Nikolaos E. Labrou

Abstract: l-Asparaginase (E.C.3.5.1.1, l-ASNase) is an enzyme extensively used as an anti-neoplastic agent in the chemotherapy of acute lymphoblastic leukemia (ALL). In the present study, we report the use of in vitro directed evolution for the creation of a new l-ASNase variant lacking glutaminase activity. A library of enzyme variants was constructed by staggered extension process (StEp) using the genes that code for the l-ASNases from Erwinia chrysanthemy (ErL-ASNase) and Erwinia carotovora (Ecal-ASNase) and screened using activity assays. A variant of the E. carotovora enzyme lacking detectable glutaminase activity was identified. Sequence analysis showed that this variant contained a single point mutation (Leu71Ile). Steady-state kinetic measurements and the analysis of the pH dependence of Vmax and Vmax /Km of l-Asn hydrolysis showed that the mutation causes significant alterations in binding and catalytic properties. Analysis of the molecular model of the mutant enzyme showed that Ile71 may perturb the conformation of important amino acid residues in the linker region which directly affects the catalytic function. The Leu71Ile mutant enzyme was used to assemble a cuvette-based biosensor specific for l-Asn. The enzyme was immobilized by crosslinking with glutaraldehyde on the side of a transparent plastic cuvette. The sensing scheme was based on the colorimetric measurement of ammonia formation using the Nessler's reagent. Calibration curve was obtained for l-Asn, with useful concentration range of 0-100 μM for l-Asn. The method's reproducibility was in the order of ±2-5% and l-Asn recoveries were 102.1%. © 2011 Elsevier B.V. All rights reserved.

Abstract: Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris.

Abstract: This unit describes a method for site-saturation mutagenesis (SSM) using PCR amplification with degenerate synthetic oligonucleotides as primers. SSM allows the substitution of predetermined protein sites against all twenty possible amino acids at once. Therefore, SSM is a powerful approach in protein engineering to characterize structure-function relationships, as well as to create improved protein variants. The procedure accepts double-stranded plasmid isolated from the dam(+) E. coli strain. The procedure is simple, fast, efficient, and eliminates time-consuming subcloning and ligation steps.

Abstract: In the present work, we describe the characterisation of the glutathione transferase (GST) gene family from Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST-like proteins in A. tumefaciens (AtuGSTs). Comparison by multiple sequence alignment generated a dendrogram revealing the phylogenetic relationships of AtuGSTs-like proteins. The beta and theta classes identified in other bacterial species are represented by five members in A. tumefaciens C58. In addition, there are three "orphan" sequences that do not fit into any previously recognised GST classes. The eight GST-like genes were cloned, expressed in Escherichia coli and their substrate specificity was determined towards 17 different substrates. The results showed that AtuGSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The 3D structures of AtuGSTs were predicted using molecular modelling. The use of comparative sequence and structural analysis of the AtuGST isoenzymes allowed us to identify local sequence and structural characteristics between different GST isoenzymes and classes. Gene expression profiling was conducted under normal culture conditions as well as under abiotic stress conditions (addition of xenobiotics, osmotic stress and cold and heat shock) to induce and monitor early stress-response mechanisms. The results reveal the constitutive expression of GSTs in A. tumefaciens and a modulation of GST activity after treatments, indicating that AtuGSTs presumably participate in a wide range of functions, many of which are important in counteracting stress conditions. These functions may be relevant to maintaining cellular homeostasis as well as in the direct detoxification of toxic compounds.

Abstract: Plant cytosolic glutathione transferases (GSTs) are an ancient enzyme superfamily with multiple and diverse functions which are important in counteracting biotic and abiotic stresses. GSTs play an important role in catalyzing the conjugation of xenobiotics and endogenous electrophilic compounds with glutathione (GSH), such as pesticides, chemical carcinogens, environmental pollutants, which leads to their detoxification. GSTs not only catalyze detoxification reactions but they are also involved in GSH-dependent isomerization reactions, in GSH-dependent reduction of organic hydroperoxides formed during oxidative stress, biosynthesis of sulfur-containing secondary metabolites, and exhibit thioltransferase and dehydroascorbate reductase activity. This review focuses on plant GSTs, and attempts to give an overview of the new insights into the catalytic function and structural biology of these enzymes. © 2011 Bentham Science Publishers Ltd.

Abstract: Two cDNA clones coding for α-type carbonic anhydrases (CA; EC 4.2.1.1) in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the full-length proteins was confirmed by heterologous expression in Escherichia coli and purification of the encoded polypeptides. The developmental expression pattern of LjCAA1 and LjCAA2 revealed that both genes code for nodule enhanced carbonic anhydrase isoforms, which are induced early during nodule development. The genes were slightly to moderately down-regulated in ineffective nodules formed by mutant Mesorhizobium loti strains, indicating that these genes may also be involved in biochemical and physiological processes not directly linked to nitrogen fixation/assimilation. The spatial expression profiling revealed that both genes were expressed in nodule inner cortical cells, vascular bundles and central tissue. These results are discussed in the context of the possible roles of CA in nodule carbon dioxide (CO(2)) metabolism.

Abstract: Random mutagenesis is a powerful tool for generating enzymes, proteins, entire metabolic pathways, or even entire genomes with desired or improved properties. This technology is used to evolve genes in vitro through an iterative process consisting of recombinant generation. Coupled with the development of powerful high-throughput screening or selection methods, this technique has been successfully used to solve problems in protein engineering. There are many methods to generate genetic diversity by random mutagenesis and to create combinatorial libraries. This can be achieved by treating DNA or whole bacteria with various chemical mutagens, by passing cloned genes through mutator strains, by "error-prone" PCR mutagenesis, by rolling circle error-prone PCR, or by saturation mutagenesis. The next sections of this review article focus on recent advances in techniques and methods used for in vitro directed evolution of enzymes using random mutagenesis. Selected examples, highlighting successful applications of these methods, are also presented and discussed.

Abstract: L-Asparaginase (L-ASNase, EC 3.5.1.1) catalyzes the hydrolysis of the non-essential amino acid L-Asn to LAsp and ammonia and is widely used for the treatment of haematopoetic diseases such as acute lymphoblastic leukaemia (ALL) and lymphomas. Therapeutic forms of L-ASNase come from different biological sources (primarily E. coli and Erwinia chrysanthemi). It is well established that the various preparations have different biochemical pharmacology properties, and different tendency to induce side-effects. This is due to different structural, physicochemical and kinetic properties of L-ASNases from the various biological sources. Understanding these properties of various L-ASNases would allow a better decipherment of their catalytic and therapeutic features, thus enabling more accurate predictions of the behaviour of these enzymes under a variety of therapeutic conditions. In addition, detailed understanding of the catalytic mechanism of L-ASNases might permit the design of new forms of L-ASNases with optimal biochemical properties for clinical applications. In this paper we review the available biochemical and pharmacokinetic information of the therapeutic forms of bacterial L-ASNases, and focus on a detailed description of structure, function and clinical applications of these enzymes.

Abstract: Plant glutathione transferases (GSTs) play a key role in the metabolism of various xenobiotics. In this report, the catalytic mechanism of the tau class GSTU4-4 isoenzyme from Glycine max (GmGSTU4-4) was investigated by site-directed mutagenesis and steady-state kinetic analysis. The catalytic properties of the wild-type enzyme and three mutants of strictly conserved residues (Ser13Ala, Asn48Ala and Pro49Ala) were studied in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction. The results showed that the mutations significantly affect substrate binding and specificity. The effect of Ser13Ala mutation on the catalytic efficiency of the enzyme could be explained by assuming the direct involvement of Ser13 to the reaction chemistry and the correct positioning of GSH and CDNB in the ternary catalytic complex. Asn48 and Pro49 were found to have a direct role on the structural integrity of the GSH-binding site (G-site). Moreover, mutation of Asn48 and Pro49 residues may bring about secondary effects altering the thermal stability and the catalytic activity (k(cat)) of the enzyme without affecting the nature of the rate-limiting step of the catalytic reaction.

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Abstract: Reverse transcriptase (RT) catalyzes the formation of dsDNA from single-stranded retroviral RNA genome. This enzyme is unique among DNA polymerases in its ability to use either RNA or DNA as a template. Moloney Murine Leukemia virus reverse transcriptase lacking RNase H activity (M-MLVH- RT) especially holds particular interest because of its ability to eliminate the deleterious effect of RNase H, which results in more efficient synthesis of full-length cDNA from mRNA. Therefore, the development of a simple purification method attracts the attention of retroviral drug and enzyme researchers and manufacturers. The present work is the first purification example of a non-tagged (native) RT by affinity chromatography using synthetic affinity ligands. In this study, the ligand was selected from a structure-biased combinatorial library of dNTP-mimetic ligands, and it was evaluated for its ability to bind and purify M-MLVH- RT from inclusion bodies of recombinant E. coli. The selected ligand (AEAd), bearing 9-aminoethyladenine and 1,6-diamine-hexane both linked on the same triazine scaffold, displayed the highest enzyme purifying ability after applying mild desorption conditions (6 mM MnCl(2) in 20 mM Tris-HCl buffer, pH 7.5). The binding capacity of immobilized AEAd with M-MLVH- RT was determined to be equal to approximately 1 mg enzyme/g moist weight gel. Adsorption studies with immobilized AEAd and soluble M-MLVH- RT demonstrated that the formation of the respective complex was perturbed by ATP. Quality control tests of the purified M-MLVH- RT essentially showed a single band (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and absence of nucleic acids and contaminating nuclease activities.

Abstract: Bacterial cytochrome P450s (CYPs) constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. In the present work we report the characterization of CYP102A2 from B. subtilis with a focus on its substrate specificity. CYP102A2 is more active in oxidation of sodium dodecyl sulphate (SDS) than any other characterized CYP. The effect of SDS and NADPH concentration on reaction rate showed nonhyperbolic and hyperbolic dependence, respectively. The enzyme was found to exhibit a bell-shaped curve for plots of activity versus pH, over pH values 5.9-8.5. The rate of SDS oxidation reached the maximum value approximately at pH 7.2 and the pH transition observed controlled by two pK(a)s in the acidic (pK(a) = 6.7 ± 0.08) and basic (pK(a) = 7.3 ± 0.06) pH range. The results are discussed in relation to the future biotechnology applications of CYPs.

Abstract: Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes catalyzing the conjugation of a broad range of toxicologically important halogenated compounds to the tripeptide glutathione (GSH) with concomitant halogen ion release. In the present work, a rapid quantitative screening method for GSTs based on colorimetric measurement of halogen ions released from halogenated xenobiotics was developed. The assay is based on the color formation resulting from the reaction of Hg(SCN)(2) with the released halogen ion of the substrate in the presence of Fe(3+). The color intensity is proportional to the extent of the catalytic reaction, allowing a quantitative measurement of the GST catalytic activity. The assay can be performed using purified recombinant enzyme (the isoenzyme GmGSTU4-4 from Glycine max) or crude recombinant Escherichia coli cell lysates in 96-well microtiter plates. The suitability of the colorimetric assay for screening mutant GST variants derived from a directed evolution library was successfully evaluated. In addition, the assay was also used for screening GST synthetic inhibitors. It was concluded that the proposed colorimetric assay is selective and sensitive and allows the screening of large numbers of samples within a few minutes.

Abstract: Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L-Phe/beta-Ala bi-substituted 1,3,5-triazine (Trz) scaffold (beta-Ala-Trz-L-Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10-binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K(D)) of 0.41 +/- 0.05 microM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60-80%). Analysis of the antibody preparation by SDS-PAGE, enzyme-linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids.

Abstract: Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.

Abstract: Cytosolic glutathione transferases (GSTs) are a diverse family of enzymes involved in a wide range of biological processes, many of which involve the conjugation of the tripeptide glutathione (GSH) to an electrophilic substrate. Detailed studies of GSTs are justified because of the considerable interest of these enzymes in medicine, agriculture and analytical biotechnology. For example, in medicine, GSTs are explored as molecular targets for the design of new anticancer drugs as a plausible means to sensitize drug-resistant tumors that overexpress GSTs. In agriculture, GSTs are exploited in the development of transgenic plants with increased resistance to biotic and abiotic stresses. Recently, selected isoenzymes of GSTs have found successful applications in the development of enzyme biosensors for the direct monitoring of environmental pollutants, such as herbicides and insecticides. This review article summarizes recent representative patents related to GSTs and their applications in biotechnology.

Abstract: The cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, although important in many biological processes in both prokaryotes and eukaryotes. In vivo, this isomerization is catalyzed by peptidyl-prolyl cis/trans-isomerases (PPIases). Here, we present the molecular and biochemical characterization of parvulin-type PPIase family members of the model legume Lotus japonicus, annotated as LjPar1, LjPar2, and LjPar3. Although LjPar1 and LjPar2 were found to be homologous to PIN1 (Protein Interacting with NIMA)-type parvulins and hPar14 from human, respectively, LjPar3 represents a novel multidomain parvulin, apparently present only in plants, that contains an active carboxyl-terminal sulfurtransferase domain. All Lotus parvulins were heterologously expressed and purified from Escherichia coli, and purified protein verification measurements used a liquid chromatography-mass spectrometry-based proteomic method. The biochemical characterization of the recombinant Lotus parvulins revealed that they possess PPIase activity toward synthetic tetrapeptides, although they exhibited different substrate specificities depending on the amino acid amino terminal to proline. These differences were also studied in a structural context using molecular modeling of the encoded polypeptides. Real-time reverse transcription-polymerase chain reaction revealed that the three parvulin genes of Lotus are ubiquitously expressed in all plant organs. LjPar1 was found to be up-regulated during the later stages of nodule development. Subcellular localization of LjPar-enhanced Yellow Fluorescence Protein (eYFP) fusions expressed in Arabidopsis (Arabidopsis thaliana) leaf epidermal cells revealed that LjPar1- and LjPar2-eYFP fusions were localized in the cytoplasm and in the nucleus, in contrast to LjPar3-eYFP, which was clearly localized in plastids. Divergent substrate specificities, expression profiles, and subcellular localization indicate that plant parvulin-type PPIases are probably involved in a wide range of biochemical and physiological processes.

Abstract: Formate dehydrogenases (FDHs, EC 1.2.1.2) comprise a group of enzymes found in both prokaryotes and eukaryotes that catalyse the oxidation of formate to CO(2). FDH1 from the model legume Lotus japonicus (LjFDH1) was cloned and expressed in E. coli BL21(DE3) as soluble active protein. The enzyme was purified using affinity chromatography on Cibacron blue 3GA-Sepharose. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. Molecular modelling studies were also employed to create a model of LjFDH1, based on the known structure of the Pseudomonas sp. 101 enzyme. The molecular model was used to help interpret biochemical data concerning substrate specificity and catalytic mechanism of the enzyme. The temporal expression pattern of LjFDH1 gene was studied by real-time RT-PCR in various plant organs and during the development of nitrogen-fixing nodules. Furthermore, the spatial transcript accumulation during nodule development and in young seedpods was determined by in situ RNA-RNA hybridization. These results considered together indicate a possible role of formate oxidation by LjFDH1 in plant tissues characterized by relative hypoxia.

Abstract: Cytosolic GSTs (glutathione transferases) are a multifunctional group of enzymes widely distributed in Nature and involved in cellular detoxification processes. The three-dimensional structure of GmGSTU4-4 (Glycine max GST Tau 4-4) complexed with GSH was determined by the molecular replacement method at 2.7 A (1 A=0.1 nm) resolution. The bound GSH is located in a region formed by the beginning of alpha-helices H1, H2 and H3 in the N-terminal domain of the enzyme. Significant differences in the G-site (GSH-binding site) as compared with the structure determined in complex with Nb-GSH [S-(p-nitrobenzyl)-glutathione] were found. These differences were identified in the hydrogen-bonding and electrostatic interaction pattern and, consequently, GSH was found bound in two different conformations. In one subunit, the enzyme forms a complex with the ionized form of GSH, whereas in the other subunit it can form a complex with the non-ionized form. However, only the ionized form of GSH may form a productive and catalytically competent complex. Furthermore, a comparison of the GSH-bound structure with the Nb-GSH-bound structure shows a significant movement of the upper part of alpha-helix H4 and the C-terminal. This indicates an intrasubunit modulation between the G-site and the H-site (electrophile-binding site), suggesting that the enzyme recognizes the xenobiotic substrates by an induced-fit mechanism. The reorganization of Arg111 and Tyr107 upon xenobiotic substrate binding appears to govern the intrasubunit structural communication between the G- and H-site and the binding of GSH. The structural observations were further verified by steady-state kinetic analysis and site-directed mutagenesis studies.

Abstract: Haloalkane dehalogenases (DHAs, E.C. 3.8.1.5) are very promising biocatalytic tools for the bioremediation of environmental pollutants which consists of haloalkanes. In the present work, we investigated the DHA from Bradyrhizobium japonicum USDA110 (BjDHA). The dehalogenase activity of B. japonicum USDA110 and RT-PCR analysis revealed that the BjDHA gene expression is induced by 1,2-dibromoethane (1,2-DBE) during the early exponential phase. The BjDHA gene was cloned, expressed in Escherichia coli BL21 (DE3) and characterized. The enzyme catalyzes the irreversible hydrolysis of a variety of haloalkanes to the corresponding alcohol, halide, and a hydrogen ion. The catalytic properties of the recombinant enzyme were investigated and the kinetic parameters (Km, kcat) for a number of substrates were determined. The results showed that the BjDHA displays wide substrate specificity towards haloalkanes and particular high activity towards 1,2-DBE. The enzyme has a different catalytic triad topology compared to the Xanthobacter haloalkane dehalogenase and is more similar to the Rhodococcus enzyme. In addition, consistent with its broad specificity, the BjDHA has a substantially larger and more polar active site cavity compared to the Xanthobacter and Rhodococcus enzymes and as a consequence, BjDHA is able to dehalogenate longer and polar compounds. These properties make this enzyme very promising bioremediation tool for environmental applications.

Abstract: The successful commercialization of high-purity proteins and enzymes parallels the development and improvement of downstream processing technology. Biomimetic ligands find wide application in protein separation and purification, being the most promising affinity ligands of large-scale potential. In the present paper we evaluate the ability of the biomimetic ligand 4-amino-phenyl-oxanilic acid coupled to Sepharose CL-6B via 1,3,5-trichloro-2,4,6-triazine to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize extracts and recombinant influenza virus neuraminidase expressed in insect cells. Under selected conditions the affinity adsorbent exhibited high selectivity and purifying ability for both proteins.

Abstract: Glutathione transferases (GSTs) are enzymes involved in cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of toxic compounds and xenobiotics, including certain chemotherapeutic drugs. The encountered chemotherapeutic resistant of tumour cells, thus, has been associated with the increase of total GST expression. GSTs, in addition to GSH-conjugating activity, exhibit sulphonamidase activity, catalyzing the GSH-mediated hydrolysis of sulphonamide bonds. Such reactions are of interest as potential tumour-directed prodrug activation strategies. In the present work we report the design and synthesis of novel chimaeric sulphonamide derivatives of bombesin, able to be activated by the model human isoenzyme GSTA1-1 (hGSTA1-1). These derivatives bear a peptidyl-moiety (analogues of bombesin peptide: R-[Lue(13)]-bombesin, R-[Phe(13)]-bombesin and R-[Ser(3),Arg(10),Phe(13)]-bombesin, where R=C(6)H(5)SO(2)NH-) as molecular recognition element for targeting the drug selectively to tumour cells. The released S-alkyl-glutathione, after hGSTA1-1-mediated cleavage of the sulphonamide bond, provides an inhibitor of varied strength against GSTs from different sources. These prodrugs are envisaged as a plausible means to sensitize drug-resistant tumours that overexpress GSTs.

Abstract: L-asparaginase (EC 3.5.1.1, L-ASNase) catalyses the hydrolysis of l-Asn, producing L-Asp and ammonia. This enzyme is an anti-neoplastic agent; it is used extensively in the chemotherapy of acute lymphoblastic leukaemia. In this study, we describe the use of in vitro directed evolution to create a new enzyme variant with improved thermal stability. A library of enzyme variants was created by a staggered extension process using the genes that code for the L-ASNases from Erwinia chrysanthemi and Erwinia carotovora. The amino acid sequences of the parental L-ASNases show 77% identity, but their half-inactivation temperature (T(m)) differs by 10 degrees C. A thermostable variant of the E. chrysamthemi enzyme was identified that contained a single point mutation (Asp133Val). The T(m) of this variant was 55.8 degrees C, whereas the wild-type enzyme has a T(m) of 46.4 degrees C. At 50 degrees C, the half-life values for the wild-type and mutant enzymes were 2.7 and 159.7 h, respectively. Analysis of the electrostatic potential of the wild-type enzyme showed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrostatic contribution to overall stability. Site-saturation mutagenesis at position 133 was used to further analyse the contribution of this position on thermostability. Screening of a library of random Asp133 mutants confirmed that this position is indeed involved in thermostability and showed that the Asp133Leu mutation confers optimal thermostability.

Abstract: Glutathione transferases (GSTs) from the tau class (GSTU) are unique to plants and have important roles in stress tolerance and the detoxification of herbicides in crops and weeds. A fluorodifen-induced GST isoezyme (GmGSTU4-4) belonging to the tau class was purified from Glycine max by affinity chromatography. This isoenzyme was cloned and expressed in Escherichia coli, and its structural and catalytic properties were investigated. The structure of GmGSTU4-4 was determined at 1.75 A resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). The enzyme adopts the canonical GST fold but with a number of functionally important differences. Compared with other plant GSTs, the three-dimensional structure of GmGSTU4-4 primarily shows structural differences in the hydrophobic substrate binding site, the linker segment and the C-terminal region. The X-ray structure identifies key amino acid residues in the hydrophobic binding site (H-site) and provides insights into the substrate specificity and catalytic mechanism of the enzyme. The isoenzyme was highly active in conjugating the diphenylether herbicide fluorodifen. A possible reaction pathway involving the conjugation of glutathione with fluorodifen is described based on site-directed mutagenesis and molecular modeling studies. A serine residue (Ser13) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione and enhance its nucleophilicity. Tyr107 and Arg111 present in the active site are important structural moieties that modulate the catalytic efficiency and specificity of the enzyme, and participate in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. A hitherto undescribed ligand-binding site (L-site) located in a surface pocket of the enzyme was also found. This site is formed by conserved residues, suggesting it may have an important functional role in the transfer and delivery of bound ligands, presumably to specific protein receptors.

Abstract: Cytosolic glutathione transferases (GSTs) are a major reserve of high-capacity ligand binding proteins which recognise a large variety of hydrophobic compounds. In the present study, the binding of non-substrate xenobiotic compounds (herbicides and insecticides) to maize GST I was investigated by employing kinetic inhibition studies, site-directed mutagenesis and molecular modelling studies. The results showed that the xenobiotics bind at the substrate binding site. Based on in silico docking analysis, two residues were selected for assessing their contribution to xenobiotic binding. The mutant Gln53Ala of GST I Exhibits 9.2-fold higher inhibition potency for the insecticide malathion, compared to the wild-type enzyme. A potentiometric assay was developed for the determination of malathion using the Gln53Ala mutant enzyme. The assay explores the ability of the xenobiotic to promote inhibition of the GST-catalysing 1-chloro-2,4-dinitrobenzene (CDNB)/glutathione (GSH) conjugation reaction. The sensing scheme is based on the pH change occurring in a low buffer system by the GST reaction, which is measured potentiometrically using a pH electrode. Calibration curve was obtained for malathion, with useful concentration range 0-20 microM. The method's reproducibility was in the order of +/-3-5% and malathion recoveries were 96.7+/-2.8%. Immobilized Gln53Ala mutant GST was used to assemble a biosensor for malathion. The enzyme was immobilized by crosslinking with glutaraldehyde and trapped behind a semipermeable membrane in front of the pH electrode. The results demonstrated that the immobilized enzyme behaved similar to free enzyme.

Abstract: Affinity chromatography on immobilized Protein A is the current method of choice for the purification of monoclonal antibodies (mAbs). Despite its widespread use it presents certain drawbacks, such as ligand instability, leaching, toxicity and high cost. In the present work, we report a new procedure for the purification of two human monoclonal anti-HIV (human immunodeficiency virus) antibodies (mAbs 2G12 and 4E10) from transgenic tobacco plants using stable and low cost chromatographic materials. The first step of the mAb 2G12 purification procedure is comprised of an aqueous two-phase partition system (ATPS) for the removal of polyphenols while providing an essential initial purification boost (2.01-fold purification). In the second step, mAb 2G12 was purified using cation-exchange chromatography (CEX) on S-Sepharose FF, by elution with 20mM sodium phosphate buffer pH 7.5, containing 0.1M NaCl. The eluted mAb was directly loaded onto an immobilized metal affinity chromatography column (IMAC, Zn(2+)-iminodiacetic acid-Sepharose 6B) and eluted by stepwise pH gradient. The proposed method offered 162-fold purification with 97.2% purity and 63% yield. Analysis of the antibody preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme immunosorbent assay (ELISA) and western blot showed that the mAb 2G12 was fully active and free of degraded variants, polyphenols and alkaloids. The effectiveness of the present purification protocol was evaluated by using a second transgenic human monoclonal anti-HIV mAb 4E10. The results showed that the same procedure can be successfully used for the purification of mAb 4E10. In the case of mAb 4E10, the proposed method offered 148-fold purification with 96.2% purity and 36% yield. Therefore, the proposed protocol may be of generic use for the purification of mAbs from transgenic tobacco plants.

Abstract: Formate dehydrogenase from Candida boidinii (CboFDH) catalyses the oxidation of formate anion to carbon dioxide with concomitant reduction of NAD(+) to NADH. CboFDH is highly specific to NAD(+) and virtually fails to catalyze the reaction with NADP(+). Based on structural information for CboFDH, the loop region between beta-sheet 7 and alpha-helix 10 in the dinucleotide-binding fold was predicted as a principal determinant of coenzyme specificity. Sequence alignment with other formate dehydrogenases revealed two residues (Asp195 and Tyr196) that could account for the observed coenzyme specificity. Positions 195 and 196 were subjected to two rounds of site-saturation mutagenesis and screening and enabled the identification of a double mutant Asp195Gln/Tyr196His, which showed a more than 2 x 10(7)-fold improvement in overall catalytic efficiency with NADP(+) and a more than 900-fold decrease in the efficiency with NAD(+) as cofactors. The results demonstrate that the combined polar interactions and steric factors comprise the main structural determinants responsible for coenzyme specificity. The double mutant Asp195Gln/Tyr196His was tested for practical applicability in a cofactor recycling system composed of cytochrome P450 monooxygenase from Bacillus subtilis, (CYP102A2), NADP(+), formic acid and omega-(p-nitrophenyl)dodecanoic acid (12-pNCA). Using a 1250-fold excess of 12-pNCA over NADP(+) the first order rate constant was determined to be equal to k(obs) = 0.059 +/- 0.004 min(-1).

Abstract: A series of small-molecule microbicides has been developed for vaginal delivery to prevent heterosexual HIV transmission, but results from human clinical trials have been disappointing. Protein-based microbicides, such as HIV-specific monoclonal antibodies, have been considered as an alternative approach. Despite their promising safety profile and efficacy, the major drawback of such molecules is the economy of large-scale production in mammalian cells, the current system of choice. Here, we show that an alternative biomanufacturing platform is now available for one of the most promising anti-HIV antibodies (2G12). Our data show that the HIV-neutralization capability of the antibody is equal to or superior to that of the same antibody produced in CHO cells. We conclude that this protein production system may provide a means to achieve microbicide ingredient manufacture at costs that would allow product introduction and manufacture in the developing world.

Abstract: A glutathione S-transferase (GST) from the mosquito Aedes aegypti (aagste2), selected in the field as a major metabolic resistance enzyme for this parasite vector, was employed to produce a highly specific assay for the determination of DDT [1,1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene]. Detection is based on the pH change occurring in an appropriate buffer system by the concomitant release of H(+) during the aagste2-catalyzed dehydrochlorination reaction and is monitored potentiometrically or colorimetrically in the presence of a pH marker. The theoretical limit of detection (LOD) of the assay is 3.8 microg/ml, and the linear range of quantification is 12 to 250 microg/ml. The method does not recognize biologically inactive DDT analogues or major DDT photodegradants and breakdown molecules, and it is highly specific for the insecticidal p.p'DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane]. The biosensor was validated with a number of insecticide swabs from DDT-sprayed surfaces and found to be reproducible and reliable as compared with high-performance liquid chromatography (HPLC) (correlation coefficient R(2)=0.98). Given the current expansion of DDT residual sprayings in many regions of Africa as a key strategic intervention for malaria vector control, this simple assay to monitor DDT levels for vector control spraying programs could have an important impact on malaria control.

Abstract: Over the last decade there has been significant progress in understanding the molecular basis of disease processes. At the same time the technological advances in the area of genomics and the efforts in proteomics research have increased the possibility of discovering many proteins with defined therapeutic functions. A large number of these proteins have found clinical application. Despite the importance of proteins as therapeutic agents, they have a number of disadvantages in comparison to small-molecule drugs, including immunogenicity and antigenicity, poor efficacy and oral bioavailability as well as, in many cases, short serum half-lives. To date, the most promising approaches for improving protein therapeutics rely on the use of genetic engineering and site-specific chemical synthesis/modification techniques. Improving the potency of protein drugs by employing modern recombinant DNA technologies and novel chemical synthesis techniques is of primary importance, not only because of the enormous medicinal benefit but also because of the significant economic edge an improved drug can provide in today's competitive market.

Abstract: Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.

Abstract: Bacterial L-asparaginases (L-ASNases) catalyze the conversion of L-asparagine to L-aspartate and ammonia. In the present work, we report the cloning and expression of L-asparaginase from Erwinia chrysanthemi 3937 (ErL-ASNase) in Escherichia coli BL21(DE3)pLysS. The enzyme was purified to homogeneity in a single-step procedure involving cation exchange chromatography on an S-Sepharose FF column. The enzymatic and structural properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. In addition, we found that the enzyme can be efficiently immobilized on epoxy-activated Sepharose CL-6B. The immobilized enzyme retains most of its activity (60%) and shows high stability at 4 degrees C. The approach offers the possibility of designing an ErL-ASNase bioreactor that can be operated over a long period of time with high efficiency, which can be used in leukaemia therapy.

Abstract: We investigated a possible relationship between the levels of reactive oxygen species (ROS) and the stimulation of frond division of the aquatic plant Spirodela polyrrhiza (duckweed) during a 7-day experimental culture period. In particular, we monitored superoxide concentration using a state-of-the-art cell biosensor. A considerable reduction in ROS and superoxide concentration was observed during the first 2 days of culture, whereas duckweed cultures achieved near exponential growth rates after the second day. In addition, apoptotic markers such as the cytoplasmic concentration of cytochrome c, mitochondrial membrane depolarization and the activity of caspase-3 declined during the culture period and at least before daughter frond maturation. We suggest that S. polyrrhiza frond division may have been stimulated by the observed reduction of free radicals and the associated avoidance of cell apoptotic pathways in cultured plants.

Abstract: The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (KD) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, approximately 61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities.

Abstract: Bacterial L-ASNases (L-asparaginases) catalyse the conversion of L-asparagine into L-aspartate and ammonia, and are widely used for the treatment of ALL (acute lymphoblastic leukaemia). In the present paper, we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsin-resistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at Arg206-Ser207 of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to histidine followed by covalent coupling of mPEG-SNHS [methoxypoly(ethylene glycol) succinate N-hydroxysuccinimide ester] to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared with the wild-type enzyme.

Abstract: In the present study, an aqueous two-phase partitioning system (ATPS) was developed and evaluated as an initial fractionation step for therapeutic antibodies and enzymes from tobacco extracts. A detailed study has been performed to analyze the effect of pH, ionic composition of the system, types of polymers and their molecular weight and concentration, on the partitioning behavior of tobacco proteins and human anti-human immunodeficiency virus (HIV) monoclonal antibody 2F5 (mAb 2F5). A polyethyleneglycol/phosphate (PEG/Pi) aqueous two-phase system composed of 12% (w/w) PEG 1500 and 13% (w/w) phosphate buffer, pH 5, was selected as the system with the highest selectivity of antibody over native tobacco proteins. Under selected conditions, sufficient purification (3-4-fold) with high recovery at the bottom phase (approximately 95%) was achieved for mAb 2F5. In addition, the system allows removal of plant-derived compounds, such as phenolics and toxic alkaloids. The antibody fraction may be directly applied to a Protein A affinity column without any further pre-treatment, thus allowing homogenous antibody preparation. Analysis of the purified antibody fraction by enzyme-linked immunosorbent assay (ELISA) and western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was further demonstrated using additional proteins and enzymes of therapeutic importance, such as neuraminidase (NA) from influenza virus and human anti-HIV monoclonal antibody 2G12 (mAb 2G12), and showed that the system may find wide applicability as an economic extraction strategy for the initial fractionation of biopharmaceuticals from transgenic tobacco plants.

Abstract: Influenza NA (neuraminidase) is an antiviral target of high pharmaceutical interest because of its essential role in cleaving sialic acid residues from cell surface glycoproteins and facilitating release of virions from infected cells. The present paper describes the use of structural information in the progressive design from a lead binding ion (a sulfate) to a potent submicromolor inhibitor (K(i) 0.13 microM). Structural information derived from the X-ray structure of an NA complexed with several sulfate ions, in combination with results derived from affinity labelling and molecular modelling studies, was used to guide design of potent sulfonic acid-based inhibitors. These inhibitors are structural fragments of the polysulfonate triazine dye Cibacron Blue 3GA and represent novel lead scaffolds for designing non-carbohydrate inhibitors for influenza neuraminidases.

Abstract: The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathione S-transferases (GSTs), plays an important role in biomolecular recognition and quaternary structure integrity. Inspection of the key structural features of the LAK motif prompted the de novo design and combinatorial synthesis of a 13-membered solid-phase ligand library, employing as a lead ligand the Phe-Trz-X structure, mimicking the LAK motif. 1,3,5-Triazine (Trz) was used as the scaffold for assembly, substituted with different LAK-mimetic amino acids. De novo ligand design was effected using bioinformatics and molecular modeling and based on mimicking the interactions of the LAK motif. The library of affinity adsorbents was assessed for binding corn and human serum proteomes and purified proteins of different structure and ligand binding specificity. The results showed remarkable differences in the binding specificity of LAK-mimetic adsorbents for a wide range of proteins, as a consequence of minor changes in ligand structure. One LAK-mimetic adsorbent was integrated in a single-step purification protocol for human monoclonal anti-human immunodeficiency virus 2F5 antibody (mAb 2F5) from spiked corn extract, affording high recovery and purity. The results demonstrate that the principle of natural recognition found in the lock-and-key motif, in combination with de novo combinatorial design, may lead to synthetic affinity ligands, useful in downstream processing and proteomic research.

Abstract: The influenza virus surface glycoprotein antigen neuraminidase (NA) is a crucial viral enzyme with many potential medical applications; therefore, the development of efficient upstream and downstream processing strategy for the expression and purification of NA is of high importance. In the present work the NA gene from the H1N1 influenza virus strain A/Beijing/262/95 was cloned from viral RNA and expressed in expresSF+ insect cells using the baculovirus expression vector system (BVES). A limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify the recombinant H1N1 neuraminidase. Affinity-ligand design was based on mimicking the interactions of the lock-and-key (LAK) motif (Phe-Gly-Gln), a common structural moiety found in the subunit interface of glutathione S-transferase I (GST I), and plays an important structural role in subunit-subunit recognition. Solid-phase combinatorial chemistry was used to synthesize 13 variants of the lock-and-key lead ligand (Phe-Trz-X, where X was selected alpha-amino acid) using the 1,3,5-triazine moiety (Trz) as the scaffold for assembly. One immobilized ligand, bearing phenylalanine and isoleucine linked on the chlorotriazine ring (Phe-Trz-Ile), displayed high affinity for NA. Absorption equilibrium and molecular modeling studies were carried out to provide a detailed picture of Phe-Trz-Ile interaction with NA. This LAK-mimetic affinity adsorbent was exploited in the development of a facile purification protocol for NA, which led to 335-fold purification in a single-step. The present purification procedure is the most efficient reported so far for recombinant NA.

Abstract: Formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 catalyzes oxidation of formate to NI2 with the coupled reduction of nicotinamide adenine dinucleotide (NAD+). The three-dimensional structures of the apo form (the free enzyme) and the holo form (the ternary FDH-NAD+-azide complex) of FDH have been established earlier. In the present study, the structures of FDH complexes with formate are solved at 2.19 and 2.28 Ã… resolution by the molecular replacement method and refined to the R factors of 22.3 and 20.5%, respectively. Both crystal structures contain four protein molecules per asymmetric unit. These molecules form two dimers identical to the dimer of the apo form of FDH. Two possible formatebinding sites are found in the active site of the FDH structure. In the complexes the sulfur atom of residue Cys354 exists in the oxidized state.

Abstract: The commercial availability of DNA polymerases has revolutionized molecular biotechnology and certain sectors of the bio-industry. Therefore, the development of affinity adsorbents for purification of DNA polymerases is of academic interest and practical importance. In the present study we describe the design, synthesis and evaluation of a combinatorial library of novel affinity ligands for the purification of DNA polymerases (Pols). Pyrococcus furiosus DNA polymerase (Pfu Pol) was employed as a proof-of-principle example. Affinity ligand design was based on mimicking the natural interactions between deoxynucleoside-triphosphates (dNTPs) and the B-motif, a conserved structural moiety found in Pol-I and Pol-II family of enzymes. Solid-phase 'structure-guided' combinatorial chemistry was used to construct a library of 26 variants of the B-motif-binding 'lead' ligand X-Trz-Y (X is a purine derivative and Y is an aliphatic/aromatic sulphonate or phosphonate derivative) using 1,3,5-triazine (Trz) as the scaffold for assembly. The 'lead' ligand showed complementarity against a Lys and a Tyr residue of the polymerase B-motif. The ligand library was screened for its ability to bind and purify Pfu Pol from Escherichia coli extract. One immobilized ligand (oABSAd), bearing 9-aminoethyladenine (AEAd) and sulfanilic acid (oABS) linked on the triazine scaffold, displayed the highest purifying ability and binding capacity (0,55 mg Pfu Pol/g wet gel). Adsorption equilibrium studies with this affinity ligand and Pfu Pol determined a dissociation constant (K(D)) of 83 nM for the respective complex. The oABSAd affinity adsorbent was exploited in the development of a facile Pfu Pol purification protocol, affording homogeneous enzyme (>99% purity) in a single chromatography step. Quality control tests showed that Pfu Pol purified on the B-motif-complementing ligand is free of nucleic acids and contaminating nuclease activities, therefore, suitable for experimental use.

Abstract: The X-ray structure of influenza virus neuraminidase (NA) isolated from whale, subtype N9, has been determined at 2.2 A resolution and contains a tetrameric protein in the asymmetric unit. In structures of NA determined previously, a calcium ion is observed to coordinate amino acids near the substrate-binding site. In three of the NA monomers determined here this calcium is absent, resulting in structural alterations near the substrate-binding site. These changes affect the conformation of residues that participate in several key interactions between the enzyme and substrate and provide at a molecular level the basis of the structural and functional role of calcium in substrate and inhibitor binding. Several sulfate ions were identified in complex with the protein. These are located in the active site, occupying the space reserved for the substrate (sialic acid) carboxylate, and in positions leading away from the substrate-binding site. These sites offer a new opportunity for the design of inhibitors of influenza virus NA.

Abstract: The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and epsilon-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the beta2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4 degrees C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37 degrees C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis.

Abstract: Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of multi-functional enzymes involved in biodegradation of several herbicide classes. The ability of the maize isoenzyme GST I to detoxify from the acetanilide herbicide alachlor was investigated by steady-state kinetics and site-directed mutagenesis studies. Steady-state kinetics fit well to a rapid equilibrium random sequential bi-bi mechanism with intrasubunit modulation between GSH binding site (G-site) and electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent and thermodynamic data suggest that product release is rate-limiting. Three residues of GST I (Trp12, Phe35 and Ile118), which build up the xenobiotic binding site, were mutated and their functional and structural roles during alachlor conjugation were investigated. These residues are not conserved, hence may affect substrate specificity and/or product dissociation. The work showed that the key amino acid residue Phe35 modulates xenobiotic substrate binding and specificity, and participates in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. Trp12 and Ile118 do not seem to carry out such functions but instead, regulate the K(m) for alachlor by contributing to its productive orientation in the H-site. The results of the present work have practical significance since this may provide the basis for the rational design of new engineered GSTs with altered substrate specificity towards herbicides and may facilitate the design of new, more selective herbicides.

Abstract: Bacterial L-asparaginases (E.C. 3.5.1.1) have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia. L-asparaginase from Erwinia carotovora NCYC 1526 (ErA) was cloned and expressed in E. coli. The enzyme was purified to homogeneity by a two-step procedure comprising cation-exchange chromatography and affinity chromatography on immobilised L-asparagine. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. Molecular modelling studies were also employed to create a model of ErA, based on the known structure of the Erwinia chrysanthemi enzyme. The molecular model was used to help interpret biochemical data concerning substrate specificity and catalytic mechanism of the enzyme. The kinetic parameters of selected substrates were determined at various pH values, and the pH-dependence profiles of V(max) and V(max)/K(m) were analyzed. The pH-dependence of V(max) shows one transition in the acidic pH range with pK(a)=5.4, and the pH-dependence of V(max)/K(m) exhibits two transitions with pK(a)=5.4 and 8.5. Based on analysis of alternative substrates and molecular modelling studies, it was concluded that the pK(a) at the acidic pH range corresponds to the active site residues Asp115 or Glu82, whereas the pK(a) observed at the alkaline pH range is not due to substrate amino group ionisation, but rather is the result of enzyme ionisation. The effect of temperature and viscosity on the catalytic activity of the enzyme was also investigated and it was concluded that the rate-limiting step of the catalytic reaction is relevant to structural transitions of the protein. Thermodynamic analysis of the activity data showed that the activation energies are dependent on the substrate, and entropy changes appear to be the main determinant contributing to substrate specificity.

Abstract: Glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of detoxification enzymes that biotransform a wide variety of endogenous and exogenous electrophilic substrates, including herbicides. The isozyme GST I from maize exhibits significant catalytic activity for the chloroacetanilide herbicide alachlor and appears to be involved in its detoxifying process. To establish the in planta ability of GST I to detoxify from alachlor, transgenesis studies were carried out. The gene gstI-6His, which encodes for 6His-tagged GST I, was used for the construction of a binary vector suitable for genetic engineering of tobacco plants (Nicotiana tabacum). Through biolistic method transgenic tobacco plants were obtained. Integration of gstI-6His gene in transgenic tobacco plants genome was confirmed by polymerase chain reaction and Southern blot hybridization. The expression of active GST I was established by Western blot analysis, using anti-6His antibody, and by direct purification of 6-His tagged GST I on Ni-NTA agarose. Primary transformed plants harboring the gstI-6His gene were transferred to MS medium supplemented with alachlor and their phenotype was evaluated. The transgenic plants showed substantially higher tolerance to alachlor compared to non-transgenic plants in terms of root, leaves and vigorous development. These transgenic plants are potentially useful biotechnological tools for the development of phytoremediation system for the degradation of herbicide pollutants in agricultural fields.

Abstract: The optimisation of enzymes for particular application or conditions remains an important target in all protein engineering endeavours. Here, we report a successful strategy for altering the pH-profile of kinetic parameters and to define in detail the molecular mechanism of maize glutathione S-transferase I (GST I). To accomplish this, selected residues from the glutathione binding site (His40, Ser11, Lys41, Asn49, Gln53 and Ser67) were mutated to Ala, and the pH-dependence of the catalytic parameters V(max), and V(max)/K(GSH)(m) of the mutated forms were analysed. The pH-dependence of V(max) for the wild-type enzyme exhibits two transitions in the acidic pH range with pK(a1) of 5.7 and pK(a2) of 6.6. Based on thermodynamic data, site-directed mutagenesis and UV deference spectroscopy, it was concluded that pK(a1) corresponds to GSH carboxylates, whereas the pK(a2) has a conformational origin of the protein. The pH-dependence of V(max)/K(GSH)(m) for the wild-type enzyme exhibits a single transition with pK(a) of 6.28 which was attributed to the thiol ionisation of bound GSH. These findings complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme and provide the basis for rationally designing engineered forms of GST I with valuable properties.

Abstract: S-(2,3-Dichlorotriazinyl)glutathione (SDTG) was synthesized and shown to be an effective alkylating affinity label for recombinant maize glutathione S-transferase I (GST I). Inactivation of GST I by SDTG at pH 6.5 followed biphasic pseudo-first-order saturation kinetics. The biphasic kinetics can be described in terms of a fast initial phase of inactivation followed by a slower phase, leading to 42 +/- 3% residual activity. The rate of inactivation for both phases exhibits nonlinear dependence on SDTG concentration, consistent with the formation of a reversible complex with the enzyme (K(d) 107.9 +/- 2.1 micro m for the fast phase, and 224.5 +/- 4.2 micro m for the slow phase) before irreversible modification with maximum rate constants of 0.049 +/- 0.002 min(-1) and 0.0153 +/- 0.001 min(-1) for the fast and slow phases, respectively. Protection from inactivation was afforded by substrate analogues, demonstrating the specificity of the reaction. When the enzyme was inactivated (42% residual activity), approximately 1 mol SDTG per mol dimeric enzyme was incorporated. Amino-acid analysis, molecular modelling, and site-directed mutagenesis studies suggested that the modifying residue is Met121, which is located at the end of alpha-helix H"'(3) and forms part of the xenobiotic-binding site. The results reveal an unexpected structural communication between subunits, which consists of mutually exclusive modification of Met residues across enzyme subunits. Thus, modification of Met121 on one subunit prevents modification of Met121 on the other subunit. This communication is governed by Phe51, which is located at the dimer interface and forms part of the hydrophobic lock-and-key intersubunit motif. The ability of SDTG to inactivate other glutathione-binding enzymes and GST isoenzymes was also investigated, and it was concluded that this new reagent may have general applicability as an affinity reagent for other enzymes with glutathione-binding sites.

Abstract: Glutathione S-transferases (GSTs) are a heterogeneous family of enzymes that catalyse the conjugation of glutathione (GSH) to electrophilic sites on a variety of hydrophobic substrates. In the present study three amino acid residues (Trp12, Phe35 and Ile118) of the xenobiotic binding site (H-site) of maize GST I were altered in order to evaluate their contribution to substrate binding and catalysis. These residues are not conserved and hence may affect substrate specificity and/or product dissociation. The results demonstrate that these residues are important structural moieties that modulate an enzyme's catalytic efficiency and specificity. Phe35 and Ile118 also participate in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. The effect of temperature on the catalytic activity of the wild-type and mutant enzymes was also investigated. Biphasic Arrhenius and Eyring plots for the wild-type enzyme showed an apparent transition temperature at 35 degrees C, which seems to be the result of a change in the rate-limiting step of the catalytic reaction. Thermodynamic analysis of the activity data showed that the activation energy increases at low temperatures, whereas the entropy change seems to be the main determinant that contributes to the rate-limiting step at high temperatures.

Abstract: Glutamate oxidase (GOX, EC 1.4.3.11) from Streptomyces catalyses the oxidation of L-glutamate to alpha-ketoglutarate. Its kinetic constants for L-glutamate were measured equal to 2 mM for Km and 85.8 s(-1) for kcat. BLAST search and amino acid sequence alignments revealed low homology to other L-amino acid oxidases (18-38%). Threading methodology, homology modeling and CASTp analysis resulted in certain conclusions concerning the structure of catalytic alpha-subunit and led to the prediction of a binding pocket that provides favorable conditions of accommodating negatively charged aromatic ligands, such as sulphonated triazine dyes. Eleven commercial textile dyes and four biomimetic dyes or minodyes, bearing a ketocarboxylated-structure as their terminal biomimetic moiety, immobilized on cross-linked agarose gel. The resulted mini-library of affinity adsorbents was screened for binding and eluting L-glutamate oxidase activity. All but Cibacron Blue 3GA (CB3GA) affinity adsorbents were able to bind GOX at pH 5.6. One immobilized minodye-ligand, bearing as its terminal biomimetic moiety p-aminobenzyloxanylic acid (BM1), displayed the higher affinity for GOX. Kinetic inhibition studies showed that BM1 inhibits GOX in a non-competitive manner with a Ki of 10.5 microM, indicating that the dye-enzyme interaction does not involve the substrate-binding site. Adsorption equilibrium data, obtained from a batch system with BM1 adsorbent, corresponded well to the Freundlich isotherm with a rate constant k of 2.7 mg(1/2)ml(1/2)/g and Freundlich isotherm exponent n of 1. The interaction of GOX with the BM1 adsorbent was further studied with regards to adsorption and elution conditions. The results obtained were exploited in the development of a facile purification protocol for GOX, which led to 335-fold purification in a single step with high enzyme recovery (95%). The present purification procedure is the most efficient reported so far for L-glutamate oxidase.

Abstract: Cytosolic GSTs (glutathione S-transferases) are a major reserve of high-capacity binding proteins and exhibit ligand-binding properties for a large variety of compounds. In the present study, the binding of two non-substrate anthraquinone dyes VBAR (Vilmafix Blue A-R) and CB3GA (Cibacron Blue 3GA) to maize (Zea mays) GST I was investigated. The results showed that the enzyme was specifically and irreversible inactivated by VBAR with a K(d) of 35.5+/-2.2 microM and a k(3) of 0.47 min(-1). Proteolytic cleavage of the VBAR-modified enzyme and subsequent separation of peptides gave only one modified peptide. Sequencing of the modified peptide revealed the target site of VBAR reaction to be Lys(41). CB3GA binds reversibly to GST I and behaves as a competitive inhibitor towards CDNB (1-chloro-2,4-dinitrobenzene) and glutathione. CB3GA binding to GST I is accompanied by a characteristic spectral change in the absorption at positive maximum (670 nm) which exhibited a hyperbolic dependence on dye concentration with a K(d) of 12.1+/-0.5 microM. Site-directed mutagenesis of selected residues (Trp(12), Phe(35), Lys(41), Asn(49), Gln(53), Ser(67) and Ile(118)) was employed, and the mutated enzymes were assessed for CB3GA binding. These results, together with molecular-modelling studies, established that the ligandin-binding site of GST I is located mainly in the hydrophobic binding site. The ability of VBAR to specifically inactivate GST I was exploited further to demonstrate the specific binding of several plant hormones and flavonoids to GST I. The inactivation of other GST isoenzymes by VBAR was also investigated, and it was concluded that VBAR may have wide applicability as an affinity label for probing structure-function relationships of GST isoenzymes.

Abstract: In this study we investigate the active-site structure and the catalytic mechanism of clostripain by using a combination of three separate techniques: affinity labelling, site-directed mutagenesis and molecular modelling. A benzamidinyl-diazo dichlorotriazine dye (BDD) was shown to act as an efficient active site-directed affinity label for Clostridium histolyticum clostripain. The enzyme, upon incubation with BDD in 0.1 m Hepes/NaOH buffer pH 7.6, exhibits a time-dependent loss of activity. The rate of inactivation exhibits a nonlinear dependence on the BDD concentration, which can be described by reversible binding of dye to the enzyme prior to the irreversible reaction. The dissociation constant of the reversible formation of an enzyme-BDD complex is KD = 74.6 +/- 2.1 micro m and the maximal rate constant of inactivation is k3 = 0.21 x min(-1). Effective protection against inactivation by BDD is provided by the substrate N-benzoyl-L-arginine ethyl ester (BAEE). Cleavage of BDD-modified enzyme with trypsin and subsequent separation of peptides by reverse-phase HPLC gave only one modified peptide. Amino acid sequencing of the modified tryptic peptide revealed the target site of BDD reaction to be His176. Site-directed mutagenesis was used to study further the functional role of His176. The mutant His176Ala enzyme exhibited zero activity against BAEE. Together with previous data, these results confirm that a catalytic dyad of His176 and Cys231 is responsible for cysteine peptidase activity in the C11 peptidase family. A molecular model of the catalytic domain of clostripain was constructed using a manually extended fold recognition-derived alignment with caspases. A rigorous iterative modelling scheme resulted in an objectively sound model which points to Asp229 as responsible for defining the strong substrate specificity for Arg at the P1 position. Two possible binding sites for the calcium required for auto-activation could be located. Database searches show that clostripain homologues are not confined to bacterial lineages and reveal an intriguing variety of domain architectures.

Abstract: Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. However, it suffers from problems regarding ligand stability and cost. Some of the most recent advances in this area have explored the power of rational and combinatorial approaches for designing highly selective and stable synthetic affinity ligands. Rational molecular design techniques, which are based on the ability to combine knowledge of protein structures with defined chemical synthesis and advanced computational tools, have made rational ligand design feasible and faster. Combinatorial approaches based on peptide and nucleic acid libraries have permitted the rapid synthesis of new synthetic affinity ligands of potential use in affinity chromatography. The versatility of these approaches suggests that, in the near future, they will become the dominant methods for designing and selection of novel affinity ligands with scale-up potential.

Abstract: Affinity chromatography is widely employed in laboratory and large-scale for the purification of biotherapeutics and diagnostics. Some of the most widely used ligands in affinity chromatography have been several reactive chlorotriazine dyes. In particular, immobilized anthraquinone dyes have found a plethora of applications in affinity chromatography because they are inexpensive, are resistant to chemical and biological degradation, are sterilizable and cleanable in situ, and are readily immobilized to generate affinity adsorbents which display high binding capacity for a broad spectrum of proteins. This article provides detailed protocols on the preparation of a dye-ligand affinity adsorbent. Also, detailed protocols for effective application of these media, emphasizing binding and elution conditions are presented.

Abstract: We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.

Abstract: The functional and structural role of the conserved Asn49 of theta class maize glutathione S-transferase was investigated by site-directed mutagenesis. Asn49 is located in the type I beta turn formed by residues 49-52, and is involved in extensive hydrogen-bonding interactions between alpha helix 2 and the rest of the N-terminal domain. The substitution of Asn49 with Ala induces positive cooperativity for 1-chloro-2,4-dinitrobenzene (CDNB) binding as reflected by a Hill coefficient of 1.9 (S(0.5)CDNB = 0.43 mm). The positive cooperativity is also confirmed by following the isothermic binding of 1-hydroxyl-2,4-dinitrobenzene (HDNB) by UV-difference spectroscopy. In addition, the mutated enzyme exhibits: (a) an increase in the Km(GSH) value of about 6.5-fold, and decrease in kcat value of about fourfold; (b) viscosity-independent kinetic parameters; (c) lower thermostability, and (d) increased susceptibility to proteolytic attack by trypsin, when compared to the wild-type enzyme. It is concluded that Asn49 affects the rate-limiting step of the catalytic reaction, and contributes significantly to the structural and binding characteristics of both the glutathione binding site (G-site) and the electrophile substrate binding site (H-site) by affecting the structural integrity of a type I beta turn (comprising residues 49-52) and probably the flexibility of the highly mobile short 310 helical segment of alpha helix 2 (residues 35-46). These structural perturbations are probably transmitted, via Phe51 and Phe65, to alpha helix H3" of the adjacent subunit which contains key residues that interact with the electrophile substrate and contribute to the monomer-monomer contact region. This may accounts for the positive cooperativity observed.

Abstract: The isoenzyme glutathione S-transferase (GST) I from maize (Zea mays) was cloned and expressed in Escherichia coli, and its catalytic mechanism was investigated by site-directed mutagenesis and dynamic studies. The results showed that the enzyme promotes proton dissociation from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity by lowering the pK(a) of the thiol from 8.7 to 6.2. Steady-state kinetics fit well to a rapid equilibrium, random sequential Bi Bi mechanism, with intrasubunit modulation between the GSH binding site (G-site) and the electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent, and thermodynamic data suggest that product release is rate-limiting. Five residues of GST I (Ser(11), His(40), Lys(41), Gln(53) and Ser(67)), which are located in the G-site, were individually replaced with alanine and their structural and functional roles in the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction were investigated. On the basis of steady-state kinetics, difference spectroscopy and limited proteolysis studies it is concluded that these residues: (1) contribute to the affinity of the G-site for GSH, as they are involved in side-chain interaction with GSH; (2) influence GSH thiol ionization, and thus its reactivity; (3) participate in k(cat) regulation by affecting the rate-limiting step of the reaction; and (4) in the cases of His(40), Lys(41) and Gln(53) play an important role in the structural integrity of, and probably in the flexibility of, the highly mobile short 3(10)-helical segment of alpha-helix 2 (residues 35-46), as shown by limited proteolysis experiments. These structural perturbations are probably transmitted to the H-site through changes in Phe(35) conformation. This accounts for the modulation of K(CDNB)(m) by His(40), Lys(41) and Gln(53), and also for the intrasubunit communication between the G- and H-sites. Computer simulations using CONCOORD were applied to maize GST I monomer and dimer structures, each with bound lactoylglutathione, and the results were analysed by the essential dynamics technique. Differences in dynamics were found between the monomer and the dimer simulations showing the importance of using the whole structure in dynamic analysis. The results obtained confirm that the short 3(10)-helical segment of alpha-helix 2 (residues 35-46) undergoes the most significant structural rearrangements. These rearrangements are discussed in terms of enzyme catalytic mechanism.

Abstract: NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii was cloned and expressed to a high level in Escherichia coli (20% of soluble E. coli protein). Molecular modelling studies were used to create a three-dimensional model of C. boidinii FDH, based on a known structure of the Pseudomonas sp. 101 enzyme. This model was used for investigating the catalytic mechanism by site-directed mutagenesis. Eleven forms of C. boidinii FDH were characterized by steady-state kinetic analysis: the wild type as well as 10 mutants involving single (Phe-69-Ala, Asn-119-His, Ile-175-Ala, Gln-197-Leu, Arg-258-Ala, Gln-287-Glu and His-311-Gln) and double amino acid substitutions (Asn-119-His/His-311-Gln, Gln-287-Glu/His-311-Gln and Gln-287-Glu/Pro-288-Thr). The kinetic results of the mutant enzymes provide the first experimental support that hydrophobic patches, formed by Phe-69 and Ile-175, destabilize substrates and stabilize products. Also, the key role of Arg-258 in stabilization of the negative charge on the migrating hydride was established. Asn-119, besides being an anchor group for formate, also may comprise one of the hinge regions around which the two domains shift on binding of NAD+. The more unexpected results, obtained for the His-311-Gln and Gln-287-Glu/His-311-Gln mutants, combined with molecular modelling, suggest that steric as well as electrostatic properties of His-311 are important for enzyme function. An important structural role has also been attributed to cis-Pro-288. This residue may provide the key residues Gln-287 and His-311 with the proper orientation for productive binding of formate.

Abstract: The 2',3'-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mM for the fast phase, 0.45 mM for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min-1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme-oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme-oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the MODELLER 4 program. The model confirmed that Lys360 is positioned at the NAD+-binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360-->Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360-->Ala enzyme. The data are discussed in terms of engineering coenzyme specificity.

Abstract: Affinity adsorbents based on immobilized triazine dyes offer important advantages circumventing many of the problems associated with biological ligands. The main drawback of dyes is their moderate selectivity for proteins. Rational attempts to tackle this problem are realized through the biomimetic dye concept according to which new dyes, the biomimetic dyes, are designed to mimic natural ligands. Biomimetic dyes are expected to exhibit increased affinity and purifying ability for the targeted proteins. Biocomputing offers a powerful approach to biomimetic ligand design. The successful exploitation of contemporary computational techniques in molecular design requires the knowledge of the three-dimensional structure of the target protein, or at least, the amino acid sequence of the target protein and the three-dimensional structure of a highly homologous protein. From such information one can then design, on a graphics workstation, the model of the protein and also a number of suitable synthetic ligands which mimic natural biological ligands of the protein. There are several examples of enzyme purifications (trypsin, urokinase, kallikrein, alkaline phosphatase, malate dehydrogenase, formate dehydrogenase, oxaloacetate decarboxylase and lactate dehydrogenase) where synthetic biomimetic dyes have been used successfully as affinity chromatography tools.

Abstract: The interaction of yeast alcohol dehydrogenase (ADH) with the reactive chlorotriazine dye Vilmafix Blue A-R (VBAR) was studied. VBAR was purified to homogeneity on lipophilic Sephadex LH-20 and characterised by reverse phase HPLC and analytical TLC. Incubation of ADH with purified VBAR at pH 8.0 and 37 degrees C resulted in a time-dependent inactivation of the enzyme. The observed rate of enzyme inactivation (kobs) exhibited a non-linear dependence on VBAR concentration from 22 to 106 nmol, with a maximum rate of inactivation (k3) of 0.134 min-1 and kD of 141.7 microM. The inhibition was irreversible and activity could not be recovered by gel-filtration chromatography. The inactivation of ADH by VBAR was competitively inhibited by the nucleotides NADH and NAD+. These results suggest that VBAR acts as an affinity label at the nucleotide binding site of yeast ADH.

Abstract: Formate dehydrogenase (FDH, EC 1.2.1.2) from Candida boidinii was purified to homogeneity. The two step procedure comprised anion exchange chromatography (2.9-fold purification, 85% step yield, elution with 35 mM KCl), followed by dye-ligand affinity chromatography on immobilized Cibacron Blue 3GA (1.4-fold purification, 75% step yield, elution with 0.15 mM NAD+/2 mM Na2SO3). The procedure afforded FDH at 63.8% overall yield and a specific activity of 7.2 units/mg. The purity of the final FDH preparation was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), high performance gel filtration liquid chromatography (gfHPLC) and N-terminal amino acid sequencing. The analytical techniques showed the presence of a single polypeptide chain that corresponds to the molecular weight of 41 kDa (as determined by SDS-PAGE) and 81 kDa (as determined by gfHPLC).

Abstract: The 4-aminophenyloxanilic acid and beta-mercaptopyruvic acid linked to the reactive diclorotriazine ring, were studied as active site-direct affinity labels towards oxaloacetate decarboxylase (EC 4.1.1.3, OXAD). Oxaloacetate decarboxylase when incubated with 4-aminophenyloxanilic-diclorotriazine (APOD) or beta-mercaptopyruvic-diclorotriazine (MPD) at pH 7.0 and 25 degrees C shows a time-dependent and concentration-dependent loss of enzyme activity. The inhibition was irreversible and activity cannot be recovered either by extensive dialysis or gel-filtration chromatography. The enzyme inactivation following the Kitz & Wilson kinetics for time-dependent irreversible inhibition. The observed rate of enzyme inactivation (k(obs)) exhibits a non-linear dependence on APOD or MPD concentration with maximum rate of inactivation (k3) of 0.013 min(-1) and 0.0046 min(-1) and K(D) equal to 20.3 and 156 microM respectively. The inactivation of oxaloacetate decarboxylase by APOD and MPD is competitively inhibited by OXAD substrate and inhibitors, such as oxaloacetate, ADP and oxalic acid whereas Mn+2 enhances the rate of inactivation. The rate of inactivation of OXAD by APOD shows a pH dependence with an inflection point at 6.8, indicating a possible histidine derivatization by the label. These results show that APOD and MPD demonstrate the characteristics of an active-site probe towards the oxaloacetate binding site of oxaloacetate decarboxylase.

Abstract: Oxaloacetate decarboxylase (OXAD), the enzyme that catalyzes the decarboxylation of oxaloacetate to pyruvic acid and carbon dioxide, was purified 245-fold to homogeneity from Pseudomonas stutzeri. The three-step purification procedure comprised anion-exchange chromatography, metal-chelate affinity chromatography, and biomimetic-dye affinity chromatography. Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native high-performance gel-filtration liquid chromatography were, respectively, 63 and 64 kDa, suggesting a monomeric protein. OXAD required for maximum activity divalent metal cations such as Mn2+ and Mg2+ but not monovalent cations. The enzyme is not inhibited by avidin, but is competitively inhibited by adenosine 5'-diphosphate, acetic acid, phosphoenolpyruvate, malic acid, and oxalic acid. Initial velocity, product inhibition, and dead-end inhibition studies suggested a rapid-equilibrium ordered kinetic mechanism with Mn2+ being added to the enzyme first followed by oxaloacetate, and carbon dioxide is released first followed by pyruvate. Inhibition data as well as pH-dependence profiles and kinetic parameters are reported and discussed in terms of the mechanism operating for oxaloacetate decarboxylation.

Abstract: Molecular modeling was employed for the design of a biomimetic chimeric ligand for L-lactate dehydrogenase (LDH). This ligand is an anthraquinone monochlorotriazinyl dye comprising two moieties: (a) the ketocarboxyl biomimetic moiety, 2-(4-aminophenyl)-ethyloxamic acid, linked on the monochlorotriazine ring, mimicking the natural substrate of LDH, and (b) the anthraquinone chromophore moiety, linked also on the same monochlorotriazine ring via a diaminobenzenesulfonate group, acting as pseudomimetic of the cofactor NAD+. The positioning of the dye in the enzyme's binding site is primarily achieved by the recognition and positioning of the pseudomimetic anthraquinone moiety. The positioning of the biomimetic ketocarboxylic moiety is based on a match between the polar and hydrophobic regions of the enzyme's binding site with those of the biomimetic moiety of the ligand. The length of the biomimetic moiety is predetermined for the ketoacid to approach the enzyme catalytic site and form charge-charge interactions. The biomimetic chimeric ligand and the commercial nonbiomimetic ligand Cibacron(R) blue 3GA (CB3GA), were immobilized on crosslinked beaded agarose gel via their chlorotriazine ring. The two affinity adsorbents were evaluated for their purifying ability for LDH from six sources (bovine heart and pancreas, porcine muscle, chicken liver and muscle, and pea seeds). The biomimetic adsorbent exhibited approximately twofold higher purifying ability for LDH compared to the CB3GA adsorbent; therefore, the former was integrated in the purification procedure of LDH from bovine heart extract. The LDH afforded by this two-step purification procedure shows specific activity equal to 600 U/mg (25 degrees C) and a single band after SDS-PAGE analysis.

Abstract: A complete series of analogs of tyrosine modified neurokinin A ([Tyr1]-NKA or [Tyr0]-NKA) has been synthesized by substituting each natural residue with 1-Cys. These analogs were tested for their ability to bind recombinant neurokinin-2 (NK-2) receptor. Substitution of Phe6 with Cys completely abolished binding of the analog to the receptor. Substitution of residues in the carboxyl-terminal region of the peptide (Met10, Leu9, Gly8, Val7) and Asp4 with Cys gave reductions in binding affinity of between 23- and 250-fold. Molecular dynamics simulations of these analogs suggest that changes in peptide structure and flexibility are not large contributors to the losses in receptor binding affinity. Reductions in binding affinity are therefore more confidently ascribed to losses of peptide-receptor interactions.

Abstract: L-Malate dehydrogenase (MDH) from Pseudomonas stutzeri was purified to homogeneity by a two-step procedure comprising anion-exchange chromatography and affinity chromatography on immobilized anthraquinone alpha-ketocarboxyl biomimetic dye. The enzyme has molecular mass of 66,500 Da and consists of two identical subunits of molecular mass of approximately 34,000 Da. Initial velocity, product inhibition, and binding studies were consistent with an ordered Bi-Bi mechanism for the enzyme action and the formation of a ternary complex. The enzyme is susceptible to activation and inhibition by its substrates. Thermodynamic analysis and kinetic inhibition studies were performed for determining basic equilibrium and kinetic constants. Malate dehydrogenase was covalently inactivated by a dichlorotriazine dye, Vilmafix Blue A-R (VBAR). The inactivation process follows first-order kinetics, and the results from kinetic analysis suggested the formation of a noncovalent enzyme-dye complex prior to the covalent reaction, with Kd 84.6 microM and a maximum rate constant 0.16 min(-1). The enzyme inactivation process was partially inhibited by substrates and inhibitors. Quantitatively inactivated MDH contained approximately 1 mole of dye per mole of enzyme subunit. The denatured enzyme contains 10 sulfhydryl groups per subunit, as shown after reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid), of which 5 can be titrated also in the native enzyme, exhibiting time-dependent reactivity. One sulfhydryl group is located in the coenzyme binding site. This study shows that the physical and catalytic properties of P. stutzeri MDH strongly resemble those of the mitochondrial eukariotic enzyme. This finding strengthens the existing view that, in the evolution process, the mitochondrial MDH might have appeared before the cytoplasmic.

Abstract: Two commercially important enzymes, L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (MDH) were purified simultaneously from bovine heart, on an agarose affinity adsorbent. This adsorbent bears a dye-ligand composed of an anthraquinone chlorotriazine chromophore linked to a biomimetic terminal 4-aminophenyloxanylic acid moiety. The purification protocol exploited the biomimetic affinity adsorbent, in combination with a cross-linked agarose DEAE anion-exchanger. The procedure comprised a preliminary anion-exchange first step, for the separation of the three enzyme activities, mMDH, cMDH and LDH. In the second step, that of affinity chromatography, the unbound mMDH obtained from the first step, was purified by specific elution with NAD+/sulphite (22.5-fold purification, 55% step-yield). The procedure afforded mMDH preparation of specific activity approx. 1,300ru/mg (25 °C) at 45% overall yield, free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase. The LDH activity, which, bound to the anion-exchanger during the first step, was recovered from the adsorbent in 200rmM KCl, and finally purified by biomimetic-dye affinity chromatography (NAD+/sulphite elution) and a second ion-exchange chromatography step (elution with 200rmM KCl). The LDH preparation exhibited specific activity approx. 500ru/mg at 25 °C (content of impurities: pyruvate kinase and GOT were not detected; MDH, 0.01%).

Abstract: The ability of the reactive dichlorotriazine dye Vilmafix Blue A-R (VBAR) to act as an affinity label for bovine heart L-malate dehydrogenase (MDH) was studied. VBAR binds specifically and irreversibly to MDH (k3 0.16 min-1; KD 14.4 microM). The inactivation of the NADH-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH and ADP. Quantitatively inhibited MDH contained approx. 1 mol of dye per mol of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM NAD+, 10 mM NADH or 10 mM ADP, or by extensive dialysis or gel-filtration chromatography. Data obtained from high-performance gel-filtration chromatography and analysed by Scatchard plot suggested the presence of two coenzyme-binding sites per MDH dimer. Tryptic digestion of VBAR-labelled MDH followed by reverse-phase HPLC analysis revealed one VBAR-labelled peptide. It appears that each subunit features the same peptide bearing the modifying residue involved in MDH labelling. The pKa of the modifying residue is 8.05. Both total acid hydrolysis of VBAR-labelled MDH followed by HPLC and TLC analysis, and molecular-modelling studies suggest that the modifying residue is Lys-81 and/or Lys-217.

Abstract: Molecular modelling and kinetic inhibition studies, as well as KD determinations by both difference-spectra and enzyme-inactivation studies, were employed to assess the ability of purpose-designed chimaeric biomimetic dyes (BM dyes) to act as affinity ligands for bovine heart L-malate dehydrogenase (MDH). Each BM dye was composed of two enzyme-recognition moieties. The terminal biomimetic moiety bore a carboxyl or a keto acid structure linked to the triazine ring, thus mimicking the substrate of MDH. The chromophore anthraquinone moiety remained unchanged and the same as that of the parent dye Vilmafix Blue A-R (VBAR), recognizing the nucleotide-binding site of MDH. The monochlorotriazine BM dyes did not inactivate MDH but competitively inhibited inactivation by the parent dichlorotriazine dye VBAR. Dye binding to MDH was accompanied by a characteristic spectral change in the range 500-850 nm. This phenomenon was reversed after titration with increasing amounts of NADH. When compared with VBAR, Cibacron Blue 3GA and two control non-biomimetic anthraquinone dyes, all BM dyes exhibited lower KD values and therefore higher affinity for MDH. The enzyme bound preferably to BM ligands substituted with a biomimetic aromatic moiety bearing an alpha-keto acid group and an amide linkage, rather than a monocarboxyl group. Thus the biomimetic dye bearing p-aminobenzyloxanilic acid as its terminal biomimetic moiety (BM5) exhibited the highest affinity (KD 1.3 microM, which corresponded to a 219-fold decrease over the KD of a control dye). BM5 displayed competitive inhibition with respect to both NADH (Ki 2.7 microM) and oxaloacetate (Ki 9.6 microM). A combination of molecular modelling and experimental studies has led to certain conclusions. The positioning of the dye in the enzyme is primarily achieved by the recognition and positioning of the nucleotide-pseudomimetic anthraquinone moiety. The hydrophobic groups of the dye provide the driving force for positioning of the ketocarboxyl biomimetic moiety. A match between the alternating polar and hydrophobic regions of the enzyme binding site with those of the biomimetic moiety is desirable. The length of the biomimetic moiety should be conserved in order for the keto acid to approach the enzyme active site and form charge-charge interactions.

Abstract: Seven biomimetic anthraquinone triazinyl dye-ligands, bearing as triazine-linked terminal moiety (keto)carboxylated structures mimicking substrates and inhibitors of malate dehydrogenase (MDH), were immobilised on cross-linked agarose Ultrogel A6R. These biomimetic ligands are terminal-ring analogues of commercial nonbiomimetic Cibacron blue 3GA (CB3GA) and parent Vilmafix blue A-R (VBAR). The biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing immobilised CB3GA and VBAR, were evaluated for their ability to purify mitochondrial malate dehydrogenase (mMDH) from bovine heart. All but two biomimetic-dye adsorbents displayed higher purifying ability for MDH, compared to nonbiomimetic-dye adsorbents. Furthermore, immobilised anthraquinone-dyes were able to discriminate between the mitochondrial and the cytoplasmic MDH isoenzymes, binding only to the former. One immobilised biomimetic-dye (BM5), bearing as biomimetic terminal moiety 4-aminophenyloxanylic acid, showed the highest purifying ability. This affinity adsorbent was exploited in the purification of mMDH from unpretreated bovine heart extract in one-step. The procedure afforded mMDH at 54% overall yield and of specific activity approx. 1300 U mg-1 (25 degrees C), using step-elution with a mixture containing 0.1 mM beta-nicotinamide adenine dinucleotide (NAD+) and 1.5 mM sulphite. Commercial analytical-grade bovine heart mitochondrial MDH, when assayed under identical conditions, gave a specific activity not exceeding 950 U mg-1. The well-known adsorbent Cibacron blue 3GA-agarose exhibited 8% lower recovery and 25% lower purification for mMDH. The product obtained from the procedure based on the BM5-adsorbent was free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase, and since it has also shown high specific activity, it should be suitable for analytical applications.

Abstract: The mode of interaction of two oxalate-recognizing enzymes, oxalate oxidase (OXO) and oxalate decarboxylase (OXD), with carboxyl-terminal biomimetic monochlorotriazinyl dyes (BM) was studied. Determinations of KD values of the respective dye-enzyme complexes by difference spectra, and kinetic inhibition studies, were employed. Oxalate-mimetic (biomimetic) dye-ligands bear a terminal carboxyl-moiety linked to the reactive chlorotriazine ring, thus mimicking the organic acid substrate of the enzymes. OXO preferred binding to BM dyes which exhibited carboxyl-aromatic terminal functions, whereas OXD has shown preference for dye-ligands bearing terminal aliphatic biomimetic moieties of moderate length. Dye binding to OXO and OXD was accompanied by a characteristic spectral change in the range 500–850 nm. Mixed-type forces (electrostatic and hydrophobic) are present in the dye · OXO complex, whereas electrostatic interactions play a dominant role in the dye · OXD complex. Biomimetic dyes bearing a m-aminobenzoate (BM1) and mercaptopyruvate (BM6) at the terminal biomimetic moiety, exhibited the highest affinity for oxalate oxidase and oxalate decarboxylase, respectively. These dyes, when compared with commercial Cibacron blue 3GA, show a decrease of their KD with OXO and OXD by 22- and 35-fold, respectively. The BM1 ligand behaved as non-linear mixed type inhibitor of OXO with respect to oxalate (Ki 5.1 μM for the OXO · BM1 complex, and K′i 0.2 μM for the BM1 · OXO · oxalate complex), whereas BM6 behaved as competitive inhibitor of OXD against oxalate as variable substrate (Ki 25.4 μM).

Abstract: Three biomimetic dye ligands bearing as a triazine-linked terminal moiety a carboxylated structure, which mimics substrates and inhibitors of L-lactate dehydrogenase (LDH), were immobilized on cross-linked agarose Ultrogel A6R. These biomimetic dyes are purpose-designed analogues of commercial monochlorotriazine Cibacron Blue 3GA (CB3GA) and parent dichlorotriazine Vilmafix Blue A-R (VBAR). The corresponding biomimetic adsorbents, along with non-biomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify LDH from bovine heart crude extract. When compared with non-biomimetic adsorbents, all biomimetic adsorbents exhibited a higher purifying ability. Further, one immobilized biomimetic dye, bearing mercaptopyruvic acid as biomimetic moiety, displayed the highest purifying ability. The concentration of immobilized dye affected both the capacity and the purifying ability of the affinity column, exhibiting an optimum value 2.2 mumol dye/g moist gel. This affinity adsorbent was exploited for the purification of LDH from bovine heart in a two-step procedure. The procedure consisted in a biomimetic dye affinity chromatography step (NAD+/sulphite elution, 25-fold purification, 64% step yield), followed by DEAE-agarose ion-exchange chromatography (1.4-fold purification, 78% step yield). The purified enzyme exhibited a specific activity of ca. 480 u/mg at 25 degrees C (content of impurities: pyruvate kinase and glutamic-oxaloacetic transaminase were not detected; malate dehydrogenase, 0.01%), compared with ca. 250 u/mg of commercial bovine heart LDH (malate dehydrogenase, 0.05%) suitable for analytical purposes.

Abstract: Seven chimeric biomimetic dye-ligands (BM) are purpose-designed and synthesized by specific structural modification of the parent anthraquinone dichlorotriazine dye Vilmafix blue A-R (VBAR). Each BM dye is composed of two enzyme-recognition moieties. The terminal biomimetic moiety bears a variable carboxylated structure linked to the triazine ring, thus mimicking the substrate of formate dehydrogenase (FDH). The anthraquinone moiety remains the same as that of the parent dye and recognizes the nucleotide-binding area of the target enzyme. Dyes are purified by liquid column chromatography (typically 99%), analyzed by liquid-paper chromatography, thin-layer chromatography, and high-performance liquid chromatography, and their lambda max and epsilon values are determined. The ability of dyes to act as affinity ligands versus Candida boidinii FDH is evaluated by kinetic studies and determining KD values from both difference spectra and enzyme inactivation studies. The parent dichlorotriazine dye VBAR binds specifically and irreversibly to FDH (k3 0.19 min-1; KD 19.3 microM). The inactivation of the NAD(+)-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH, and ADP. Quantitatively inhibited FDH contained approx 1 mol of dye per mole of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM each of NAD+, NADH, and ADP or by extensive dialysis or gel filtration chromatography. The monochlorotriazine BM dyes do not inactivate FDH but inhibit competitively the inactivation by VBAR. When compared to VBAR and Cibacron blue 3GA (CB3GA), all BM dye-ligands exhibited lower KD values. FDH generally preferred binding to BM ligands which bore an aromatic terminal biomimetic moiety substituted with a monocarboxyl group rather than an alpha-ketoacid. Dye binding to FDH is accompanied by a characteristic spectral change in the range 550-800 nm. This phenomenon is perturbed after titration by increasing amounts of NAD+. Electrostatic interactions appeared to play a dominant role in the dye.FDH complex. The BM dye-ligand bearing a m-aminobenzoate at its terminal biomimetic moiety (BM1) exhibited the highest affinity (KD 1.6 microM, 8.0-fold decrease over CB3GA). BM1 differentiated between the binding sites of FDH, displaying uncompetitive inhibition with respect to NAD+ (Ki 15.6 microM) and competitive with respect to formate (Ki 18.1 microM).

Abstract: The mode of interaction of the ketocarboxyl-group-recognizing enzyme oxaloacetate decarboxylase (OXAD) from Pseudonomas sp., with purpose-designed (keto)-carboxyl-terminal biomimetic monochlorotriazinyl-dyes (BM) and parent dichlorotriazinyl-dye Vilmafix blue A-R (VBAR) was investigated. Kinetic inhibition studies and determinations of KD values of the respective dye-enzyme complex from both difference spectra and enzyme inactivation studies were employed. Substratemimetic (biomimetic) dye-ligands bear a terminal (keto)carboxyl-moiety linked to the reactive chlorotriazine ring, thus mimicking the organic acid substrate of OXAD. Dichlorotriazine-dye VBAR bound specifically and irreversibly to OXAD (k3 0.22 min-1). The inactivation of OXAD by VBAR was enhanced in the presence of 1 mM Mn+2 (KD 67.2 microM) but in the absence of metal cation was decreased (KD 117 microM). The metal cation behaves as a partial competitive activator. Either of binary complexes dye.OXAD and OXAD.Mn+2 could be formed first, prior to addition of the third constituent to form the ternary complex, although the former route may be favored. The pKa of the catalytically important nucleophile, involved in the specific modification of OXAD, was calculated to 7.4. Biomimetic monochlorotriazine dyes have failed to inactivate OXAD but inhibited competitively the inactivation by VBAR. When compared to commercial VBAR and Cibacron blue 3GA (CB3GA), all BM ligands show lower KD values, therefore, higher affinity for the enzyme. OXAD preferred binding to BM dyes which exhibited a large aliphatic ketocarboxyl-terminal biomimetic moiety. Dye binding to OXAD was accompanied by a characteristic spectral change in the range 550-800 nm. Electrostatic interactions appeared to play a dominant role in the dye.OXAD complex. The BM ligand bearing an aminoethyloxamate as its terminal biomimetic moiety (BM7) displayed the highest affinity (KD 0.5 or 7.0 microM; approx 10-fold decrease over CB3GA). The BM7 ligand behaved as competitive inhibitor (Ki 98 microM) of oxaloacetate decarboxylase against oxaloacetate as variable substrate.

Abstract: Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron(R) Blue 3GA (CB3GA), and parent dichloro-triazine dye, Vilmafix Blue A-R (VBAR), bearing a car-boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press-disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4 degrees C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap-topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( K(D)) of the complex between immobilized BM6 and FDH was found to equal 0.05 microM. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single-band on sodium dodecyl sulphate poly-acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30 degrees C).

Abstract: The quality criteria imposed on several biochemicals are stringent, thus, high-separation purification technology is important to downstream processing. Affinity-based purification technologies are regarded as the finest available, and each one differs in its purifying ability, economy, processing speed and capacity. The most widely used affinity technology is classical affinity chromatography, however, other chromatography-based approaches have also been developed, for example, perfusion affinity chromatography, hyperdiffusion affinity chromatography, high-performance affinity chromatography, centrifugal affinity chromatography, affinity repulsion chromatography, heterobifunctional ligand affinity chromatography and the various chromatographic applications of 'affinity tails'. On the other hand, non-chromatographic affinity technologies aim at high throughput and seek to circumvent problems associated with diffusion limitations experienced with most chromatographic packings. Continuous affinity recycle extraction, aqueous two-phase affinity partitioning, membrane affinity filtration, affinity cross-flow ultrafiltration, reversible soluble affinity polymer separation and affinity precipitation are all non-chromatographic technologies. Several types of affinity ligands are used to different extents; antibodies and their fragments, receptors and their binding substances, avidin/biotin systems, textile and biomimetic dyes, (oligo)peptides, antisense peptides, chelated metal cations, lectins and phenylboronates, protein A and G, calmodulin, DNA, sequence-specific DNA, (oligo)nucleotides and heparin. Likewise, there are several support types developed and used; natural, synthetic, inorganic and composite materials.

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Abstract: Transmissible spongiform encephalopathies (TSE) or prion diseases are a group of neurodegenerative diseases that include Creutzfeldt—Jakob’ s disease in humans, bovine spongiform encephalopathy of cattle, and scrapie in sheep and goats. The main pathogenic event is the conversion of cellular prion protein (PrP) from the normal, enzyme-sensitive (PrPsen), to the insoluble proteinase K-resistant isoform (PrPres). In the present work we describe the development of a structure-activity profile using affinity chromatography for triazine-based (1,3,5-trichloro-2,4,6-triazine, Trz) biomimetic absorbents. The results showed differences in binding specificity of the affinity adsorbents for recombinant mouse PrP. One immobilized ligand, bearing L-Phe and L-Asp linked on the Trz ring (Phe-Trz-Asp), displayed high binding capacity and therefore high affinity for recombinant PrP. Biocomputing analysis allows the identification on PrP of a large surface pocket with positive electrostatic potential able to accommodate the Phe-Trz-Asp biomimetic ligand. PrP-binding ligands could have possible applications in TSE therapy by stabilizing the structure of PrP and inhibiting PrP aggregation. Alternatively, may be useful for detecting or removing a prion protein from a sample, such as a biological fluid.

Abstract: L-Asparaginase (E.C.3.5.1.1, L-ASNase) catalyzes the hydrolysis of L-Asn, producing L-Asp and ammonia. This enzyme is an anti-neoplastic agent; it is used extensively in the chemotherapy of acute lymphoblastic leukemia (ALL). L-asparaginase from Erwinia carotovora (EcaL-ASNase) was cloned and expressed in E. coli. The enzyme was purified to homogeneity by a two-step procedure comprising cation-exchange chromatography and affinity chromatography on immobilized L-asparagine and subjected to thermal inactivation studies. Thermodynamic parameters (Ea, ΔH≠ and ΔS≠) for the thermal inactivation process of the enzyme were determined. It was concluded that the low thermal stability of the enzyme is of entropic origin and is most likely due to structural determinants that cause a higher degree of local disorders at specific locations.

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Abstract: Glutathione transferases (GSTs) are enzymes involved in cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of electrophilic compounds of both endogenous and exogenous origins. This conjugation reaction usually makes the electrophilic substrates more water soluble and, thereby, facilitates their excretion from the body. Determination of metabolic properties of a new chemical entity (NCE) is one of the most important steps during the drug discovery and development process. Nowadays, in vitro methods are used for early estimation and prediction of in vivo metabolism of NCEs. In this review detailed descriptions are given of several biotransformation reactions catalyzed by GSTs that can be used at very early phases of drug development, thereby enabling unsuitable candidates to be eliminated from consideration much earlier in the drug discovery process. Knowledge of the structure-function relationships in classes of compounds that are substrates for GSTs enables the design of molecules that can be stable, or labile which has potential applications in drug and prodrug design.

Abstract: Plant molecular farming is a recently developed biotechnology industry that uses plants as biomolecular reactors, capable of producing large amounts of recombinant therapeutic proteins safely and inexpensively. To capitalize on the advantages of plant-based expression systems, it is necessary to design an economical downstream processing strategy. In the present study, we describe the design, synthesis and application of new biomimetic affinity adsorbents for the purification of anti-HIV monoclonal antibody 2F5 (mAb 2F5). The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathione transferases (GSTs), plays an important role in biomolecular recognition and quaternary structure integrity. This motif was used for the de novo design and combinatorial synthesis of a 13-membered solid-phase ligand library, employing as a lead ligand the Phe-Trz-X structure, mimicking the LAK motif. 1,3,5-Triazine (Trz) was used as the scaffold for assembly, substituted with different LAK-mimetic amino acids. The LAK-mimetic adsorbents were evaluated for their ability to bind and purify mAb 2F5 from corn extract. The results demonstrate that the principle of natural recognition found in the lock-and-key motif, when exploited in de novo combinatorial design, may lead to synthetic affinity ligands, useful in downstream processing and proteomic research.

Abstract: CONTENTS. A. PREFACE. B. ENZYME STRUCTURE AND MECHANISM. B.1. Nomenclature and Classification of Enzymes. B.2. Basic Elements of Enzyme Structure. B.2.1. The Primary Structure of Enzyme. B.2.2. The Three-Dimensional Structure of Enzymes. B.3. Theory of Enzyme Catalysis and Mechanism. B.4. Coenzymes, Prosthetic groups, and Metal ion Cofactors. B.5. Kinetics of Enzyme-Catalyzed Reactions. B.6. Enzyme Dynamics during Catalysis. C. ENZYME PRODUCTION. C.1. Enzyme Heterologous Expression. C.2. The Choice of Expression System. C.2.1. Bacterial Cells. C.2.2. Mammalian Cells. C.2.3. Yeast. C.2.4. Filamentous Fungi. C.2.5. Insect Cells. C.2.6. Dictyostelium discoideum. C.2.7. Trypanosomatid protozoa. C.2.8. Trangenic Plants. C.2.9. Transgenic Animals. C.3. Enzyme Purification. C.3.1. Ion-exchange Chromatography. C.3.2. Affinity Chromatography. D. ENZYME ENGINEERING. D.1. Tailor-Made Enzymes by Protein Engineering. D.2. Rational Enzyme Design. D.3. Directed Enzyme Evolution. E. IMMOBILIZED ENZYMES. E.1. Methods for Immobilization. E.2. New Approaches for Oriented Enzyme Immobilization: the Development of Enzyme Arrays. F. ENZYME UTILIZATION IN INDUSTRY. G. REFERENCE LIST.

Abstract: Natural enzymes and proteins often need to be optimized in order to be used in an industrial process or as a candidate product for diagnostics or therapeutics applications. In contrast to earlier attempts at the rational design of new enzyme variants, directed evolution is a much faster, more effective, and less expensive approach. Directed evolution can be described as the process for generating enzymes, proteins, entire metabolic pathways, or even entire genomes with desired and improved properties. Recent advances in the ability to create genetic diversity and to select or screen for improved functions in large libraries of enzyme variants are being combined aiming at solving difficult molecular design problems. With these new advances the widespread use of biocatalysts in all areas of biotechnology will be ensured in the coming decade.

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Abstract: Affinity chromatography (Clonis, 1988; 1990; Labrou & Clonis, 1994; Garg et al. 1996; Finette et al. 1997) is the most powerful technique used in protein purification, especially in cases where the quality criteria imposed on the final product are stringent. In principle, affinity chromatography exploits natural bio-recognition phenomena for the formation of specific reversible complexes between a ligand, immobilized on an insoluble porous support packed in a column, and the complementary ligand-binding sites on the biomolecule to be isolated. Some of the most widely used ligands in affinity chromatography have been several reactive chlorotriazine dyes (Clonis, 1988; 1990; 1991; Labrou & Clonis, 1994; Garg et al. 1996, Clonis et al. 2000). In particular, immobilized Cibacron Blue 3GA or F-3GA (CB3GA) has found a plethora of applications in affinity chromatography (Labrou & Clonis, 1994; Garg et al. 1996; Clonis et al. 2000), because of its broad spectrum of interaction with many proteins.

Abstract: Molecular recognition provide the basis for the selective interaction of biological macromolecules. This selective interaction is exploited in separation science by the technique commonly known as affinity chromatography [1,2,3]. This technique has provided, by far, the more selective tool to purify a large number of macromolecules from a crude biological mixture [1,2]. For biotechnology, the discovery of recognition molecules (ligand) and the understanding of their interaction with its binding sites (protein) provide the starting point to develop an affinity-based purification method. Suitable tranditional pairs of protein and ligand combinations for affinity chromatography are antigen-antibody, drug-receptor, glycoprotein-lectin and enzyme-substrate/cofactor/inhibitor [4]. More resently generic ligand supports capable of purifying proteins enginered with an affinity-tag are found wide application for the puriffication of recombinant proteins [5,6,7]. Many of the above mentioned adsorbents are ready-made commercial available products, but very often the use of affinity chromatography requires that the resercher to synthesize by himself the adsorbent which will fulfill all the necessary requirements for the particular purification. This chapter attempts to guide the researcher in the synthesis and use of affinity adsorbent as well as to present the basic chromatographic operations for protein purification.

Abstract: Affinity chromatography has proven to be the most effective technique for the purification and separation of proteins from complex mixtures (1). Although affinity adsorbents based on biological ligands such as immobilized antibodies, lectins and nucleotide cofactors appear to be highly successful, their use at a preparative scale is limited because of their instability, expense, and low capacity (1). Synthetic affinity ligands, such as reactive chlorotriazine dyes, have become an integral part of affinity-based protein purification methods for a number of reasons. The dyes are inexpensive, chemical immobilization of the dyes to the matrix is easy and the resultant dye-adsorbents are resistant to chemical or biological degradation, the protein binding capacity is high and far exceeds the binding capacity exhibited by biological ligands (1,2). The main disadvantage of reactive chlorotriazine dyes appears to be their moderate selectivity, which may limit their use. On the other hand, their lack of selectivity, in certain circumstances, may be beneficial, as it circumvents the requirement for a different adsorbent for each putative purification (3).

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Abstract: The tachykinins are a family of peptide neurotransmitters which are widely distributed in the nervous system and modulate important biological actions. All tachykinins share the highly conserved C-terminal sequence Phe-X-Gly-Leu-MetNH2 which appears to possess the biological activity, whereas their divergent N-terminal region defines receptor specificity. Mammalian tachykinins, Substance P (SP), Neurokinin A (NKA), and Neurokinin B (NKB), exert their effects by activating the G-protein-coupled receptors Neurokinin-1 (NK-1), Neurokinin-2 (NK-2), and Neurokinin-3 (NK-3), respectively. Considerable progress has been made in the elucidation of the three-dimensional structure of the tachykinin peptides either free, or bound to the receptor. These developments are useful for the rational design of novel peptide-ligands with highly specific biological properties.