Studies of the reactivity of artificial peroxidase-like hemoproteins based on antibodies elicited against a specifically designed ortho-carboxy substituted tetraarylporphyrin.

The temperature and pH dependence as well as the selectivity of the peroxidase activity of a complex associating a monoclonal antibody 13G10 with its iron(III)-alpha,alpha,alpha,beta-mesotetrakis(ortho-carboxyphenyl) porphyrin (Fe(ToCPP)) hapten have been studied and compared to those of Fe(ToCPP) alone. It first appears that the peroxidase activity of the 13G10-Fe(ToCPP) complex is remarkably thermostable and remains about 5 times higher than that of Fe(ToCPP) alone until at least 80 degrees C. Secondly, this complex is able to use not only H2O2 as oxidant but also a wide range of hydroperoxides such as alkyl, aralkyl and fatty acid hydroperoxides and catalyze their reduction 2-6-fold faster than Fe(ToCPP) alone. It is also able to catalyze the oxidation by H202 of a variety of reducing cosubstrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB) and 3,3'-dimethoxybenzidine 3-8-fold faster than Fe(ToCPP) alone, the bicyclic aromatic ABTS and TMB being the best reducing cosubstrates. Finally, a pH dependence study, between pH 4.6 and 7.5, of the oxidation of ABTS by H2O2 in the presence of either 13G10-Fe(ToCPP) or Fe(ToCPP) shows that Km(H2O2) values vary very similarly for both catalysts, whereas very different variations are found for the k(cat) values. With Fe(ToCPP) as catalyst the k(cat) value remains constant around 100 min(-1) whereas with the 13G10-Fe(ToCPP) complex, it increases sharply below pH 5 to reach 540 min -1 at pH 4.6. This could be due to the participation of a carboxylic acid side chain of the antibody protein, as a general acid-base catalyst, to the heterolytic cleavage of the O-O bond of H2O2 leading to the highly reactive iron(V)-oxo intermediate in the peroxidase mechanism. Accordingly, the modification of the carboxylic acid residues of antibody 13G10 by glycinamide leads to a 50% decrease of the peroxidase activity of the 13G10-Fe(ToCPP) complex.

Active site topology of artificial peroxidase-like hemoproteins based on antibodies constructed from a specifically designed ortho-carboxy-substituted tetraarylporphyrin.

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)-alpha,alpha,alpha,beta-meso-tetrakis(ortho-carboxyphenyl)porph yrin [alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin]], and which exhibit in the presence of this alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra-aryl-substituted porphyrin has shown that: (a) the central iron(III) atom of alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] is not recognized by either of the two antibodies; and (b) the ortho-carboxylate substituents of the meso-phenyl rings of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o-COOHPh)4-porphyrinH2 as well as mono- and di-ortho-carboxyphenyl-substituted porphyrins suggests that the three carboxylates in the alpha, alpha, beta position are recognized by both 13G10 and 14H7 with the two in the alpha, beta positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two-thirds of the alpha,alpha,alpha,beta-Fe[(o-COOHPh)4-porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of alpha,alpha,alpha, beta-Fe[(o-COOHPh)4-porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O-O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.

Hemoabzymes. Different strategies for obtaining artificial hemoproteins based on antibodies.

Besides existing models of chemical or biotechnological origin for hemoproteins like peroxidases and cytochromes P450, catalytic antibodies (Abs) with a metalloporphyrin cofactor represent a promising alternative route to catalysts tailored for selective oxidation reactions. A brief overview of the literature shows that, until now, the first strategy for obtaining such artificial hemoproteins has been to produce antiporphyrin Abs, raised against various free-base, N-substituted, Sn-, Pd-, or Fe-porphyrins. Four of them exhibited, in the presence of the corresponding Fe-porphyrin cofactor, a significant peroxidase activity, with kcat/K(m) values of 10(2) to 5 x 10(3)/M/s. This value remained low when compared to that of peroxidases, probably because neither a proximal ligand of the Fe, nor amino acid residues participating in the catalysis of the heterolytic cleavage of the O-O bond of H2O2, have been induced in those Abs. This strategy has been shown to be insufficient for the elaboration of effective models of cytochromes P450, because only one set of Abs, raised against meso-tetrakis(para-carboxyvinylphenyl)porphyrin, was reported to catalyze the nonstereoselective oxidation of styrene by iodosyl benzene using a Mn-porphyrin cofactor, and attempts to generate Abs having binding sites for both the substrate and the metalloporphyrin cofactor, using as a hapten a porphyrin covalently linked to the substrate, were not successful. A second strategy is then proposed, which involves the chemical labeling of antisubstrate Abs with a metalloporphyrin. As an example, preliminary results are presented on the covalent linkage of an Fe-porphyrin to an antiestradiol Ab, in order to obtain semisynthetic catalytic Abs able to catalyze the selective oxidation of steroids.

Artificial peroxidase-like hemoproteins based on antibodies constructed from a specifically designed ortho-carboxy substituted tetraarylporphyrin hapten and exhibiting a high affinity for iron-porphyrins.

In order to get catalytic antibodies modelling peroxidases BALB/c mice have been immunized with iron(III)-alpha,alpha,alpha,beta-mesotetrakis-orthocarboxypheny l-porphyrin (Fe-(ToCPP))-KLH conjugates. Monoclonal antibodies have been produced by the hybridoma technology. Three antibodies, 2 IgG1 and 1 IgG2a, were found to bind both Fe(ToCPP) and the free base ToCPPH2 with similar binding constants. None of those antibodies was found to bind tetraphenylporphyrin. Those results suggest that the recognition of Fe(ToCPP) by the antibodies was mainly due to the binding of the carboxylate groups to some amino acid residues of the protein. True Kd values of 2.9 x 10(-9) M and 5.5 x 10(-9) M have been determined for the two IgG1-Fe(ToCPP) complexes. Those values are the best ones ever reported for iron-porphyrin-antibody complexes. UV-vis. studies have shown that the two IgG1-Fe(ToCPP) complexes were high-spin hexacoordinate iron(III) complexes, with no amino acid residue binding the iron, whereas the IgG2a-Fe(ToCPP) complex was a low-spin hexacoordinate iron(III) complex with two strong ligands binding the iron atom. Both IgG1-Fe(ToCPP) complexes were found to catalyze the oxidation of 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) 5-fold more efficiently than Fe(ToCPP) alone whereas the binding of IgG2a to this iron-porphyrin had no effect on its catalytic activity. kcat values of 100 min(-1) and 63 min(-1) and kcat/Km values of 105 M(-1) s(-1) and 119 M(-1) s(-1) have been found respectively for the two IgG1-Fe(ToCPP) complexes.

A catalytic antibody isomerizing a delta 5-3-cétostéroïde.

Antibodies were raised against 3-fluoro-4-aza-estradiol-17-hemisuccinate to elicit abzymes capable of isomerizing delta 5-3-ketosteroids. The hapten was designed to present a planar A ring showing some analogy with the intermediate dienol and a polarity capable of inducing catalytic groups. Antibodies binding the hapten tightly were cloned and purified from either ascites or hybridoma supernatants. Catalytic activity was tested with androst-5-ene-3,17-dione. Among the five different monoclonal antibodies binding the hapten, one proved to enhance significantly the substrate isomerization rate (kcat/kuncat = 830). Initial rates followed Michaelis kinetics and the activity was competitively inhibited by the hapten.

Investigation of a 17 beta-estradiol-monoclonal antiestradiol antibody binding mechanism using dilute solutions of organic solvents.

Quantitative understanding of steroid hormone transport and receptor-mediated action requires knowledge of the bonding forces involved in each steroid-protein complex and the effects of a biological environment on these forces. An approach to these problems using dilute solutions of water-miscible organic solvents, with a range of polarity, dielectric and hydrogen bonding properties, was tested on an estradiol-antiestradiol antibody binding system on the basis that comparing the effects of the solvents would both permit the importance of hydrophobic and hydrogen bonding to be differentiated and give information on the effects of the environment on the reaction. The results were compared with thermodynamic measurements. All the solvents reduced the Gibbs free energy of binding as a function of their concentration in the medium. The decreases were virtually a monotonic function of their dielectric constant, indicating reduced hydrogen bonding. Analysis of the decreases in terms of the solvents' hydrogen bonding and polarity properties supported this. Thermodynamic measurement showed the binding reaction was enthalpy-driven with, overall, a slightly unfavorable entropy contribution. This again showed the hydrophobic effect was not the main bonding force. The most deleterious solvent, iso-propanol, not only decreased the enthalpic contribution to binding but rendered the entropic contribution more favorable. This approach still does not allow the relative importance of hydrogen bonding and van der Waals contacts in the actual binding to be differentiated but it does give indications on how a biological environment may affect a steroid-protein binding reaction in vivo.

Kinetics and stability of delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni in the system of reverse micelles of aerosol OT in isooctane.

Partially purified delta 5-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni was studied kinetically after solubilization in reverse micelles of aerosol OT (AOT) in isooctane and water, as regards its application to biotechnology. With delta 5,10-estren-17 beta-ol-3-one as a substrate, KSI displays an enzyme activity in the micellar system but a low stability. In the presence of urea, the enzyme is, however, stable. Kinetic parameters of the stabilized enzyme are highly sensitive to both the hydration degree of the surfactant and its concentration. The hypothesis of the geometric correspondence of a non-spherical enzyme and spherical micellar matrix is considered.

A direct dot-enzyme immunoassay to detect human ovulation.

This paper describes an original dot-enzyme-linked immunosorbent assay (ELISA) for predicting ovulation in women, based on the detection of the pre-ovulatory estrogen peak in urine. A monoclonal anti-estrogen antibody is used which recognizes not only free estrogens but also some of their urinary metabolites (17-glucuro- and sulfo-conjugates) allowing a direct assay on early morning urines. Antigen is immobilized as a spot on a nitrocellulose membrane which is immersed in urine in the presence of this antibody. A peroxidase-labeled second antibody allows the detection of the first antibody bound to the membrane. Antigen and anti-estrogen antibody concentrations are chosen to obtain a maximal enzymatic coloration of spots corresponding to basal urinary estrogen levels and no coloration corresponding to the pre-ovulatory surge. Six menstrual cycles were studied, comparing dot-ELISA results with patterns of: (1) urinary estrogens measured by RIA either directly or after hydrolysis and extraction, and (2) basal body temperatures. The validity of the pre-ovulatory signal obtained and the requirements for an adaptation of this methodology to a reliable home kit are discussed.

Comparison of monoclonal antibodies to estradiol obtained from structurally different immunogens.

The immunization procedure and immunogen characteristics required to optimize the production of anti-steroid monoclonal antibodies have been studied. Five different estradiol-bovine serum albumin conjugates were tested for immunizing mice, as were two different immunization protocols (high and low dose) and the effect of varying the myeloma/spleen cell ratio for cell fusion. Antibody-producing hybridomas, obtained using the spleens of 9 high anti-steroid titre mice, were detected by RIA and EIA. The latter method was less specific than the former for higher affinity anti-estrogen antibodies. All the immunogens elicited anti-estrogen antibodies and the efficiency appeared related to the steroid density on the immunogen rather than the chemical nature of the derivative or the immunization and fusion protocols. Thirty-six anti-estrogen producing hybridomas were detected. Comparison showed that all the immunogens elicited antibodies in a wide range of affinities and specificities. None of the antibodies recognized corticosteroids or progesterone. Cross reactions with testosterone and other estrogens were not clearly related to the nature of the immunogen except that estradiol coupled to the BSA via its carbon 17 yielded antibodies specific for steroids with a non-derivatized phenolic A-ring.

Improvement of estradiol enzymoimmunoassay, using a monoclonal antibody and an avidin/biotin amplification system.

A microtitre plate enzyme immunoassay for estradiol, using a purified monoclonal antibody covalently bound to peroxidase and a small amount of immobilized immunogen, was optimized. Decreasing the antibody concentration to 2 X 10(-10) M (Kd/5) gave optimum estradiol detectability. The enzymatic signal was, however, very low in this assay. A 14-fold enhancement could be obtained using an avidin-biotin system in which several biotin molecules are conjugated with the antibody, providing multiple sites for binding by an avidin-enzyme complex. Further reagent concentration optimization gave an assay in which a range of 2 to 140 pg estradiol/well could be assayed simply and reproducibly.

Murine monoclonal antibody against aldosterone: production, characterization and use for enzymoimmunoassay.

Hybridomas secreting monoclonal antibodies to aldosterone were obtained by fusion of myeloma cells and spleen cells from Balb/c mice immunized with aldosterone-3-carboxylmethyloxime-bovine serum albumin. A monoclonal antibody was purified from ascites fluid and characterized. An affinity constant of 1.61 x 10(9) M-1 has been measured and no cross-reactivity with tetrahydroaldosterone (THA), cortisol, cortisone, corticosterone, deoxycorticosterone (DOC), dehydroepiandrosterone (DHA), progesterone and estrone, could be detected. A peroxidase conjugated-antibody (1.5 mole of enzyme per mole of antibody) was obtained and used for microwell enzyme immunoassay and Immun-Blot assay. The high affinity and specificity of this antibody should make the direct determination of aldosterone in biological fluids possible at concentrations as low as 5 x 10(-10) M.

Solubilization of 17 beta-hydroxysteroid dehydrogenase from rat liver microsomes.

The conditions for the solubilization of 17 beta-hydroxysteroid dehydrogenase from a rat liver microsomal preparation with the non-ionic detergent Triton X-100 were studied. The recoveries of 17 beta-hydroxysteroid dehydrogenase activity and of proteins in the solubilized form were determined as a function of detergent concentration, of pH and temperature, of incubation time and of saline concentration. The soluble fraction obtained under the optimal conditions contained 80% of the proteins and 75% of the enzymatic activity of initial microsomes. The presence of Triton X-100 in the solubilized proteins was not essential for enzyme activity.

Protection of substrate against enzymatic action by binding to proteins. Dependence upon enzyme and binding protein.

In fetal or adult rats estradiol is carried in the plasma by alpha-fetoprotein or albumin. The protection of the carriers toward enzymatic oxidation by 17 beta-hydroxysteroid dehydrogenase from rat liver has been studied. Concentrations of carrier protein and estradiol were adjusted to give free estradiol concentrations varying from Km/10 to Km/100 and the ratio of the catalytic velocity to that observed for the same concentration of free estradiol in the absence of carrier protein were recorded. With alpha-fetoprotein the ratio fell as expected as the carrier concentration was increased, but with serum albumin the ratio was close to unity when the concentration of carrier protein was increased from 70 to 700 microM. Thus, a fetoprotein but not albumin protected the steroid against catalytic oxidation. Similar experiments were carried out replacing the mammalian enzyme by the dehydrogenase from Pseudomonas testosteroni, and in this case, both carrier proteins protected the substrate. The lack of protection by albumin against the dehydrogenation by the mammalian enzyme is discussed.

The comparative specificity of 3 oestradiol-binding proteins. Rat alpha-foetoprotein, rat liver 17beta-hydroxy steroid dehydrogenase and anti-(oestradiol-6-carboxymethyloxime-bovine serum albumin) antiserum.

1. The specificity of 3 oestradiol-binding proteins was studied. Two of these proteins are naturally occurring (rat alpha-foetoprotein and rat liver microsomal 17beta-hydroxy steroid dehydrogenase) and the third is an artificially induced model, anti-(oestradiol-6-carboxymethyloxime-bovine serum albumin) gamma-globulins. 2. A specific binding procedure for each protein model permitted a determination of its affinity for oestradiol and for 30 other steroids. 3. The results obtained have brought to light the different areas of the steroid molecule that are important for its recognition by each of the three proteins. The two naturally occurring proteins (alpha-foetoprotein and 17beta-hydroxy steroid dehydrogenase) recognize the edge of the steroid defined by C-4, C-6, C-8 and C-15. On the other hand, the gamma-globulins recognize the opposite edge, i.e. that defined by C-2, C-10, C-11 and C-17. 4. Diethylstilboestrol, whose structure is analogous to that of a steroid, is only recognized by the two naturally occurring proteins.