Les benzoxaboroles (qui sont les dérivés cycliques des acides boroniques) s’imposent comme une nouvelle classe de molécules intéressantes, pour la formulation de nouveaux agents thérapeutiques (notamment avec la commercialisation de l’AN2690, dérivé fluoré du benzoxaborole, en tant qu’antifongique), tout comme la formation de matériaux fonctionnels. A ce jour, il n’y a pas eu de mise au point de synthèse systématique de petites molécules benzoxaboroles multivalentes, limitant ainsi leur utilisation. Dans le cadre de cette thèse, différentes approches de synthèse ont été développées et ont permis l’obtention de 12 nouveaux composés bi- ou tri-valents. La principale réactivité de ces composés est leur capacité à complexer les cis-diols. Étant donné que la force de l’interaction est dépendante de la nature du substrat, il est important de pouvoir l’analyser en détail. La complexation entre les benzoxaboroles avec les diols est généralement réalisée par la méthode spectrofluorimétrique qui est controversée, particulièrement dans le cas des molécules organoborées multivalentes. Pour analyser en détail la stœchiométrie de ces complexations, une méthode RMN multinucléaire en solution a ici été développée. Les composés bi- ou tri-valents ont ensuite été testés en tant qu’agent thérapeutique. En effet, récemment, l’activité d’inhibition contre l’anhydrase carbonique de motifs benzoxaboroles monovalents a été discutée dans la littérature. La multivalence nos molécules pouvant être un avantage, leur constante d’inhibition contre l’anhydrase carbonique ont été mesurées. Enfin, dans un tout autre domaine, la réactivité avec les diols et la multivalence de nos composés ont été mises en avant par la formation de nouveaux complexes moléculaires. / Benzoxaboroles (which are cyclic derivates of boronic acids) are emerging as an interesting class of molecules for the design of news therapeutic agents (in particular with the commercialization of AN2690, which is a fluorinated derivative of benzoxaborole, as an antifungal agent), as well as for the formation of functional materials. However, to date, only few syntheses of small molecules of multivalent benzoxaborole have been described in the literature. In this thesis, different approaches were explored to isolate 12 new bi- or tri-valent benzoxaboroles. The main reactivity of these compounds is their ability to bind to cis-diols. Given that the strength of interaction between an organoboron molecule and a diol depends on the nature of the interacting counterparts, it is important to be able to measure it in detail. The complexation between benzoxaboroles and cis-diols is usually studied by a spectrofluorimetric method, which is controversial, especially in the case of multivalent benzoxaboroles. To analyse in detail the stoichiometry of these complexation, a multinuclear NMR method in solution was developed in this thesis. The di- or tri-valent benzoxaboroles were then tested as therapeutic agents. Indeed, recently, the activity of monovalent benzoxaborole against carbonic anhydrases was discussed in the literature. Since the multivalence of our molecules could be a benefit, the measurement of their inhibition constants against carbonic anhydrases was performed. Finally, in a completely different type of application, their reactivity toward cis-diols and their multivalence was put forward to create new complexes.

L'hypoxie est une caractéristique saillante de nombreuses tumeurs solides et provient d'un apport vasculaire inadéquat et immature, entraînant une diminution l'apport d'oxygène et nutriments. Ces régions hypoxiques montrent une résistance aux modalités de traitement classiques telles que la radio et la chimiothérapie et sont associées à une survie insuffisante. Dans des conditions hypoxiques,le HIF-1α améliore l'expression de nombreux gènes cibles,l'un d'eux étant l'anhydrase carbonique IX (CAIX). La CAIX est une enzyme transmembranaire,impliquée dans l'hydratation réversible du dioxyde de carbone au bicarbonate et au proton. L'objectif de cette thèse était de cibler CAIX en utilisant divers médicaments à double cible combinés avec des radiosensibilisateurs, des médicaments cytotoxiques etdes médicaments bio-réductibles. Dans cette thèse,plusieurs approches de ciblage CAIX ont été étudiées.Étant donné que les nitroimidazoles sont de bons sensibilisants cellulaires hypoxiques, nous avons conçu plusieurs composés à double cible existant par combinaison d'un nitroimidazole et d'une fraction inhibitrice d'anhydrase carbonique IX (chapitre 3). Auparavant, notre groupe a montré que le dérivé à base de sulfamide 7 a amélioré l'efficacité thérapeutique de l'irradiation de manière dépendante la CAIX avec un rapport d'amélioration de la sensibilisation (SER) de 1,50, ce qui est plus élevé que plusieurs radiosensibilisateurs testés cliniquement tels que le misonidazole et le nimorazole. L'efficacité de la chimiosensibilisation a été observée lors de la combinaison de 7 avec doxorubicine chez des souris porteurs de tumeur HT29 (chapitre 3).Une approche à double cible similaire peut être exploitable pour délivrer des médicaments cytotoxiques vers des cellules exprimant CAIX, ce qui entraîne une administration ciblée spécifique de la tumeur et par conséquent réduit la toxicité normale des tissus. Le chapitre 4 décrit la conception et la synthèse de nouvelles séries de composés à double cible combinant plusieurs médicaments anti-cancéreux. Un seul composé,c'est-à-dire un dérivé ATRi, a montré une efficacité supérieure en association avec un rayonnement dans des cellules surexprimant CAIX par rapport aux cellules dépourvues d'expression CAIX.Le chapitre 5 décrit la conception et la synthèse de divers dérivés de nitroimidazole bio-réductibles,des agents alkylants de moutarde d'azote et des dérivés de N-oxyde combinés avec une fraction inhibitrice de l'anhydrase carbonique IX. Tous ces composés présentent des profils d'inhibition faibles à modérés vers plusieurs isoformes CA testées. Nous avons observé que différentes substitutions et liens dans la même famille de composés influencent la capacité de liaison à CAIX.Pour élargir notre étude sur les médicaments bio-réductibles, le chapitre 6 décrit la conception et la synthèse de 2, 5-nitroimdazole et moutarde d'azote combinées avec des inhibiteurs de l'anhydrase carbonique IX par un agent de liaison de carbamate. Le dérivé 2-nitroimidazole 1b a révélé une cytotoxicité dans les lignées cellulaires HT29 et HCT116 et pourrait être expliqué par le potentiel de réduction des 2-nitroimidazoles par rapport aux 5-nitroimidazoles, car nos résultats n'ont montré aucune cytotoxicité des dérivés du 5-nitroimidazole. Nos futures études visent à optimiser l'efficacité radiosensibilisante de 2b et 2c explorent davantage les propriétés cytotoxiques de 1b.En conclusion, cette thèse a montré que le double nitroimidazole cible combiné à la CAIXi augmente l'efficacité des modalités de traitement standard telles que la chimiothérapie et la radiothérapie. Le ciblage de la CAIX avec la combinaison de médicaments cytotoxiques continue d'être une approche intéressante pour les tumeurs hypoxiques cibles à l'avenir. Les médicaments bio-réductibles avec des potentiels de réduction plus élevés serviraient d'agents cytotoxiques potentiels aux tumeurs hypoxiques, ce qui réduirait la toxicité tissulaire normale. / Hypoxia is a salient feature in many solid tumors and arises due to an inadequate and immature vascular supply resulting in a decreased delivery of oxygen and nutrients. These hypoxic regions show resistance towards conventional treatment modalities such as radio- and chemotherapy and are associated with poor survival. Under hypoxic conditions HIF-1α enhances the expression of many target genes, one of them being carbonic anhydrase IX (CAIX).CAIX is a transmembrane enzyme, which is involved in reversible hydration of carbon dioxide to bicarbonate and a proton.The aim of this thesis was to target CAIX using various dual target drugs combined with radiosensitizers, cytotoxic drugs and bio-reducible drugs.In this thesis several CAIX targeting approaches have been investigated.Since nitroimidazoles are good hypoxic cell sensitizers,we have designed several dual target compounds existing out of a combination of a nitroimidazole and a carbonic anhydrase IX inhibitory moiety (Chapter 3). It has been shown that extracellular acidity limits the uptake of weak basic chemotherapeutic drugs, such as doxorubicin, and thereby decreases its efficacy. We hypothesized that combining these nitroimidazole moieties with a sulfonamide/sulfamide/sulfamate to target CAIX results in a decrease in extracellular acidosis and sensitizes hypoxic tumors to chemo- and radiotherapy. Previously, our group has shown that the sulfamide-based derivative 7 enhanced the therapeutic efficacy of irradiation in a CAIX dependent manner with a sensitization enhancement ratio of 1.50,which is higher than several clinically tested radiosensitizers such as misonidazole and nimorazole. Chemosensitization efficacy was observed upon combination of 7 with doxorubicin in HT29 tumor-bearing mice (Chapter 3).A similar dual target approach may be exploitable to deliver cytotoxic drugs towards CAIX expressing cells, resulting in a specific tumor targeted delivery and consequently reduced normal tissue toxicity. Chapter 4 describes the design and synthesis of new series of dual target compounds combining several anti-cancer drugs, Only one compound, i.e. an ATRi derivative, showed a higher efficacy in combination with radiation in CAIX overexpressing cells as compared to cells lacking CAIX expression.Chapter 5 describes the design and synthesis of various bio-reducible nitroimidazole derivatives, nitrogen mustard alkylating agents and N-oxide derivatives combined with a carbonic anhydrase IX inhibiting moiety. All these compounds showed weak to moderate inhibition profiles towards several tested CA isoforms. We have observed that different substitutions and linkers within the same family of compounds influence the binding capacity to CAIX. For example derivatives 17 and 20 belong to same family, but have a different linker and substitution of the aromatic ring, leading to a different binding capacity towards CAIX.To expand our study on bio-reducible drugs, Chapter 6 describes the design and synthesis of 2-, 5-nitroimdazole and nitrogen mustards combined with carbonic anhydrase IX inhibitors by a carbamate linker. The 2-nitroimidazole derivative 1b revealed cytotoxicity in HT29 and HCT116 cell lines and might be explain by the higher reduction potential of 2-nitroimidazoles compared to 5-nitroimidazoles, since our results did show no cytotoxicity of the 5-nitroimidazole derivatives. Our future studies aim to optimize the radiosensitizing efficacy of 2b and 2c and further explore the cytotoxic properties of 1b.In conclusion, this thesis showed that the dual target nitroimidazole combined with CAIXi increases the efficacy of standard treatment modalities such as chemo and radiotherapy. Targeting CAIX with combination of cytotoxic drugs continues to be an interesting approach to target hypoxic tumors in future. Bio-reducible drugs with higher reduction potentials would serve as potential cytotoxic agents to hypoxic tumors thereby decreasing the normal tissue toxicity.

Les anhydrases carboniques (CAs, EC4.2.1.1) sont une famille ubiquitaire de métalloenzymes à zinc. Ces enzymes catalysent la réaction réversible d'hydratation du dioxyde de carbone en bicarbonate avec la formation d'un proton. Elles jouent ainsi un important rôle dans de nombreux processus physiologiques tels que la respiration, le transport des ions entre les tissus, l´homéostasie et la régulation du pH. Chez l'homme, seize isoformes différents ont été décrits et certains d'entre eux sont impliqués dans divers troubles pathologiques comme le cancer avec les isoformes CA IX et CA XII. Par ailleurs, des deux enzymes, la CA IX est non seulement l'isoforme le plus actif pour la réaction précédemment décrite mais, elle est aussi la plus largement exprimée dans les tumeurs sous hypoxie (carcinome du sein, du colon…). Grâce à son rôle dans l'acidification du microenvironnement tumoral, la CA IX est associée au phénomène de métastases. Il a été démontré que l'inhibition de son activité catalytique permet de réduire non seulement la croissance et la prolifération tumorale mais aussi la résistance de ces tumeurs aux traitements anticancéreux conventionnels. Dans le cadre d'une approche pharmacologique, cette inhibition se fait via des petites molécules possédant en leur sein une fonction liant l'atome de zinc du site actif de l'enzyme. Dans ce manuscrit, nous avons décrit la synthèse de glycoinhibiteurs insaturés inédits à travers le développement d'une nouvelle méthodologie de synthèse, la sulfamidoglycosylation, basée sur le réarrangement de Ferrier puis le développement d'une nouvelle fonction liant l'atome de zinc, l'hydroxylamine-O-sulfonamide, qui a ensuite servi pour la synthèse d'autres glycoinhibiteurs insaturés par sulfonamidoglycosylation. Ces composés ont montré des activités inhibitrices de l'ordre du nanomolaire sur la CA IX et la CA XII. / The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc enzymes. These enzymes catalyse the reversible hydration reaction of carbon dioxide to bicarbonate, releasing a proton in the process. The enzymes are thus key players in numerous physiological processes such as respiration, ion transport between tissues, homeostasis and pH regulation. In humans, sixteen different isoforms have been described and some of them are involved in diverses pathological conditions such as the CA IX and CA XII isoforms in cancer. Furthermore, from the two enzymes, CA IX is not only the most active isoform for the previously described reaction, but also the most widely expressed under hypoxia in hypoxic tumors (breast carcinoma, colon ...). Through its role in the acidification of the tumoral microenvironment, the CA IX is associated with metastases. It has been demonstrated that inhibition of its catalytic activity reduces not only the tumoral growth and proliferation, but also the resistance of these tumors to conventional cancer treatments. As part of a pharmacological approach, the known inhibitors are small molecules bearing a zinc binding function. In this manuscript, we described the synthesis of novel unsaturated glycoinhibitors through the development of a new synthetic methodology, the sulfamidoglycosylation based on the Ferrier rearrangement, and the development of a new zinc binding function, the hydroxylamine-O-sulfonamide which was then used for the synthesis of other unsaturated glycoinhibitors by sulfonamidoglycosylation. These compounds showed nanomolar inhibitory activities against the CA IX and CA XII.

Les anhydrases carboniques (AC) sont des enzymes qui catalysent les réactions d'hydratation du CO2 et d'hydrolyse de l’OCS. L’AC présente dans les plantes et les microorganismes du sol influence le bilan atmosphérique d'OCS ainsi que celui du CO18O car les isotopes de l’oxygène sont échangés avec ceux des pools d'eau pendant l'hydratation duCO2. L’utilisation de l’OCS et du CO18O comme traceurs du cycle du C global ouvre une nouvelle voie pour estimer les contributions de la photosynthèse et de la respiration à grande échelle. Ceci requiert néanmoins une meilleure compréhension des facteurs contrôlant l'activité de l’AC des sols. Nous avons étudié le rôle du pH du sol et des communautés microbiennes sur l'activité de l’AC. Nous avons testé l’hypothèse que l'activité de l’AC serait (H1) inhibée dans les sols acides, et que (H2) les échanges isotopiques CO2-H2O seraient réduits dans les sols alcalins. Nous avons également présumé que l'activité de l’AC serait (H3) positivement corrélée à l'abondance des microorganismes phototrophes, et que (H4) la structure des communautés affecterait différemment les flux de CO18O et d’OCS. Nos résultats valident H1 et H2. Ils montrent aussi que les flux de CO2 dans le sol et l'activité d’AC associée sont positivement corrélés à l'abondance des microorganismes phototrophes (H3), tandis que le dépôt d'OCS dans les sols dépend de l'abondance des champignons (H4). Ces résultats sont en cours d’intégration dans un modèle de l'activité de l’AC des sols mondiaux, ce qui permettra une estimation robuste des flux globaux de photosynthèse et de respiration à partir de bilans atmosphériques de COS et CO18O. / Carbonic anhydrases (CA) are a group of enzymes that catalyse CO2 hydration and OCS hydrolysis. The presence of CA in plants and soil microorganisms is responsible for the largest atmosphere-biosphere exchange of OCS but also CO18O, because oxygen isotopes are exchanged with soil and plant water pools during CO2 hydration. Consequently, CO18O and OCS atmospheric mixing ratios have been proposed as complementary tracers of the global C cycle that could open avenues to estimate the contribution of photosynthesis and respiration at global scales. However, a mechanistic understanding of the drivers of CA activity is required. We investigated the role of soil pH and microbial community on soil CA activity. We hypothesised that CA activity should be(H1) inhibited in acidic soils but that (H2) the associated CO2-H2O exchange would also be reduced in alkaline soils. We further assumed that (H3) soil CA activity would be enhanced by an increase in soil phototrophs abundance, but that (H4) soil community structure would affect differently CO18O and OCS fluxes. Our results confirmed H1 and H2. We also confirmed that soil CO2 fluxes and the associated CA activity were positively correlated with phototrophic communities abundance (H3), while soil OCS uptake and the associated CA activity seemed driven by fungal abundance (H4). These findings are now being incorporated into a model of soil CA activity worldwide that will allow robust estimates of photosynthesis and respiration at large scales from the atmospheric budgets of OCS and CO18O.

In this exploratory study, we investigated total erythrocyte carbonic anhydrase (CA) estrase activity as well as CA I isozyme concentration in patients with diabetes mellitus type II (DM) and healthy individuals of Howard University Hospital community. Total estrase activity of CA was measured spectrophotometrically using p-nitrophenol acetate before and after inhibition with acetazolamide. CA I isozyme was measured by radial immunodiffusion using monoclonal antibody (CA I) in agarose plates. The study involved 20 consented participants; 10 normal (N) and 10 (DM), 21 to 84 years of age. The study was approved by the Howard University Institution Review Board. The CA activity was measured following lysis of cells as U/min/mL and CA I concentration as mg/l. We observed CA activity as 46.3±4(N) and 25±2.1 (DM) whereas CA I concentration as 1896±125 (N) and 1104 ±63 (DM). We speculate that the change in the CA activity may of fundamental importance in the regulation of intracellular; pHi for the basic control of metabolism in diabetes mellitus. Further, we propose that CA activity is a good candidate for a biomarker of diabetes mellitus for the early detection of insulin resistance because the CA activity variation was proportional to the severity of the diabetes.

The enzyme carbonic anhydrase (CA), catalysing the interconversion between CO2 and HCO3', has long been known to be present in plants as well as in animals. Several of the animal isozymes, but none of the plant CAs, have been extensively studied. When the first plant CA cDNA sequences were published in 1990, it was obvious that the animal and plant CAs represent evolutionarily distinct families with no significant sequence homology between the families. Pea CA is synthesised as a precursor and subsequently processed at the import into the chloroplast. When we purified CA from pea leaves two oligomeric forms with molecular masses around 230 kDa were obtained. One form was homogenous while the other form contained subunits of two different sizes. The larger subunit has an acidic and highly charged N-terminal extension, consisting of 37 residues. We propose that the sequence that precedes the cleavage site resulting in the large subunit represents the functional transit peptide, directing CA to the chloroplast. Neither the transit peptide nor the acidic 37-residue peptide were found to affect the folding, activity or oligomerisation of pea CA. Kinetic investigations showed that pea CA requires a reduced environment and high concentrations of buffer for maximal catalytic activity. High buffer concentrations result in a faster turnover of the enzyme (kcat) while the efficiency (kcatlKm) is not affected. This is consistent with a ping-pong mechanism with the buffer as the second substrate. Both kcat and kcatlKm increase with pH but the dependences cannot be described by simple titration curves. SCN' is an uncompetitive inhibitor at high pH and a noncompetitive inhibitor at neutral and low pH. This is in accordance with the mechanistic model, previously proposed for human CAM, involving a zincbound water molecule as a catalytic group. In this model, the carbon dioxide - bicarbonate interconversion, reflected by kcatlKm, is temporally separated from a rate limiting proton-transfer step. At high pH, solvent hydrogen isotope effects obtained for pea CA agree with this scheme, while they do not fit at neutral and low pH. Site-specific mutations of cysteine residues at positions 165, 269 and 272 were difficult to study, either because strong deviations from Michaelis-Menten kinetics were observed, or because the mutants were found in inclusion bodies. However, the mutant H208A was found to be a very efficient enzyme with the highest kcatlKm value obtained for any CA so far, 2.9-108 M'1s '1. With the H208A mutant an increased dependence on high buffer concentrations at low pH was obtained. At high pH, the mutant is more efficient than the unmutated enzyme. The H208A mutant is also more prone to oxidation than the wild-type enzyme. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 4 uppsatser</p> / digitalisering@umu

<p><p>The adsorption of proteins to solid surfaces has been extensively investigated during the past 20-30 years. The knowledge can be applied in biotechnological applications in for example immunoassays and biosensors. Human carbonic anhydrase II is a widely studied protein and the CO₂-activity makes it an interesting candidate for biotechnological purposes. To make this possible, the factors affecting the adsorption of proteins have to be mapped. The stability of the protein is under great influence of the adsorption and the protein tends to undergo conformational changes leading to a molten globule like state upon adsorption. The stability of a protein also affects the extent of conformational changes and the nature of the adsorption. A more stable protein, adsorbs with less structural changes as a consequence of adsorption, and desorbs from the surface more rapidly than a less stable one. Also the hydrophobicity, charge and area of the surface are affecting the interaction with the protein. Still, the same adsorption pattern is noticed for the same protein at different surfaces, leading to the conclusion that the properties of the protein affect the interaction, rather than the properties of the surface. Biosensors containing carbonic anhydrase have been developed. These make measurement and detection of zinc ions possible. To be able to use carbonic anhydrase as a potential agent in biotechnology, attached to solid surfaces, the protein has to be biotechnologically engineered to get a more stable structure, or else the denaturation will destroy this possibility.</p></p>

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories: Loss-of-function diseases Gain-of-toxic-function diseases Infectious misfolding diseases   Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.   The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease. Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

Malaria is one of the biggest current global health problems, and with the increasing occurance of drug resistant Plasmodium falciparum strains, there is an urgent need for new antimalarial drugs. Given the important role of carbonic anhydrase in Plasmodium falciparum (PfCA), it is a potential novel drug target. Heterologous expression of malaria proteins is problematic due to the unusual codon usage of the Plasmodium genome, so to overcome this problem a synthetic PfCA gene was designed, optimized for expression in Pichia pastoris. This gene was also modified to avoid glycosylation, and cloned into the vector pPICZαA under the control of the methanol inducible promoter AOX1. To facilitate export of the protein into the growth medium, the gene was fused in-frame with the α-factor secretion signal from Saccharomyces cerevisiae. The construct was successfully integrated in the genome of P. pastoris GS115, and attempts were made to express the protein and purify it using immobilized metal ion affinity chromatography.In this work, no expression of the PfCA protein could be detected, so further research should focus on optimization of expression conditions, or redesign of the expression vector.

The adsorption of proteins to solid surfaces has been extensively investigated during the past 20-30 years. The knowledge can be applied in biotechnological applications in for example immunoassays and biosensors. Human carbonic anhydrase II is a widely studied protein and the CO2-activity makes it an interesting candidate for biotechnological purposes. To make this possible, the factors affecting the adsorption of proteins have to be mapped. The stability of the protein is under great influence of the adsorption and the protein tends to undergo conformational changes leading to a molten globule like state upon adsorption. The stability of a protein also affects the extent of conformational changes and the nature of the adsorption. A more stable protein, adsorbs with less structural changes as a consequence of adsorption, and desorbs from the surface more rapidly than a less stable one. Also the hydrophobicity, charge and area of the surface are affecting the interaction with the protein. Still, the same adsorption pattern is noticed for the same protein at different surfaces, leading to the conclusion that the properties of the protein affect the interaction, rather than the properties of the surface. Biosensors containing carbonic anhydrase have been developed. These make measurement and detection of zinc ions possible. To be able to use carbonic anhydrase as a potential agent in biotechnology, attached to solid surfaces, the protein has to be biotechnologically engineered to get a more stable structure, or else the denaturation will destroy this possibility.