Adsorption behavior of bovine serum albumin on lowly activated anionic exchangers suggests a new strategy for solid-phase proteomics

Diluted solutions of bovine serum albumin (BSA) (e.g., 0.1 mg /mL) do not form detectable protein large aggregates. Using gel-filtration experiments, we determined that a diluted solution of BSA is 97% monomeric BSA and 3% dimeric. The adsorption of this diluted BSA on highly activated anionic exchangers (e,g., having 40 micromol/wet g) keeps this mainly monomeric form. When supports activated with 2 micromol/wet g are used, only dimers become adsorbed to the support, accounting for 100% of the offered BSA. When the diluted BSA solution is offered to very mildly activated anionic exchangers (even only 0.125 micromol/wet g), an unexpected adsorption of most of the BSA on the support was also observed. These very slightly activated supports are only able to adsorb very large proteins or very large protein-protein complexes, larger than BSA dimers. In fact, a rapid cross-linking of the adsorbed BSA with dextran-aldehyde reveals the formation of very large BSA-BSA complexes with molecular mass higher than 500 000 Da, complexes that may be observed for soluble BSA with very high concentrations but are not detectable at 0.1 mg/mL. Moreover, the size of the aggregates strongly depends on the concentration of the ionized groups on the support: the less activated the supports are, the higher the sizes of the complexes. It seems that the interaction of the BSA molecules on the margins of the BSA aggregate with the groups on the support may stabilize the whole protein aggregate, although some components are not interacting with the support. Aggregates could account for more than 40% of the BSA in the solution after 50 h of incubation. However, only these large BSA aggregates were adsorbed in the support.     

Purification, stabilization, and concentration of very weak protein-protein complexes: Shifting the association equilibrium via complex selective adsorption on lowly activated supports

Very weak protein-protein interactions are very difficult to detect because these complexes could be under the detection limit or they tend to dissociate. Here, using as a model the antibody-antigen interaction weaken by the presence of dioxane, we have shown a strategy for the protein complexes purification by selective adsorption of the associated proteins. This strategy is based on the use of poorly activated anionic exchanger supports to selectively adsorb large complexes. This selective adsorption of the associated proteins shifted the association equilibrium of the soluble proteins toward the associated form. Thus, in the presence of 15% v/v dioxane, a concentration that is able to almost fully break the immunocomplex (less that 3% of the immunocomplex appeared associated when soluble antigen-antibody mixture was cross-linked with aldehyde-dextran), we can obtain more than 90% of the fully pure immunocomplex from the non-associated protein, adsorbed on anionic exchanger supports having a very low activation. This simple strategy may be a very useful tool to solve one of the most relevant challenges in the modern proteomics, the detection of very weak protein-protein interactions.     

Simple purification of immunoglobulins from whey proteins concentrate

We have developed a new protocol with only two steps for purification of immunoglobulins (Ig) from a protein concentrate of whey. Following this protocol, we have an 80% recovery of immunoglobulins, fairly pure. The purification was achieved by eliminating the BSA, via a strong adsorption on DEAE-agarose. Full desoprtion of the other serum proteins could be achieved without contamination with BSA. Thus, a protein solution containing only Ig and very small proteins (e.g., beta-lactoglobulins and alpha-lactalbumin) was obtained. Offering this protein mixture to a lowly activated aminated support, only Ig adsorbed on the support. It has been shown that BSA is able to interact with other proteins (including Ig and lactalbumins). This ability to form complexes with other proteins prevented the success of the direct adsorption of Ig on this mildly activated support, even although Ig should be the largest protein presented in dairy whey.     

The adsorption of multimeric enzymes on very lowly activated supports involves more enzyme subunits: Stabilization of a glutamate dehydrogenase from Thermus thermophilus by immobilization on heterofunctional supports

Glutamate dehydrogenase (GDH) from Thermus thermophilus is a homotrimeric enzyme that tends to dissociate at acidic pH values. GDH is readily adsorbed on highly activated anionic exchangers (HAAE), but hardly adsorbed on lowly activated supports (LAAE) or on highly activated epoxy supports. When using amino-epoxy supports, GDH immobilized on HAAE-epoxy and more slowly on LAAE-epoxy supports. Both immobilized biocatalysts were incubated at pH 10 for different times to increase the multipoint covalent attachment. LAAE-epoxy-GDH was stable at pH 4 and 25 °C, the enzyme stability did not depend on the enzyme concentration and did not release any subunit to the supernatant, in opposition to the results obtained using HAAE-epoxy supports. The general application of this strategy to stabilize multimeric enzymes was verified by immobilizing a crude protein extract. It seems that proteins adsorb on LAAE by the larger region of their surface (that is the one that involves the highest number of enzyme subunits), since it is the only area large enough to permit a multipoint ionic exchange on this LAAE. On the contrary, using HAAE, some proteins may become adsorbed by clusters that were rich in anionic groups and located in a corner of the multimer, involving only some of the subunits in the enzyme immobilization. That way, a careful design of the design of the support permits to take full advantage of the immobilization on heterofunctional supports.     

The co-operative effect of physical and covalent protein adsorption on heterofunctional supports

It has been found that the enzymes penicillin G acylase from Escherichia coli (PGA) and lipase from Bacillus thermocatenulatus (BTL) did not significantly adsorb on highly activated amino-agarose beads at pH 7 (a support where 85–90% of a crude extract of proteins become adsorbed). Moreover, it has been found that these enzymes do not covalently immobilize on highly activated epoxy-agarose beads at pH 7. However, both enzymes slowly immobilize on heterofunctional supports having a high density of amino–epoxy groups. The immobilized enzymes retain a high percentage of activity (more than 90% for PGA and 60% for BTL). On the other hand, the immobilization of a crude extract of proteins on amino–epoxy supports under conditions where only a limited protein ionic exchange was permitted (by using high ionic strength or lowly activated supports), also permitted a similar high immobilization yield of the proteins. Similarly, glutamate dehydrogenase (GDH) and β-galactosidase from Thermus thermophilus can be fully immobilized under conditions where less than 20% of these enzymes can be ionically exchanged in the aminated support. The results suggested that the percentage of proteins that may be physically adsorbed on the support becomes irreversibly immobilized by the covalent reaction between the nucleophilic groups in the protein surface and the very near epoxy groups of the support (in an almost intramolecular reaction). Thus, using these supports, it is possible to immobilize almost all the proteins by anionic exchange, that is, the area with the highest density in anionic groups. In many cases, this region could not correspond to the protein regions usually utilized to immobilize proteins. This way, it is possible to achieve, in a very simple fashion and without modifying the protein, new orientations of some immobilized enzymes and proteins.     

A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling

Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.     

Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate-dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate-dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic-dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (beta-galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermussp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.     

Mixed ion exchange supports as useful ion exchangers for protein purification: purification of penicillin G acylase from Escherichia coli

A support having similar amounts of carboxymethyl and amino groups has been prepared and evaluated as an ion exchanger. It has been found that this support was able to adsorb a high amount of protein from a crude extract of proteins (approximately 55%) at pH 5. Moreover, it was able to adsorb approximately 60% of the protein that did not become adsorbed on supports bearing just one kind of ionic groups. The use of divalent cations reinforced the adsorption of proteins on these supports. These results suggest that the adsorption of proteins on supports bearing almost neutral charge is not driven by the existence of opposite charges between the adsorbent and the biomacromolecule but just by the possibility of forming a high number of enzyme-support ionic bonds. This support has been used to purify the enzyme penicillin G acylase (PGA) from Escherichia coli. PGA was not significantly adsorbed at any pH value on either amino- or carboxyl-activated supports, while it can be fully adsorbed at pH 5 on this new carboxyl-amino matrix. Thus, we have been able to almost fully purify PGA from crude extracts with a very high yield by using these new supports.     

Genetic modification of the penicillin G acylase surface to improve its reversible immobilization on ionic exchangers

A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.     

Genetic Modification of the Penicillin G Acylase Surface To Improve Its Reversible Immobilization on Ionic Exchangers

A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.     

Overproduction of Thermus sp. Strain T2 beta-galactosidase in Escherichia coli and preparation by using tailor-made metal chelate supports

A novel thermostable chimeric beta-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the beta-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the beta-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.     

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports

In this work, we have used supports activated with m-amino-phenylboronic groups to “reversibly” immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.     

Determination of the contact energies between a regulator of G protein signaling and G protein subunits and phospholipase C beta 1

Cell signaling proteins may form functional complexes that are capable of rapid signal turnover. These contacts may be stabilized by either scaffolding proteins or multiple interactions between members of the complex. In this study, we have determined the affinities between a regulator of G protein signaling protein, RGS4, and three members of the G protein-phospholipase Cbeta (PLC-beta) signaling cascade which may allow for rapid deactivation of intracellular Ca(2+) release and activation of protein kinase C. Specifically, using fluorescence methods, we have determined the interaction energies between the RGS4, PLC-beta, G-betagamma, and both deactivated (GDP-bound) and activated (GTPgammaS-bound) Galpha(q). We find that RGS4 not only binds to activated Galpha(q), as predicted, but also to Gbetagamma and PLCbeta(1). These interactions occur through protein-protein contacts since the intrinsic membrane affinity of RGS4 was found to be very weak in the absence of the protein partner PLCbeta(1) or a lipid regulator, phosphatidylinositol-3,4,5 trisphosphate. Ternary complexes between Galpha(q), Gbetagamma and phospholipase Cbeta(1) will form, but only at relatively high protein concentrations. We propose that these interactions allow RGS4 to remain anchored to the signaling complex even in the quiescent state and allow rapid transfer to activated Galpha(q) to shut down the signal. Comparison of the relative affinities between these interacting proteins will ultimately allow us to determine whether certain complexes can form and where signals will be directed.     

Retention behaviour of proteins on poly(vinylimidazole)-copper(II) complexes supported on silica: application to the fractionation of desialylated human α1-acid glycoprotein variants

The retention behaviour of various amino acids, peptides and proteins on poly(vinylimidazole)-Cu(II) complexes supported on silica was investigated. Free amino acids and peptides containing one histidine and in some instances one additional tryptophan residue in their primary structure were found to elute from the supports only after addition of a competing complexing agent to the mobile phase. However, the results obtained with proteins containing metal binding groups suggested that, in addition to the presence of donor-acceptor interactions between the macromolecules and the immobilized metal, other additional (essentially ionic and/or hydrophobic) interactions took place between the proteins and the surrounding of the metal. When donor-acceptor interactions were predominant, proteins were strongly adsorbed on the stationary phase and their elution required the addition of a competing complexing agent in the mobile phase. However, when the binding between the proteins and the supports via donor-acceptor interactions was less favourable, proteins were eluted from the columns without the addition of a competing agent in the mobile phase. With respect to the binding of these proteins, ionic and/or hydrophobic interactions were no longer negligible during the chromatographic process and the retention of the macromolecules by the stationary phase depended on the elution conditions (ionic strength, pH, etc.). These supports were used in the fractionation of the three main genetic variants of desialylated α₁-acid glycoprotein.     

Improvement of the functional properties of a thermostable lipase from alcaligenes sp. via strong adsorption on hydrophobic supports

Lipase QL from Alcaligenes sp. is a quite thermostable enzyme. For example, it retains 75% of catalytic activity after incubation for 100 h at 55 °C and pH 7.0. Nevertheless, an improvement of the enzyme properties was intended via immobilization by covalent attachment to different activated supports and by adsorption on hydrophobic supports (octadecyl-sepabeads). This latter immobilization technique promotes the most interesting improvement of enzyme properties: (a) the enzyme is hyperactivated after immobilization: the immobilized preparation exhibits a 135% of catalytic activity for the hydrolysis of p-nitrophenyl propionate as compared to the soluble enzyme; (b) the thermal stability of the immobilized enzyme is highly improved: the immobilized preparation exhibits a half-life time of 12 h when incubated at 80 °C, pH 8.5 (a 25-fold stabilizing factor regarding to the soluble enzyme); (c) the optimal temperature was increased from 50 °C (soluble enzyme) up to 70 °C (hydrophobic support enzyme immobilized preparations); (d) the enantioselectivity of the enzyme for the hydrolysis of glycidyl butyrate and its dependence on the experimental conditions was significantly altered. Moreover, because the enzyme becomes reversibly but very strongly adsorbed on these highly hydrophobic supports, the lipase may be desorbed after its inactivation and the support may be reused. Very likely, adsorption occurs via interfacial activation of the lipase on the hydrophobic supports at very low ionic strength. On the other hand, all the covalent immobilization protocols used to immobilize the enzyme hardly improved the properties of the lipase.     

Oriented covalent immobilization of antibodies on physically inert and hydrophilic support surfaces through their glycosidic chains

Immobilization of antibodies by their oxidized sugar chain on aminated supports is a very efficient methodology to have a properly oriented antibody. However, these supports may behave as anionic exchangers, producing the unspecific adsorption of other proteins and reducing the selectivity of the system. To overcome this problem, we have proposed two solutions based in tailor-made support surfaces to immobilize antihorseradish peroxidase (HRP). The first solution was the use of supports having a very low amount of amino groups. These amino groups need to be very reactive with the aldehyde groups generated in the protein sugar chains to be efficient. Using supports having 7 micromol EDA/g (e.g., ethylenediamine modified glyoxyl-agarose), the antibody may be immobilized, keeping over 90% of the anti-HRP functionality. Second, by mixing amino groups and carboxylic groups, a neutral surface of the support has been generated. Again, this support has been unable to adsorb proteins while oxidized anti-HRP could be immobilized, giving functional anti-HRP antibodies. Both preparations retained 100% functionality after 2 months of storage at 4 degrees C. This way, the tailoring of the support surfaces has permitted solving some limitations of the immobilization of sugar-chain oxidized antibodies on primary amino supports.     

A novel heterofunctional epoxy-amino sepabeads for a new enzyme immobilization protocol: immobilization-stabilization of beta-galactosidase from Aspergillus oryzae

The properties of a new and commercially available amino-epoxy support (amino-epoxy-Sepabeads) have been compared to conventional epoxy supports to immobilize enzymes, using the beta-galactosidase from Aspergillus oryzae as a model enzyme. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. This support has both a great anionic exchanger strength and a high density of epoxy groups. Epoxy supports require the physical adsorption of the proteins onto the support before the covalent binding of the enzyme to the epoxy groups. Using conventional supports the immobilization rate is slow, because the adsorption is of hydrophobic nature, and immobilization must be performed using high ionic strength (over 0.5 M sodium phosphate) and a support with a fairly hydrophobic nature. Using the new support, immobilization may be performed at moderately low ionic strength, it occurs very rapidly, and it is not necessary to use a hydrophobic support. Therefore, this support should be specially recommended for immobilization of enzymes that cannot be submitted to high ionic strength. Also, both supports may be expected to yield different orientations of the proteins on the support, and that may result in some advantages in specific cases. For example, the model enzyme became almost fully inactivated when using the conventional support, while it exhibited an almost intact activity after immobilization on the new support. Furthermore, enzyme stability was significantly improved by the immobilization on this support (by more than a 12-fold factor), suggesting the promotion of some multipoint covalent attachment between the enzyme and the support (in fact the enzyme adsorbed on an equivalent cationic support without epoxy groups was even slightly less stable than the soluble enzyme).     

Preparation of inert magnetic nano-particles for the directed immobilization of antibodies

Various activated supports (cyanogen bromide, glutaraldehyde, epoxy-chelates, primary amino) were evaluated for the immobilization of IgG anti-horseradish peroxidase. Cyanogen bromide and glutaraldehyde supports greatly reduced the recognition capacity of the antigen, probably due to the incorrect orientation of the antibody on the support. Hetero-functional epoxy-chelate and immobilization by the sugar chain on primary amino groups had little effect on high recognition of the antigen (near to the theoretically expected value). However, the immobilization by the sugar chain resulted in a higher adsorption rate of horseradish peroxidase, possibly due to a favourable orientation on a flexible spacer arm). Antibodies immobilized on aminated surfaces showed two major drawbacks. Firstly, the biological activity of the immobilized antibody sharply decreased over several days when stored at low ionic strength, although this effect could be partially reversed by incubation at high ionic strength. Secondly, a high level of non-specific proteins adsorption on the support surface was observed. Both problems could be successfully resolved by controlling the coating of the support with aldehyde-aspartic-dextran. We propose that the loss of biological activity was related to the ionic adsorption of the immobilized antibody on the support surface, leading to a blocking of the recognition areas. This optimized protocol was applied to the immobilization of IgG anti-horseradish peroxidase from rabbit on magnetic nano-particles. A 10 microg preparation of nano-particles was able to capture more than 75% of the 0.1 microgram of recombinant horseradish peroxidase present in 10 L of crude protein extract (1g/L) from Escherichia coli.     

Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports

A novel thermostable chimeric β-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the β-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the β-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.     

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites

New tailor-made anionic exchange resins have been prepared, based on films of large polyethylenimine polymers (e.g., MW 25,000) completely coating, via covalent immobilization, the surface of different porous supports (agarose, silica, polymeric resins). Most proteins contained in crude extracts from different sources have been very strongly adsorbed on them. Ionic exchange properties of such composites strongly depend on the size of polyethylenimine polymers as well as on the exact conditions of the covalent coating of the solids with the polymer. On the contrary, similar coating protocols yield similar matrices by using different porous supports as starting material. For example, 77% of all proteins contained in crude extracts from Escherichia coli were adsorbed, at low ionic strength, on the best matrices, and less than 15% of the adsorbed proteins were eluted from the support in the presence of 0.3 M NaCl. Under these conditions, 100% of the adsorbed proteins were eluted from conventional DEAE supports. Such polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes. For example, lipase from Candida rugosa (CRL), beta-galactosidase from Aspergillus oryzae and D-amino acid oxidase (DAAO) from Rhodotorula gracilis, were adsorbed on such matrices in a few minutes at pH 7.0 and 4 degrees C. Immobilized enzymes preserved 100% of catalytic activity and remained fully immobilized in 0.2 M NaCl. In addition to that, CRL and DAAO were highly stabilized upon immobilization. Stabilization of DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption.