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Resume L'albumine normale et les fractions bisalbuminiques issues de variants genetiques heterozygotes d'origine europeenne ont ete etudiees par focalisation isoelectrique en couche mince realisee sur le serum brut ou sur les fractions albuminiques purifiees par chromatographie d'affinite sur Blue-Trisacryl. Des gammes restreintes d'ampholytes procurant des gradients de pH entre 5 et 8, utilisees en presence d'uree 8 M, ont permis d'obtenir des profils de focalisation autorisant une discrimination des differents variants analyses. Dans de telles conditions, l'albumine normale et les variants presentent une microheterogeneite avec un certain nombre de bandes majeures. L'albumine normale se repartit en quatre bandes principales tandis que les variants presentent des bandes additionnelles differant par le nombre et le pI. La position des bandes supplementaires, par comparaison avec celles de l'albumine normale est en bonne correlation avec le comportement electrophoretique des variants, les allotypes rapides exhibant des bandes additionnelles de pI plus anodiques, et les allotypes lents des bandes de pI plus cathodiques. Le nombre et la position de ces bandes a permis de caracteriser un certain nombre de variants alors que d'autres presentaient des profils similaires, cette similitude etant en relation avec des substitutions d'aminoacides identiques intervenues a des endroits differents de la molecule d'albumine. Ces observations indiquent que la focalisation isoelectrique peut, d'une part permettre une distinction claire des variants genetiques de l'albumine humaine, a la condition que les mutations de ceux-ci soient de nature differente, d'autre part confirmer l'occurence de mutations de nature identique lorsque les profils de bandes sont indistinguables. Parallelement a l'etude des mobilites electrophoretiques relatives a trois pH, la focalisation isolectrique analytique devient une technique tres utile, a employer comme deuxieme etape dans l'identification des allotypes et ce, prealablement ou a la place de la determination par sequencage, de la modification structurale caracterisant le variant.

We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is co-purified with paraoxonase (PON1). The association between HPON1 and HPBP is modulated by phosphate and calcium concentrations. The HPBP X-ray structure solved at 1.9 A resolution is similar to the prokaryotic phosphate solute-binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation of genes between evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the first identified transporter capable of binding phosphate ions in human plasma. Thus it is thought to become a new predictor and a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.

Among the different areas of biotechnology, enzyme engineering represents a growing field where major progress has been recently made. Indeed, chemical, pharmaceutical or food industries have increased needs for enzymes. This increase requires enzyme optimization in order to achieve, together or separately, greater operational stability, better specificity, increased solubility or preferential enantioselectivity. Directed and random mutagenesis, the classical methods of enzymatic engineering, have proved to be efficient in some cases, but are quite tricky. Directed evolution is a hybrid method recently developed in order to reproduce the random mechanisms of evolution in vitro. This method has now been used to optimise an increasing number of enzymes. In our research group, a directed evolution project has been initiated on a bacterial phosphotriesterase, a promising enzyme, capable of efficiently detoxifying organophosphorus nerve agents.

The concept of antigen internal image was applied to the production of catalytic antibodies. An antibody raised in rabbits to DNase (Ab1), acted as a competitive inhibitor of the catalysis, and thus was assumed to contain anti-active site Ab. This Ab1 was used to elicit a polyclonal anti-idiotypic antibody (Ab2). This later exhibited a DNA recognition specificity, suggesting the existence of structural internal images mimicking the conformation of the active site. Moreover, Ab2 were able to hydrolyse DNA, indicating the existence of internal images mimicking the enzymatic activity of DNase. Consequently, a strategy can thus be considered using the idiotypic way, for the production of abzymes in the form of internal images of enzyme active sites.

Until recently, the characterization of genetic variants of human serum albumin was performed by electrophoretic typing prior to the determination of their amino acid substitutions. We describe a procedure using isoelectric focusing in the presence of urea for the analysis of the genetic variation of albumin. This procedure allowed a clear distinction of a new variant, previously found to be identical with albumin Sondrio according to its relative electrophoretic mobilities at 3 pHs. This new variant, the third rare albumin allotype identified in the Ile-de-France region, was called albumin Paris 2.

The diversity of a polyclonal anti-idiotypic response (Ab2) to a murine monoclonal anti-A (Ab1) was investigated after purification of two Ab2 populations. One was eluted from human polyclonal anti-A column and the other from Ab1. Analysis of the Ab specificity, as well as screening of the clonotypic distribution, were achieved after splitting Ab by IEF; this was followed by immunoblotting and probing with various anti-ABH mAb. The first population reacted with almost all the murine anti-ABH mAb, as well as with four human anti-A mAb, and consequently consisted of Ab2 beta. The second was composed of "true" Ab2 directed against Ab1. In the first population internal images mimicked either A Ag, or H Ag, or some epitopes common to both. This study demonstrates the plurality of internal images-bearing Ig molecules, some mimicking completely, and some only partially or even unfaithfully the nominal A determinant. The analysis of this idiotypic cascade proves the existence of a degeneracy of the initial restricted antigenic specificity. The consequences of such a process are discussed.

Normal human serum albumin and bisalbuminic fractions from genetic variants of european origin have been studied by ultrathin-layer isoelectric focusing performed on whole sera or after purification of albumin fractions by affinity chromatography on Blue-Trisacryl. Narrow range ampholytes giving pH gradient between 5 and 8, together with the use of 8 M urea, provided suitable patterns allowing to discriminate the various allotypes. In these conditions, both normal albumin and heterozygous variants were microheterogeneous with several main bands; in the case of normal albumin, four major bands were found, while variants exhibited additional bands differing in number and pI. The position of additional bands comparatively to that of normal albumin was consistent with the electrophoretic behavior of variants. Fast moving allotypes exhibited additional bands with more anodal pIs, whereas slow moving variants were characterized by cathodal additional bands. The number and position of these bands allowed to characterize some variants, when they failed to distinguish between some others, identical patterns being correlated to identical mutations arising at different locations on the albumin molecule. These observations indicate that isoelectric focusing could allow a clear distinction of albumin variants, provided that their mutation were different, or could confirm the occurrence of identical mutation when IEF patterns are indistinguishable. In addition to electrophoretic mobilities at various pH, analytical isoelectric focusing could be a useful technique employed as a second step in the identification of allotypes prior to the determination of the structural change characterizing the variant.

The idiotypy of antibodies (Ab) specific for oligosaccharide determinants of blood groups of the human ABO system was studied through a cascade. Xenogenic antiidiotypic Ab (Ab2) raised in rabbits to the murine monoclonal anti-A61 (Ab1) were screened for reactivity with various anti-ABH Ab. Three anti-A and three anti-A,B monoclonal antibodies (mAb) which were developed in the same mouse strain as that producing Ab1, as well as a human polyclonal anti-A, were found to share cross-reactive idiotopes (CRI) with Ab1. CRI on murine mAb could be due to a Biozzi recurrent Id on anti-A Ab reacting with anti-Id "a la Oudin", while CRI on human anti-A Ab suggested the presence of paratope-induced anti-Id. Inhibition by Ab2 of haemagglutination of A, B or O human red blood cells by many murine anti-ABH mAb, and by polyclonal or monoclonal human anti-A, strongly supported the occurrence of anti-Id mimicking ABH epitopes belonging to type 2 determinants carried by human erythrocytes. Furthermore, a rabbit immunized with Ab2 produced a potent Ab3 response characterized by anti-H-type-2 specificity. Altogether, these results are consistent with the first successful production of anti-Id Ab that mimics the tridimensional shape of a well defined and strictly carbohydrate epitope, eliciting a haemagglutinating Ab3.

Isolated albumin almost always contains polymerized forms which appear during preparation and storage of the protein. The proportion of polymerized forms reflects the degree of stability of the solution. The quantitative estimation of the polymers is usually performed by gel chromatography. In this work, the high resolution power of polyacrylamide gradient gel electrophoresis (Gradient PAGE) was used to separate the polymers present in the preparations of human serum albumin. The analysis of the different peaks obtained by gel chromatography allows to conclude that peak 1 contains aggregates and high polymers, peak 2 trimer and dimer and peak 3 the monomer of albumin. The aggregates of the peak 1 can be dissociated by SDS and correspond, in gradient PAGE, to the high polymers. By using gradient PAGE in the presence of SDS under the conditions described in this paper, it is possible to estimate the proportion of high polymers and aggregates present in albumin preparations. These results are similar to those obtained by chromatography followed by a protein assay but noticeably inferior to those resulting from measurements performed by absorbance at 280 nm.

1. 1. Available molecular weights data for Arthropod hemocyanin subunits as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate are analyzed. 2. 2. Relationship between buffer composition and subunit mobility in SDS-PAGE is shown by studying Cancer pagurus hemocyanin. 3. 3. Tris buffers are suspected to give erroneous molecular weight estimations for Arthropod hemocyanin subunits.

1. 1. The subunit composition of Cancer pagurus hemocyanin hexamers and dodecamers, previously isolated, are analyzed by immunochemical techniques. 2. 2. Subunits I and II, and homodimer of I are observed in both hexameric and dodecameric forms whereas an additional subunit III is only present in dodecamers. 3. 3. Molecular models are proposed for the functional polymeric forms. 4. 4. Conservative domains in hemocyanin subunits of two close related species, Cancer pagurus and Macropipus puber, are evidenced by immunochemical comparison.

1. 1. By using electrophoretic and immunochemical techniques, we have identified several monomeric subunits in the hemocyanin of the crab Cancer pagurus. 2. 2. Three antigenically distinct monomeric subunits, termed I, II, III, were identified by crossed immunoelectrophoresis. 3. 3. Crossed immunoelectrophoresis of subunits separated by polyacrylamide gradient gel electrophoresis instead of cellulose acetate, has shown the existence of a dimer which has no cross-reactivity with subunit II. 4. 4. The combination of subunits I and II allows hexamers to be formed. 5. 5. The formation of higher polymers requires the presence of subunit III (joining piece).

1. 1. The hemocyanin subunits of the crab Cancer pagurus is studied with regard to its electrophoretic and antigenic heterogeneity. 2. 2. Two types of subunits, F (fast moving) and S (slow moving) are found by electrophoretic analysis in cellulose acetate with appropriate buffers. 3. 3. There is no cross-reactivity between the F and S subunits. 4. 4. Electrophoresis with sieving effect reveals a third type of subunit. 5. 5. Differences are observed in the ability of these subunits to form hexamers and high molecular weight polymers.

1. Soluble caviar (oocyte) proteins of four sturgeon species from the Caspian sea, Acipenser stellatus (Sevrouga), Acipenser guldenstadti (Ossietre), Acipenser nudiventris (Chipe) and Huso huso (Belouga) were studied by isoelectric focusing. 2. Isoelectric focusing patterns of these proteins differ from one species to another and allow the identification of the specific origin of caviar. 3. Moreover, this technique allowed the discrimination of a subspecies, Acipenser guldenstadti persicus from the species. 4. This biochemical characterization of caviar proteins allowing identification of sturgeon species producing caviar could be added to data used in fraud tests.

Resume L'alpha-1-antitrypsine est le constituant majeur de la fraction alpha-1-globulinique du serum humain. Des variations d'origine genetique sont parfois associees a une pathologie. Lors du controle systematique des proteines seriques, pratique chez les donneurs preleves par plasmapherese, nous avons pu observer un dedoublement de la bande des alpha-1-globulines du a l'expression a l'etat heterozygote, d'un variant genetique du systeme PI. Dans l'eventualite d'un allotype lie a une pathologie hematologique, nous avons caracterise celui-ci. Il s'agit de l'allotype tres rare, PI*X, dont la frequence genique dans la population etudiee est de l'ordre de 1 pour 10 000.

Resume La proalbumine humaine differe de l'albumine par un hexapeptide N-terminal supplementaire Arg-Gly-Val-Phe-Arg-Arg. Cet hexapeptide est libere par une proteolyse tryptique lors de la transformation de la proalbumine en albumine. Recemment, deux variants genetiques de la proalbumine ont ete decrits : la proalbumine Christchurch ( Brennan et Carrel , 1980 ) caracterisee par un hexapeptide N-terminal Arg-Gly-Val-Phe-Arg-Gln et la proalbumine Lille ( Abdo et coll., 1981 ) caracterisee par un hexapeptide Arg-Gly-Val-Phe-His-Arg. Ces deux variants genetiques de la proalbumine sont le resultat d'une mutation ponctuelle Gln/Arg (Christchurch) ou His/Arg (Lille). L'existence de ces variants genetiques de la proalbumine humaine, caracterises par une mobilite electrophoretique plus lente que l'albumine, et identique a celle de certains variants « lentsde l'albumine nous a incite a rechercher une identite possible entre ces variants et ceux de la proalbumine. Dans ce travail, l'identification de la structure « proalbuminea ete realisee sur deux caracteres : clivage de l'hexapeptide supplementaire par addition de trypsine entrainant la liberation d'albumine de mobilite normale et l'impossibilite pour le site N-terminal de la proalbumine de fixer du Ni (II) radioactif contrairement a l'albumine puisque le site de fixation N-terminal est bloque par l'hexapeptide supplementaire. L'etude du serum de 20 sujets presentant une bisalbuminemie d'origine genetique et correspondant aux variants lents de l'albumine de type B. SO/BS, Pollibauer et Gainesville, nous a montre que les types Pollibauer et Gainesville sont en fait des variants genetiques de la proalbumine humaine. La sequence des amino-acides de l'extremite N-terminale realisee sur le variant albumine Gainesville apres isolement de cette proteine, nous a montre l'existence d'un hexapeptide additionnel Arg-Gly-Val-Phe-Arg-Gln identique a l'hexapeptide decrit dans la proalbumine Christchurch, identifiant ainsi le variant albumine Gainesville avec la proalbumine Christchurch. L'isolement et la sequence N-terminale du variant albumine Pollibauer sont actuellement en cours afin de comparer la structure de l'hexapeptide du variant Pollibauer avec celle de la proalbumine Lille.

1. 1. The hemocyanin subunits of the crab Macropipus puber are characterized with regard to their electrophoretic and antigenic heterogeneity. 2. 2. Four antigenically distinct monomeric subunits referred to as I, II, III, IV, are identified by crossed immunoelectrophoresis of the whole hemocyanin. 3. 3. There is no evidence for a covalent dimeric component in the hemocyanin of M. puber . 4. 4. Comparative analysis of previously isolated hexamers and dodecamers shows that hexamers are devoid of subunit III, whereas this one is required for the assembly of dodecamers.

1. 1. Isolated hemocyanins from six species of Decapod Crustacea (Panulirus regius. Maia squinado, Cancer pagurus, Neptunus validas, Carcinus moenas and Macropipus puber) are analyzed by immunodiffusion techniques using rabbit-specific anti-hemocyanin antisera. 2. 2. Immunodiffusion shows the existence of cross-reactivities between the hemocyanins of all these species except Panulirus regius hemocyanin. 3. 3. Selective absorptions of the anti-hemocyanin antisera show the existence of multiple antigenic determinants: specific antigenic determinants, distinctive of the species, and determinants common to several hemocyanins. 4. 4. There is a good correlation between the number of common antigenic determinants observed by immunodiffusion and the number of common tryptic peptides of these hemocyanins.

Resume Huit types de variants genetiques de l'albumine sont observes dans la population francaise. L'analyse des profils electrophoretiques des serums contenant ces variants, realises a 3 pH differents (8,6, 5,0 et 6,9) apres addition d'une proteine temoin, la transferrine, permet d'identifier chacun des variants par l'estimation quantitative de ses mobilites relatives. La precision et la reproductibilite de la technique en a fait une methode de reference couramment utilisee pour l'etude des variants europeens. Les echantillons servant de temoins de reference de cinq types de variants genetiques, les proalbumines Christchurch et Lille, les albumines Vanves, B et Reading ont ete soumis a des analyses de sequences afin de determiner la nature et la localisation de leur modification structurale. Ces donnees, completees par la mutation deja decrite des variants d'albumine Gent et Roma, permettent de disposer de sept temoins de reference, sur les huit existants, dont la mutation a ete caracterisee et confirmee.

Using agarose gel coated on GelBond film sheets, and using Coomassie blue stain followed by silver stain, a sensitive microassay has been developed for detecting small amounts of antigen-antibody precipitates, and for quantitating low concentrations of antigen. In order to obtain a high sensitivity, antigen-antibody ratios were adjusted imperatively close to the equivalence in double-diffusion, and for quantitative estimation, single radial immunodiffusion was performed, according to Mancini, by measuring circles at the end point. The use of a double staining procedure allows to detect as little as 8 micrograms/ml antigen by Ouchterlony and 200 ng/ml by Mancini technique. The sensitivity of the method is 100 times greater than classical techniques and other advantages such as the need for minimal amounts of unconcentrated samples, the absence of radioactive labelling, and the absence of interference due to a second or a third antibody coat, make this assay useful for analyzing and quantitating monoclonal antibodies obtained by hybridoma or B-cell immortalization.

1. 1. Molecular weights of the hemocyanin subunit have been determined in six species of Decapods, using SDS polyacrylamide electrophoresis in a linear gradient gel. The data so obtained are 72,500 for Neptunus validus hemocyanin subunit, 76,000 for Panulirus regius and Cancer pagurus, 77,000 for Macropipus puber, 78,000 for Carcinus moenas and 79,500 for Maia squinado. 2. 2. In these species, hemocyanin polymeric forms result respectively from the association of 5, 12, 24 or 48 subunits evocating the existence of a common process of polymers formation in Decapod Crustacea. 3. 3. Molecular homogeneity or heterogeneity of the hemocyanin subunit is investigated by immunochemical technics.

Circulating proalbumin in humans has been described in two distinct events: genetic variants of proalbumin related to mutations at the cleavage site, and normal proalbumin related to an abnormal cleaving enzyme system. We report a case of acquired proalbuminemia that appeared after an acute episode of hepatitis related to the delta agent, in a chronic carrier of hepatitis B virus. This component, not present in normal plasma, was identified as proalbumin by immunological methods. It was indistinguishable from the molecule normally present in hepatocytes as judged by electrophoretic mobility, limited susceptibility to tryptic digestion, and its inability to bind labeled Ni. We suggest that this release of proalbumin is related to the concurrent presence of both hepatitis B and delta virus in some of the infected hepatocytes.

Pst I endonuclease fragment length polymorphism of the antithrombin III probe was studied in a French population. Among 25 subjects tested, 15 were found heterozygous at the Pst I site, so that this polymorphism might serve as a useful marker of antithrombin III gene in families with antithrombin III deficiency.

Alpha-1 antitrypsin is the major component responsible for the normal alpha 1 band in human serum. Some genetic variants giving double alpha-1 band, may be associated with pathological process. In the course of a systematic screening of blood donors a double-band alpha-1 pattern was observed in a serum, due to the heterozygous expression of a genetic variant of the PI system. A possible clinical significance of the variant was investigated by characterizing it. The very rare allotype PI*X was identified and its frequency in the population of french blood donors was estimated around to one for 10,000.

Eight different types of genetic variants of albumin are observed in the French population. The analysis of electrophoretic patterns of sera containing these variants, performed a three different pHs (8.6, 5.0 and 6.9) after addition of a reference protein (transferrin), allows the identification each variant by a quantitative estimation of its relative mobilities. The accuracy and reproducibility of the technique make it a useful reference method, commonly employed for studying European variants. The samples used as references for five genetic variant types, proalbumins Christchurch and Lille, albumins Vanves, B and Reading, were subjected to sequence analysis to determine the nature and localization of their structural change. Together with the mutations of albumins Gent and Roma previously described, the data presented here make available seven reference specimens for which the structural changes are characterized out of the eight variants known to exist in France.

Isolated albumin almost always contains polymerized forms which appear during preparation and storage of the protein. The proportion of polymerized forms reflects the degree of stability of the solution. The quantitative estimation of the polymers is usually performed by gel chromatography. In this work, the high resolution power of polyacrylamide gradient gel electrophoresis (Gradient PAGE) was used to separate the polymers present in the preparations of human serum albumin. The analysis of the different peaks obtained by gel chromatography allows to conclude that peak 1 contains aggregates and high polymers, peak 2 trimer and dimer and peak 3 the monomer of albumin. The aggregates of the peak 1 can be dissociated by SDS and correspond, in gradient PAGE, to the high polymers. By using gradient PAGE in the presence of SDS under the conditions described in this paper, it is possible to estimate the proportion of high polymers and aggregates present in albumin preparations. These results are similar to those obtained by chromatography followed by a protein assay but noticeably inferior to those resulting from measurements performed by absorbance at 280 nm.

This paper reports a unique familial occurrence of Waldenstrom's macroglobulinemia (WM) in monozygotic twins. The determination of twin monozygosity has been performed by electrophoretic and immunological typing of genetic systems (erythrocyte blood groups, leucocyte antigens and serum protein polymorphism). The two monoclonal IgM differ one from the other by their light chain type and their idiotypic determinants. Although a genetic predisposition to WM exists in these twins, the gene recombination leading to idiotypic specificity and light chain assortment occurs independently of the monoclonal malignant involvement.

Circulating proalbumin in humans has been described in two distinct events: genetic variants of proalbumin related to mutations at the cleavage site, and normal proalbumin related to an abnormal cleaving enzyme system. We report a case of acquired proalbuminemia that appeared after an acute episode of hepatitis related to the delta agent, in a chronic carrier of hepatitis B virus. This component, not present in normal plasma, was identified as proalbumin by immunological methods. It was indistinguishable from the molecule normally present in hepatocytes as judged by electrophoretic mobility, limited susceptibility to tryptic digestion, and its inability to bind labeled Ni. We suggest that this release of proalbumin is related to the concurrent presence of both hepatitis B and delta virus in some of the infected hepatocytes.

A linear relationship was established between the log of the molecular weight of a protein incubated with 1% sodium dodecyl sulfate and the log of the polyacrylamide concentration reached by this protein after electrophoresis across a 3 to 30% polyacrylamide gradient gel. This relationship can be used to determine protein molecular weights from 20,000 to 1,000,000 with good accuracy.

Using agarose gel coated on GelBond film sheets, and using Coomassie blue stain followed by silver stain, a sensitive microassay has been developed for detecting small amounts of antigen-antibody precipitates, and for quantitating low concentrations of antigen. In order to obtain a high sensitivity, antigen-antibody ratios were adjusted imperatively close to the equivalence in double-diffusion, and for quantitative estimation, single radial immunodiffusion was performed, according to Mancini, by measuring circles at the end point. The use of a double staining procedure allows to detect as little as 8 micrograms/ml antigen by Ouchterlony and 200 ng/ml by Mancini technique. The sensitivity of the method is 100 times greater than classical techniques and other advantages such as the need for minimal amounts of unconcentrated samples, the absence of radioactive labelling, and the absence of interference due to a second or a third antibody coat, make this assay useful for analyzing and quantitating monoclonal antibodies obtained by hybridoma or B-cell immortalization.

The possibility that a plasmid carried by Klebsiella pneumoniae plays a role in the pathogenesis of ankylosing spondylitis was explored. K. pneumoniae K21 contains a congruent to 25-kb plasmid, but this plasmid is not present in lymphocyte DNAs of ankylosing spondylitis HLA-B27 patients, as demonstrated by molecular hybridization experiments.

Organophosphates (OPs) constitute the largest class of insecticides used worldwide and certain of them are potent nerve agents. Consequently, enzymes degrading organophosphates are of paramount interest, as they could be used as bioscavengers and biodecontaminants. Looking for a stable OPs catalyst, able to support industrial process constraints, a hyperthermophilic phosphotriesterase (known as SsoPox) was isolated from the archaeon Sulfolobus solfataricus and was found to be highly thermostable. The solved 3D structure revealed that SsoPox is a noncovalent dimer, with lactonase activity against “quorum sensing signals”, and therefore could represent also a potential weapon against certain pathogens. The structural basis of the high thermostability of SsoPox has been investigated by performing a careful comparison between its structure and that of two mesophilic phosphotriesterases (PTEs) from P. diminuta and A. radiobacter. In addition, the conformational stability of SsoPox against the denaturing action of temperature and GuHCl has been determined by means of circular dichroism and fluorescence measurements. The data suggest that the two fundamental differences between SsoPox and the mesophilic counterparts are: (a) a larger number of surface salt bridges, also involved in complex networks; (b) a tighter quaternary structure due to an optimization of the interactions at the interface between the two monomers.