Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques

Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi-subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross-linking of previously immobilized enzyme with tailor-made dextran-aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl-agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl-agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross-linked with dextran-aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity.     

Monitoring enzyme catalysis in the multimeric state: Direct observation of Arthrobacter 4-hydroxybenzoyl-coenzyme A thioesterase catalytic complexes using time-resolved electrospray ionization mass spectrometry

The ability to examine real-time reaction kinetics for multimeric enzymes in their native state may offer unique insights into understanding the catalytic mechanism and its interplay with three-dimensional structure. In this study, we have used a time-resolved electrospray mass spectrometry approach to probe the kinetic mechanism of 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase from Arthrobacter sp. strain SU in the millisecond time domain. Intact tetrameric complexes of 4-HBA-CoA thioesterase with up to four natural substrate (4-HBA-CoA) molecules bound were detected at times as early as 6 ms using an online rapid-mixing device directly coupled to an electrospray ionization time-of-flight mass spectrometer. Species corresponding to the formation of a folded tetramer of the thioesterase at charge states 16+, 17+, 18+, and 19+ around m/z 3800 were observed and assigned as individual tetramers of thioesterase and noncovalent complexes of the tetramers with up to four substrate and/or product molecules. Real-time evaluation of the reaction kinetics was accomplished by monitoring change in peak intensity corresponding to the substrate and product complexes of the tetrameric protein. The mass spectral data suggest that product 4-HBA is released from the active site of the enzyme prior to the release of product CoA following catalytic turnover. This study demonstrates the utility of this technique to provide additional molecular details for an understanding of the individual enzyme states during the thioesterase catalysis and ability to observe real-time interactions between enzyme and substrates and/or products in the millisecond time range.     

Heterofunctional supports for the one-step purification,immobilization and stabilization of large multimeric enzymes: Amino-glyoxyl versus amino-epoxy supports

For the immobilization-stabilization of multimeric enzymes, we propose a novel heterofunctional support containing a very low concentration of ionized amino groups and a very high concentration of very poorly reactive glyoxyl (aldehyde) groups. A large tetrameric enzyme, β-galactosidase from Thermus sp., was purified and dramatically stabilized with this novel support. The enzyme was first immobilized by physical adsorption via selective multipoint anionic exchange involving the largest region of the enzyme containing all enzyme subunits. Then, an additional long incubation of the immobilized derivative under alkaline conditions was performed in order to promote an intense intramolecular multipoint covalent attachment between amino groups of the adsorbed enzyme and the very stable glyoxyl groups on the support. This novel β-galactosidase derivative is the first one in which the four subunits of this enzyme become attached to a pre-existing support. Additionally, the novel amino-glyoxyl supports were much more suitable than amino-epoxy supports for intramolecular multipoint covalent immobilization of the adsorbed enzyme onto the support. In fact, at pH 7.0, the new supports covalently immobilize the physically adsorbed protein 24-fold more rapidly than epoxy supports. Furthermore, derivatives prepared on amino-glyoxyl supports preserved 85% of catalytic activity and were 5-fold more stable than derivatives prepared on amino-epoxy supports and more than 1000-fold more stable than soluble enzyme.     

Structural and functional stabilization of L-asparaginase via multisubunit immobilization onto highly activated supports

A new protocol for the stabilization of the quaternary structure of multimeric enzymes has been attempted using as model enzyme (tetrameric) L-asparaginase from Escherichia coli. Such strategy is based upon multisubunit covalent immobilization of the enzyme onto activated supports (agarose-glutaraldehyde). Supports activated with different densities of reactive groups were used; the higher the density of groups, the higher the stabilization attained. However, because of the complexity of that enzyme, even the use of the highest densities of reactive groups was not enough to encompass all four subunits in the immobilization process. Therefore, a further chemical intersubunit cross-linking with aldehyde-dextran was pursued; these derivatives displayed a fully stabilized multimeric structure. In fact, boiling the modified enzyme derivative in the presence of sodium dodecyl sulfate and beta-mercaptoethanol did not lead to release of any enzyme subunit into the medium. Such a derivative, prepared under optimal conditions, retained ca. 40% of the intrinsic activity of the free enzyme and was also functionally stabilized, with thermostabilization enhancements of ca. 3 orders of magnitude when compared with its soluble counterpart. This type of derivative may be appropriate for extracorporeal devices in the clinical treatment of acute leukemia and might thus bring about inherent advantages in that all subunits are covalently bound to the support, with a longer half-life and a virtually nil risk of subunit release into the circulating blood stream.     

A simple strategy for the purification of large thermophilic proteins overexpressed in mesophilic microorganisms: application to multimeric enzymes from Thermus sp. strain T2 expressed in Escherichia coli

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an alpha-galactosidase and a beta-galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.     

Destabilization of the Homotetrameric Assembly of 3-Deoxy-d-Arabino-Heptulosonate-7-Phosphate Synthase from the Hyperthermophile Pyrococcus furiosus Enhances Enzymatic Activity

Many proteins adopt homomeric quaternary structures to support their biological function, including the first enzyme of the shikimate pathway that is ultimately responsible for the biosynthesis of the aromatic amino acids in plants and microorganisms. This enzyme, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), adopts a variety of different quaternary structures depending on the organism in which it is found. The DAH7PS from the hyperthermophilic archaebacterium Pyrococcus furiosus was previously shown to be tetrameric in its crystalline form, and this quaternary association is confirmed in an improved structure in a different crystal system. This tetramer is also present in solution as revealed by small-angle X-ray scattering and analytical ultracentrifugation. This homotetrameric form has two distinct interfaces, both of which bury over 10% each of the surface area of a single monomer. Substitution of Ile for Asp in the hydrophobic region of one interface gives a protein with a remarkable 4-fold higher maximum catalytic rate than the wild-type enzyme. Analytical ultracentrifugation at pH 7.5 reveals that the tetrameric form is destabilized; although the protein crystallizes as a tetramer, equilibrium exists between tetrameric and dimeric forms with a dissociation constant of 22 μM. Thus, under the conditions of kinetic assay, the enzyme is primarily dimeric, revealing that the dimeric form is a fully functional catalyst. However, in comparison to the wild-type protein, the thermal stability of the dimeric protein is significantly compromised. Thus, an unusual compromise of enzymatic activity versus stability is observed for this DAH7PS from an organism that favors a hyperthermophilic environment.     

Disulfiram irreversibly aggregates betaine aldehyde dehydrogenase--a potential target for antimicrobial agents against Pseudomonas aeruginosa

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacterium against the high-osmolality stress prevalent in the infected tissues. This tetrameric enzyme contains four cysteine residues per subunit and is a potential drug target. In our search for specific inhibitors, we mutated the catalytic Cys286 to alanine and chemically modified the recombinant wild-type and the four Cys-->Ala single mutants with thiol reagents. The small methyl-methanethiosulfonate inactivated the enzymes without affecting their stability while the bulkier dithionitrobenzoic acid (DTNB) and bis[diethylthiocarbamyl] disulfide (disulfiram) induced enzyme dissociation--at 23 degrees C--and irreversible aggregation--at 37 degrees C. Of the four Cys-->Ala mutants only C286A retained its tetrameric structure after DTNB or disulfiram treatments, suggesting that steric constraints arising upon the covalent attachment of a bulky group to C286 resulted in distortion of the backbone configuration in the active site region followed by a severe decrease in enzyme stability. Since neither NAD(P)H nor betaine aldehyde prevented disulfiram-induced PaBADH inactivation or aggregation, and reduced glutathione was unable to restore the activity of the modified enzyme, we propose that disulfiram could be a useful drug to combat infection by P. aeruginosa.     

Structural aspects of aldehyde dehydrogenase that influence dimer-tetramer formation

Aldehyde dehydrogenases are isolated as dimers or tetramers but have essentially identical structures. The homotetramer (ALDH1 or ALDH2) is a dimer of dimers (A-B + C-D). In the tetrameric enzyme, Ser500 from subunit "D" interacts with Arg84, a conserved residue, from subunit "A". In the dimeric ALDH3 form, the interaction cannot exist. It has been proposed that the formation of the tetramer is prevented by the presence of a C-terminal tail in ALDH3 that is not present in ALDH1 or 2. To understand the forces that maintain the tetramer, deletion of the tail in ALDH3, addition of different tails in ALDH1, and mutations of different residues located in the dimer-dimer interface were made. Gel filtration of the recombinantly expressed enzymes demonstrated that no change in their oligomerization occurred. Urea denaturation showed a diminution to the stability of the ALDH1 mutants. The K(m) for propionaldehyde was similar to that of the wild-type enzyme, but the K(m) for NAD was altered. A double mutant of D80G and S82A produced an enzyme with the ability to form dimers and tetramers in a protein-concentration-dependent manner. Though stable, this dimeric form was inactive. The tetramer exhibited 10% activity compared with the wild type. Sequence alignment demonstrated that the hydrophobic surface area is increased in the tetrameric enzymes. The hydrophobic surface seems to be the main force that drives the formation of tetramers. The results indicated that residues 80 and 82 are involved in maintaining the tetramer after its assembly but the C-terminal extension contributes to the overall stability of the assembled protein.     

Synthetic peptides as inactivators of multimeric enzymes: inhibition of Plasmodium falciparum triosephosphate isomerase by interface peptides.

Synthetic peptides corresponding to two distinct segments of the subunit interface of the homodimeric enzyme triosephosphate isomerase (residues 9-18, ANWKCNGTLE, peptide I; residues 68-79, KFGNGSYTGEVS, peptide II) from Plasmodium falciparum (PfTIM) have been investigated for their ability to act as inhibitors by interfering with the quaternary structure of the enzyme. An analog of peptide II containing cysteine at the site corresponding to position 74 and tyrosine at position 69 in the protein sequence KYGNGSCTGEVS (peptide III) was also investigated. A substantial fall in enzyme activity was observed following incubation of the enzyme with peptide II, whereas peptide I did not show any appreciable inhibition. The inhibitory effect was more pronounced on two mutants of PfTIM (Y74C and Y74G), both of which have reduced stability compared to the wild-type protein due to an interface cavity. The IC50 value determined for peptide II is in the range of 0.6-0.8 microM. This study suggests that interface peptides of oligomeric enzymes can be used to inhibit dimeric enzymes by disrupting their native multimeric states and may provide lead structures for potential inhibitor design.     

Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans.

The aldehyde dehydrogenases (ALDHs) are a superfamily of multimeric enzymes which catalyse the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the reduction of their cofactor, NAD or NADP, into NADH or NADPH. At present, the only known structures concern NAD-dependent ALDHs. Three structures are available in the Protein Data Bank: two are tetrameric and the other is a dimer. We solved by molecular replacement the first structure of an NADP-dependent ALDH isolated from Streptococcus mutans, in its apo form and holo form in complex with NADP, at 1.8 and 2.6 A resolution, respectively. Although the protein sequence shares only approximately 30 % identity with the other solved tetrameric ALDHs, the structures are very similar. However, a large local conformational change in the region surrounding the 2' phosphate group of the adenosine moiety is observed when the enzyme binds NADP, in contrast to the NAD-dependent ALDHs.Structure and sequence analyses reveal several properties. A small number of residues seem to determine the oligomeric state. Likewise, the nature (charge and volume) of the residue at position 180 (Thr in ALDH from S. mutans) determines the cofactor specificity in comparison with the structures of NAD-dependent ALDHs. The presence of a hydrogen bond network around the cofactor not only allows it to bind to the enzyme but also directs the side-chains in a correct orientation for the catalytic reaction to take place. Moreover, a specific part of this network appears to be important in substrate binding. Since the enzyme oxidises the same substrate, glyceraldehyde-3-phosphate (G3P), as NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the active site of GAPDH was compared with that of the S. mutans ALDH. It was found that Arg103, Arg283 and Asp440 might be key residues for substrate binding.     

Time-dependent effects of trimethylamine-N-oxide/urea on lactate dehydrogenase activity: an unexplored dimension of the adaptation paradigm.

Given that enzymes in urea-rich cells are believed to be just as sensitive to urea effects as enzymes in non-urea-rich cells, it is argued that time-dependent inactivation of enzymes by urea could become a factor of overriding importance in the biology of urea-rich cells. Time-independent parameters (e.g. Tm, k(cat), and Km) involving protein stability and enzyme function have generally been the focus of inquiries into the efficacy of naturally occurring osmolytes like trimethylamine-N-oxide (TMAO), to offset the deleterious effects of urea on the intracellular proteins in the urea-rich cells of elasmobranchs. However, using urea concentrations found in urea-rich cells of elasmobranches, we have found time-dependent effects on lactate dehydrogenase activity which indicate that TMAO plays the important biological role of slowing urea-induced dissociation of multimeric intracellular proteins. TMAO greatly diminishes the rate of lactate dehydrogenase dissociation and affords significant protection of the enzyme against urea-induced time-dependent inactivation. The effects of TMAO on enzyme inactivation by urea adds a temporal dimension that is an important part of the biology of the adaptation paradigm.     

Highly active dimeric and low-activity tetrameric forms of selenium-containing rat thioredoxin reductase 1

Mammalian thioredoxin reductase 1 (TrxR1) is a selenoprotein that contains a selenocysteine (Sec) residue at the penultimate C-terminal position. When rat TrxR1 is expressed recombinantly in Escherichia coli, partial truncation at the Sec-encoding UGA gives rise to additional protein species that lack Sec. Phenylarsine oxide (PAO) Sepharose can subsequently be used to enrich the Sec-containing protein and yield activity corresponding to that of native enzyme. Herein we extensively purified recombinant rat TrxR1 over PAO Sepharose, which gave an enzyme with about 53 U/mg specific activity. Surprisingly, only about 65% of the subunits of this TrxR1 preparation contained Sec, whereas about 35% were protein products derived from UGA truncation. Further analyses revealed a theoretical maximal specific activity of 70–80 U/mg for the homodimeric enzyme with full Sec content, i.e., significantly higher than that reported for native TrxR1. We also discovered the formation of highly stable noncovalently linked tetrameric forms of TrxR1, having full FAD content but about half the specific activity in relation to the selenium content compared to the dimeric protein. The characterization of these different forms of recombinant TrxR1 revealed that inherent turnover capacity of the enzyme must be revised, that multimeric states of the protein may be formed, and that the yield of bacterial selenoprotein production may be lower than earlier reported. The biological significance of the hitherto unsurpassed high specific activity of the enzyme involves the capacity to support a higher turnover in vivo than previously believed. The tetrameric forms of the protein could represent hitherto unknown regulatory states of the enzyme.     

Altered dynamics upon oligomerization corresponds to key functional sites

It is known that over half of the proteins encoded by most organisms function as oligomeric complexes. Oligomerization confers structural stability and dynamics changes in proteins. We investigate the effects of oligomerization on protein dynamics and its functional significance for a set of 145 multimeric proteins. Using coarse‐grained elastic network models, we inspect the changes in residue fluctuations upon oligomerization and then compare with residue conservation scores to identify the functional significance of these changes. Our study reveals conservation of about ½ of the fluctuations, with ¼ of the residues increasing in their mobilities and ¼ having reduced fluctuations. The residues with dampened fluctuations are evolutionarily more conserved and can serve as orthosteric binding sites, indicating their importance. We also use triosephosphate isomerase as a test case to understand why certain enzymes function only in their oligomeric forms despite the monomer including all required catalytic residues. To this end, we compare the residue communities (groups of residues which are highly correlated in their fluctuations) in the monomeric and dimeric forms of the enzyme. We observe significant changes to the dynamical community architecture of the catalytic core of this enzyme. This relates to its functional mechanism and is seen only in the oligomeric form of the protein, answering why proteins are oligomeric structures. Proteins 2017; 85:1422–1434. © 2017 Wiley Periodicals, Inc.     

Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation.

How can enzymes be protected against denaturation and proteolysis while keeping them in a fully functional state? One solution is to encapsulate the enzymes into liposomes, which enhances their stability against denaturation and proteases. However, the permeability barrier of the lipid membrane drastically reduces the activity of enzyme entrapped in the liposome by reducing the internal concentration of the substrate. To overcome this problem, we permeabilized the wall of the liposome by reconstitution of a porin from Escherichia coli. In this way, we recovered the full functionality of the enzyme while retaining the protection against denaturation and proteolytic enzymes. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 615–618, 2001.     

Mass spectrometry as a novel approach to probe cooperativity in multimeric enzymatic systems

Investigating cooperativity in multimeric enzymes is of utmost interest to improve our understanding of the mechanism of enzymatic regulation. In the present article, we propose a novel approach based on mass spectrometry to probe cooperativity in the binding of a ligand to a multisubunit enzyme. This approach presents the selective advantage of giving a direct insight into all the subsequent ligation states that are formed in solution as the ligand is added to the enzyme. A quantitative interpretation of the electrospray ionization (ESI) mass spectra gives the relative abundance of all the distinct enzymatic species, which allows one to directly deduce the cooperativity of the system. The overall method is described for the addition of the oxidized cofactor nicotinamide adenine dinucleotide (NAD(+)) to a dimeric mutant of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase (GPDH). It is then applied to four tetrameric enzymes: sturgeon muscle GPDH, wild type and S48G mutant of GPDH from B. stearothermophilus, and alcohol dehydrogenase (ADH) from Bakers yeast. The results illustrate the possibilities offered by this new technique. First, mass spectrometry allows a control of the enzymes before the addition of NAD(+). Second, the cooperative behavior can be drawn from one single ESI mass spectrum, which makes the method highly attractive in terms of the amount of biological material required. Above all, the major benefit lies in the direct visualization of all the enzymatic species that are in equilibrium in solution. The direct measurement of cooperativity readily resolve the inconvenience of the classical approaches employed in this field, which all need to model the experimental data in order to get the cooperative behavior of the system.     

Subunit assembly for DNA cleavage by restriction endonuclease SgrAI

The SgrAI endonuclease usually cleaves DNA with two recognition sites more rapidly than DNA with one site, often converting the former directly to the products cut at both sites. In this respect, SgrAI acts like the tetrameric restriction enzymes that bind two copies of their target sites before cleaving both sites concertedly. However, by analytical ultracentrifugation, SgrAI is a dimer in solution though it aggregates to high molecular mass species when bound to its specific DNA sequence. Its reaction kinetics indicate that it uses different mechanisms to cleave DNA with one and with two SgrAI sites. It cleaves the one-site DNA in the style of a dimeric restriction enzyme acting at an individual site, mediating neither interactions in trans, as seen with the tetrameric enzymes, nor subunit associations, as seen with the monomeric enzymes. In contrast, its optimal reaction on DNA with two sites involves an association of protein subunits: two dimers bound to sites in cis may associate to form a tetramer that has enhanced activity, which then cleaves both sites concurrently. The mode of action of SgrAI differs from all restriction enzymes characterised previously, so this study extends the range of mechanisms known for restriction endonucleases.     

Arc1p organizes the yeast aminoacyl-tRNA synthetase complex and stabilizes its interaction with the cognate tRNAs

Eukaryotic aminoacyl-tRNA synthetases, in contrast to their prokaryotic counterparts, are often part of high molecular weight complexes. In yeast, two enzymes, the methionyl- and glutamyl-tRNA synthetases associate in vivo with the tRNA-binding protein Arc1p. To study the assembly and function of this complex, we have reconstituted it in vitro from individually purified recombinant proteins. Our results show that Arc1p can readily bind to either or both of the two enzymes, mediating the formation of the respective binary or ternary complexes. Under competition conditions, Arc1p alone exhibits broad specificity and interacts with a defined set of tRNA species. Nevertheless, the in vitro reconstituted Arc1p-containing enzyme complexes can bind only to their cognate tRNAs and tighter than the corresponding monomeric enzymes. These results demonstrate that the organization of aminoacyl-tRNA synthetases with general tRNA-binding proteins into multimeric complexes can stimulate their catalytic efficiency and, therefore, offer a significant advantage to the eukaryotic cell.     

Endothelin-1 stimulates catalase activity through the PKCδ-mediated phosphorylation of serine 167

Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H₂O₂) levels. These changes correlated with an increase in serine-phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be protein kinase Cδ (PKCδ) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H₂O₂ compared to the wild-type enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist tezosentan. S167 is located on the dimeric interface, suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel filtration to examine the multimeric structure of recombinant wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKCδ was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity.     

Stabilization of multimeric enzymes: Strategies to prevent subunit dissociation

The moderate stability of enzymes is one of the main drawbacks that hinder general implementation of these interesting biocatalysts at industrial scale. An especially complex problem is the stabilization of multimeric proteins, where dissociation of the subunits produces enzyme inactivation and even product contamination. In this review, different strategies to stabilize multimeric enzymes at different levels are revised. First, the use of proper experimental conditions may facilitate the handling of the enzymes (ions, polymers, etc.). Second, genetic tools may be used to crosslink (via disulfide bonds) or just to reinforce the subunit–subunit interactions. The physical or chemical crosslinking of the enzyme subunits will be also discussed. Finally, the use of immobilization strategies (with or without pre-existing supports) will be discussed. Special emphasis will be put on the new immobilization strategies specifically designed to involve the maximum amount of enzyme subunits in the immobilization (and thus, in the further multipoint covalent attachment).