specific activity and an overall recovery of 46% were achieved. The most effective step in its purification is the successful use of immobilized metal ion affinity chromatography (IMAC). This method was very selective, reproducible and requires a minimum of sample pre-treatment prior to chromatography.

Animals↗
PubMed1985

Thiophilic adsorption--a new method for protein fractionation.

Divinylsulphone-activated agarose to which mercaptoethanol is coupled showed very selective group adsorption of human serum proteins, in particular the immunoglobulins. The adsorption increases markedly in the presence of high concentrations of neutral water-structure forming salts and is distinct from adsorptions based on hydrophobic interaction. A characteristic feature of this new type of adsorbent is the structure of the groups attached to the polymer, P, i.e., R-S-CH2-CH2-SO2-CH2-CH2-O-P, where R is a small aliphatic residue. Our results indicate that the thioether sulphur and the adjacent sulphone group act cooperatively and are apparently necessary to maintain the distinct behaviour of such absorbents.

Adsorption↗
PubMed1985

Synthesis of new hydrophobic adsorbents based on homologous series of uncharged alkyl sulphide agarose derivatives.

A homologous series of uncharged thioalkyl derivatives of agarose were prepared by a simplified synthetic route and their adsorption behaviour towards human serum proteins was evaluated and compared with that of a commercially available alkyl ether derivative of agarose. The influence of the spacer arm length on the adsorption efficiency was also investigated. The degree of substitution of the derivatives can be estimated conveniently by sulphur analysis. The four different types of thiolkyl derivatives (C6, C8, C12 and C14) investigated here behave in all respects like hydrophobic adsorbents. The coupling yield obtained is high (75% or more) and is better than that obtained by alternative synthetic routes reported so far. The adsorption capacity towards serum proteins of the various derivatives increases with increasing alkyl chain length and degree of substitution. Desorption is achieved by a progressive decrease in the polarity of the eluent and the recovery of the applied material is in the range 80-90%. The role played by the thioether as a possible modulator of the observed hydrophobic adsorption is discussed. For the group separation of serum proteins the optimum adsorbent, as regards capacity combined with ease of elution of adsorbed material, should be substituted with chains of six or eight carbon atoms and have a ligand concentration in the range 80-120 mumole g-1 dry gel.

Adsorption↗
PubMed1985

Fe3+-hydroxamate as immobilized metal affinity-adsorbent for protein chromatography.

The adsorbent glycinehydroxamate-Sepharose 6B, charged with Fe3+ under specified conditions, is reported. It was used at various pH values for chromatography of the following proteins: lysozyme, cytochrome c, avidin, bovine pancreatic RNase, myoglobin, ovalbumin and human serum albumin. The common naturally occurring amino acids were tested for their interactions with the new sorbent under neutral conditions.

Adsorption↗
PubMed1985

Separation of serum proteins on a Fe3+-monohydroxamate adsorbent.

The Fe3+-monohydroxamate adsorbent was tested by chromatography of increasing volumes of serum proteins. The specifically bound proteins were displaced in a stepwise manner by increasing the sodium chloride concentration. The resolved chromatographic fractions were further analysed by gel electrophoresis. A comparison between the properties of Fe3+-monohydroxamate and commercially available adsorbents is presented.

Blood Proteins↗
PubMed1985

A high-capacity hydrophobic adsorbent for human serum albumin.

A simple method, based on salting out hydrophobic interaction chromatography, for the efficient removal of trace amounts of serum albumin from partially purified protein preparations is described. The method is also successfully applied for the purification of albumin from Cohn fraction IV, a by-product obtained from the commercial fractionation of human serum proteins by the ethanol precipitation procedure. About 70% of the adsorbed albumin can be eluted by buffer of low ionic strength and can thus be lyophilized directly, if required. The adsorbent can be used for several cycles of adsorption and desorption without affecting its selectivity or capacity. Its adsorption properties and capacity for serum albumin are compared with those of the commercially available adsorbent Blue Sepharose CL-6B.

Adsorption↗
PubMed1983

Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions.

Immobilized metal ion affinity adsorption (IMA adsorption) is a collective term that is proposed to include all kinds of adsorptions whereby metal atoms or ions immobilized on a polymer cause or dominate the interaction at the sorption sites. IMA chromatography is one of the most powerful methods available to date for protein fractionation although this is not as yet widely recognized. This study deals with the theoretical aspects of IMA adsorption and its practical applications as exemplified by the various results reported here. The synthesis of iminodiacetate-substituted agarose (IDA-agarose) and tris(carboxymethyl)ethylenediamine-agarose (TED-agarose) is described. Many types of metal ions can easily be immobilized on these gel derivatives to form IMA adsorbents. We have not observed any damage to the proteins during the adsorption-desorption process. After performance of an experiment, the gels can easily be regenerated and can be loaded with the same or a different metal ion for an ensuing experiment. Specific adsorption is demonstrated for serum proteins on immobilized Ni(II) and Fe(III). Ligand-specific desorption (affinity elution) is also demonstrated by including in the buffer system certain solutes which are similar to or identical with some particular amino acids found in proteins. High concentrations of certain salts that affect the structure of water, such as Na2SO4, promote coordinate covalent bonding of proteins by a mechanism that is apparently similar to that found in hydrophobic interactions. Neutral detergents and aquoorganic solvents may be used. This opens up the possibility for the fractionation of membrane components. The IMA-adsorption method could also be expanded to other areas besides protein fractionation.

Adsorption↗