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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.

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(1) A new concept for producing soluble polymeric affinity ligands is proposed and exemplified. By solid-phase synthesis, an insoluble hydrophilic polymer is converted into an affinity gel. The gel is hydrolytically degraded to water-soluble affinity polymeric ligands which are recovered and purified. (2) A water-soluble biopolymeric metal-affinity carrier based on an iminodiacetic acid (IDA) derivative of dextran has been synthesized through the modification of Sephadex G-200 by IDA, followed by hydrolysis with dextranase and size-exclusion-chromatographic purification of the high-molecular-mass fragments. (3) The molecular size of the soluble products as a function of hydrolysis time with dextranase from Penicillium sp. was determined. The range of molecular size of the biopolymeric chelating ligand varies from around 200 Da to greater than 580 kDa. (4) The influence of three metal ions chelated with the Sephadex derivative on the hydrolysis rate and the molecular-size distribution of end products was studied. Eu3+ was found to improve the rate of solubilization. Ni2+ and Cu2+ decreased the hydrolysis rate, as compared with that of the metal-free IDA-Sephadex. (5) The method introduced here has the potential of being developed and applied as a general technology for synthesis of soluble multifunctional affinity ligands. Such ligands should be useful for liquid-phase extraction as well as for the synthesis of adsorbents with localized multiple binding sites. Other possible fields of applications are to be found in medicine, where they could be used for slow drug delivery or detoxification, and in analytical chemistry, where they could be used in various assays.

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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).

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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.

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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.

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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.

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The interaction of immobilized metal-chelating adsorbents with a dual heterobifunctional soluble polyethylene glycol (PEG) of the form X-PEG-Y is described, where X represents an affinity ligand and Y a chelating agent. The bifunctional PEG derivative used in this study was biotin-PEG-iminodiacetic acid (IDA). Affinity and metal binding constants of this conjugate for copper and avidin were found to be in excellent agreement with the binding affinities of the corresponding unconjugated groups IDA and biotin, respectively. The characteristics of the interaction of this bifunctional derivative is described in terms of its adsorption in immobilized metal affinity chromatographic (IMAC) adsorbents. The results show that this derivative can be reversibly and selectively bound to specific IMAC adsorbents under certain experimental conditions. This immobilized scheme resembles a system where an IMAC adsorbent was transformed into an affinity adsorbent as a result of the interactions of both chelating derivatives, one in solution (biotin-PEG-IDA) and the other on the solid matrix (IMAC adsorbent). Apparently the modified IMAC adsorbents, once the affinity chelating ligands are attached, exhibit characteristics similar to those of covalently bound affinity ligands in affinity chromatographic systems.

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Vertebrate transferrin is a well characterized iron transport protein. In contrast, little is known concerning the role of transferrin in insects. Yet, study of iron metabolism in insects could give insights into strategies for insect control, particularly for insects that transmit disease.

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We report the fractionation of calcium-binding proteins using immobilized metal ion affinity chromatography (IMAC) with hard metal ions. Various hard metal ions (Mn2+, La3+, Nd3+, Eu(3 were immobilized on cross-linked agarose substituted with Tris(carboxymethyl)ethylenediamine (TED) and used as an adsorbent. After systematic studies, europium was selected for further work on the fractionation of calcium-binding proteins. It was found that the presence of Ca2+ in the sample and the solvent strongly promoted the adsorption and selectivity. Selective elution was accomplished in stepwise mode by the addition of calcium chelators such as malonate, citrate and phosphate. Calmodulin of high purity was isolated from a crude extract. Similar behavior of other calcium-binding proteins indicates that the reported chromatographic procedure can be generally applied to such proteins.

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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.

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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.

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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.

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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.

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An adsorbent for metal ions has been prepared by reacting high molecular weight polyethyleneimine (PEI) with a crosslinked and activated agarose gel, Novarose. The synthesis variables, i.e. time, temperature, pH, PEI concentration and PEI/Novarose ratio, were optimized in order to obtain a high metal binding capacity of the adsorbent. The binding capacity for Cu(2+) is 500 micromol/ml packed adsorbent. A number of properties of the adsorbent relevant for metal ion accumulation has been investigated for Cu(2+), Ni(2+), Cd(2+) and Zn(2+). Adsorption capacities, adsorption isotherms, distribution coefficients, recoveries and relative rates of accumulation were determined. The adsorbent can be used for preconcentration and for separation of interfering alkali and alkaline earth metals in analytical applications.

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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.

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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.

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The DNA sequence of a 359-bp fragment of nifH was determined for the heterocystous strains Anabaena sp. strain CA (ATCC 33047), Nostoc muscorum UTEX 1933, a Nostoc sp., Gloeothece sp. strain ATCC 27152, Lyngbya lagerheimii UTEX 1930, and Plectonema boryanum IU 594. Results confirmed that the DNA sequence of the 359-bp segment is sufficiently variable to distinguish cyanobacterial nifH genes from other eubacterial and arachaeobacterial nifH genes, as well as to distinguish heterocystous from nonheterocystous nifH genes. Nonheterocystous cyanobacterial nifH sequences were greater than 70 and 82% identical on the DNA and amino acid levels, respectively, whereas corresponding values for heterocystous cyanobacterial nifH sequences were 84 and 91%. The amplified nifH fragments can be used as DNA probes to differentiate between species, although there was substantial cross-reactivity between the nifH amplification products of some strains. However, an oligonucleotide designed from a sequence conserved within the heterocystous cyanobacteria hybridized primarily with the amplification product from heterocystous strains. The use of oligonucleotides designed from amplified nifH sequences shows great promise for characterizing assemblages of diazotrophs.

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Amyloglucosidase from Halobacterium sodomense was purified by a combination of hydrophobic interaction chromatography and immobilized metal ion affinity chromatography at analytical and preparative scale with 75% recovery. The enzyme was found to be a dimer of two different subunits with molecular weights of 72,000 and 82,000 D, respectively, combining in a 175,000 D native protein. The specific activity, KM, and amino acid composition of the enzyme was determined.

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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.

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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.

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