Mechanism of action of non-synonymous single nucleotide variations associated with α-carbonic anhydrases II, IV and VIII
- Authors: Sanyanga, T. Allan
- Date: 2020
- Subjects: Carbonic anhydrase , Carbonic anhydrase -- Therapeutic use , Nucleotides
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/167346 , vital:41470
- Description: The carbonic anhydrase (CA) group of enzymes are Zinc (Zn2+) metalloproteins responsible for the reversible hydration of CO2 to bicarbonate (BCT or HCO− 3 ) and protons (H+) for the facilitation of acid-base balance and homeostasis within the body. Across all organisms, a minimum of six CA families exist, including, α (alpha), β (beta), γ (gamma), δ (delta), η (eta) and ζ (zeta). Some organisms can have more than one family, with exception to humans that contain the α family solely. The α-CA family comprises of 16 isoforms (CA-I to CA-XV) including the CA-VIII, CA-X and CA-XI acatalytic isoforms. Of the catalytic isoforms, CA-II and CA-IV possess one of the fastest rates of reaction, and any disturbances to the function of these enzymes results in CA deficiencies and undesirable phenotypes. CA-II deficiencies result in osteopetrosis with renal tubular acidosis and cerebral calcification, whereas CA-IV deficiencies result in retinitis pigmentosa 17 (RP17). Phenotypic effects generally manifest as a result of poor protein folding and function due to the presence of non-synonymous single nucleotide variations (nsSNVs). Even within the acatalytic isoforms such as CA-VIII that llosterically regulates the affinity of inositol triphosphate (IP3) for the IP3 receptor type 1 (ITPR1) and regulates calcium (Ca2+) signalling, the presence of SNVs also causes phenotypes cerebellar ataxia, mental retardation, and dysequilibrium syndrome 3 (CAMRQ3). Currently the majority of research into the CAs is focused on the inhibition of these proteins to achieve therapeutic effects in patients via the control of HCO− production or reabsorption as observed in glaucoma and diuretic medications. Little research has therefore been devoted into the identification of stabilising or activating compound that could rescue protein function in the case of deficiencies. The main aim of this research was to identify and characterise the effects of nsSNVs on the structure and function of CA-II, CA-IV and CA-VIII to set a foundation for rare disease studies into the CA group of proteins. Combined bioinformatics approaches divided into four main objectives were implemented. These included variant identification, sequence analysis and protein characterisation, force field (FF) parameter generation, molecular dynamics (MD) simulation and dynamic residue network analysis (DRN). Six variants for each of the CA-II, CA-IV and CA-VIII proteins with pathogenic annotations were identified from the HUMA and Ensembl databases. These included the pathogenic variants K18E, K18Q, H107Y, P236H, P236R and N252D for CA-II. CA-IV included the pathogenic R69H, R219C and R219S, and benign N86K, N177K and V234I variants. CA-VIII included pathogenic S100A, S100P, G162R and R237Q, and benign S100L and E109D variants. CA-II has been more extensively studied than CA-IV and CA-VIII, therefore residues essential to its function and stability are known. To discover important residues and regions within the CA-IV and CA-VIII proteins sequence and motif analysis was performed across the α-CA family, using CA-II as a reference. Sequence analysis identified multiple conserved residues between the two acatalytic CA-II and CA-IV, and the acatalytic CA-VIII isoforms that were proposed to be essential for protein stability. With exception to the benign N86K CA-IV variant, none of the other pathogenic or benign CA-II, CA-IV and CA-VIII SNVs were located at functionally or structurally important residues. Motif analysis identified 11 conserved and important motifs within the α-CA family. Several of the identified variants were located on these motifs including K18E, K18Q, H107Y and N252D (CA-II); N86K, R219C, R219S and V234I (CA-IV); and E109D, G162R and R237Q (CA-VIII). As there were no x-ray crystal structures of the variant proteins, homology modelling was performed to calculate the protein structures for characterisation. In CA-VIII, the substitution of Ser for Pro at position 100 (variant S100P) resulted in destruction of the β-sheet that the SNV was located on. Little is known about the mechanism of interaction between CA-VIII and ITPR1, and residues involved. SiteMap and CPORT were used to identify binding site amino for CA-VIII and results identified 38 potential residues. Traditional FFs are incapable of performing MD simulations of metalloproteins. The AMBER ff14SB FF was extended and Zn2+ FF parameters calculated to add support for metalloprotein MD simulations. In the protein, Zn2+ was noted to have a charge less than +1. Variant effects on protein structure were then investigated using MD simulations. Root mean square deviation (RMSD) and radius of gyration (Rg) results indicated subtle SNV effects to the variant global structure in CA-II and CA-IV. However, with regards to CA-VIII RMSD analysis highlighted that variant presence was associated with increases to the structural rigidity of the protein. Principal component analysis (PCA) in conjunction with free energy analysis was performed to observe variant effects on protein conformational sampling in 3D space. The binding of BCT to CA-II induced greater protein conformational sampling and was associated with higher free energy. In CA-IV and CA-VIII PCA analysis revealed key differences in the mechanism of action of pathogenic and benign SNVs. In CA-IV, wild-type (WT) and benign variant protein structures clustered into single low energy well hinting at the presence of more stable structures. Pathogenic variants were associated with higher free energy and proteins sampled more conformations without settling into a low energy well. PCA analysis of CA-VIII indicated the opposite to CA-IV. Pathogenic variants were clustered into low energy wells, while the WT and benign variants showed greater conformational sampling. Dynamic cross correlation (DCC) analysis was performed using the MD-TASK suite to determine variant effects on residue movement. CA-II WT protein revealed that BCT and CO2 were associated with anti-correlated and correlated residue movement, highlighting at opposite mechanisms. In CA-IV and CA-VIII variant presence resulted in a change to residue correlation compared to the WT proteins. DRN analysis was performed to investigate SNV effects of residue accessibility and communication. Results demonstrated that SNVs are associated with allosteric effects on the CA protein structures, and effects are located on the stability assisting residues of the aromatic clusters and the active site of the proteins. CA-II studies discovered that Glu117 is the most important residue for communication, and variant presence results in a decrease to the usage of the residue. This effect was greatest in the CA-II H107Y SNV, and suggests that variants could have an effect on Zn2+ dissociation from the active site. Decreases to the usage of Zn2+ coordinating residues were also noted. Where this occurred, compensatory increases to the usage of other primary and secondary coordination residues were observed, that could possibly assist with the maintenance of Zn2+ within the active site. The CA-IV variants R69H and R219C highlighted potentially similar pathogenic mechanisms, whereas N86K and N177K hinted at potentially similar benign mechanisms. Within CA-VIII, variant presence was associated with changes to the accessibility of the N-terminal binding site residues. The benign CA-VIII variants highlighted possible compensatory mechanisms, whereby as one group of N-terminal residues loses accessibility, there was an increase to the accessibility of other binding site residues to possibly balance the effect. Catalytically, the proton shuttle residue His64 in CA-II was found to occupy a novel conformation named the “faux in” that brought the imidazole group even closer to the Zn2+ compared to the “in” conformation. Overall, compared to traditional MD simulations the incorporation of DRN allowed more detailed investigations into the variant mechanisms of action. This highlights the importance of network analysis in the study of the effects of missense mutations on the structure and function of proteins. Investigations of diseases at the molecular level is essential in the identification of disease pathogenesis and assists with the development of specifically tailored and better treatment options especially in the cases of genetically associated rare diseases.
- Full Text:
- Date Issued: 2020
- Authors: Sanyanga, T. Allan
- Date: 2020
- Subjects: Carbonic anhydrase , Carbonic anhydrase -- Therapeutic use , Nucleotides
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/167346 , vital:41470
- Description: The carbonic anhydrase (CA) group of enzymes are Zinc (Zn2+) metalloproteins responsible for the reversible hydration of CO2 to bicarbonate (BCT or HCO− 3 ) and protons (H+) for the facilitation of acid-base balance and homeostasis within the body. Across all organisms, a minimum of six CA families exist, including, α (alpha), β (beta), γ (gamma), δ (delta), η (eta) and ζ (zeta). Some organisms can have more than one family, with exception to humans that contain the α family solely. The α-CA family comprises of 16 isoforms (CA-I to CA-XV) including the CA-VIII, CA-X and CA-XI acatalytic isoforms. Of the catalytic isoforms, CA-II and CA-IV possess one of the fastest rates of reaction, and any disturbances to the function of these enzymes results in CA deficiencies and undesirable phenotypes. CA-II deficiencies result in osteopetrosis with renal tubular acidosis and cerebral calcification, whereas CA-IV deficiencies result in retinitis pigmentosa 17 (RP17). Phenotypic effects generally manifest as a result of poor protein folding and function due to the presence of non-synonymous single nucleotide variations (nsSNVs). Even within the acatalytic isoforms such as CA-VIII that llosterically regulates the affinity of inositol triphosphate (IP3) for the IP3 receptor type 1 (ITPR1) and regulates calcium (Ca2+) signalling, the presence of SNVs also causes phenotypes cerebellar ataxia, mental retardation, and dysequilibrium syndrome 3 (CAMRQ3). Currently the majority of research into the CAs is focused on the inhibition of these proteins to achieve therapeutic effects in patients via the control of HCO− production or reabsorption as observed in glaucoma and diuretic medications. Little research has therefore been devoted into the identification of stabilising or activating compound that could rescue protein function in the case of deficiencies. The main aim of this research was to identify and characterise the effects of nsSNVs on the structure and function of CA-II, CA-IV and CA-VIII to set a foundation for rare disease studies into the CA group of proteins. Combined bioinformatics approaches divided into four main objectives were implemented. These included variant identification, sequence analysis and protein characterisation, force field (FF) parameter generation, molecular dynamics (MD) simulation and dynamic residue network analysis (DRN). Six variants for each of the CA-II, CA-IV and CA-VIII proteins with pathogenic annotations were identified from the HUMA and Ensembl databases. These included the pathogenic variants K18E, K18Q, H107Y, P236H, P236R and N252D for CA-II. CA-IV included the pathogenic R69H, R219C and R219S, and benign N86K, N177K and V234I variants. CA-VIII included pathogenic S100A, S100P, G162R and R237Q, and benign S100L and E109D variants. CA-II has been more extensively studied than CA-IV and CA-VIII, therefore residues essential to its function and stability are known. To discover important residues and regions within the CA-IV and CA-VIII proteins sequence and motif analysis was performed across the α-CA family, using CA-II as a reference. Sequence analysis identified multiple conserved residues between the two acatalytic CA-II and CA-IV, and the acatalytic CA-VIII isoforms that were proposed to be essential for protein stability. With exception to the benign N86K CA-IV variant, none of the other pathogenic or benign CA-II, CA-IV and CA-VIII SNVs were located at functionally or structurally important residues. Motif analysis identified 11 conserved and important motifs within the α-CA family. Several of the identified variants were located on these motifs including K18E, K18Q, H107Y and N252D (CA-II); N86K, R219C, R219S and V234I (CA-IV); and E109D, G162R and R237Q (CA-VIII). As there were no x-ray crystal structures of the variant proteins, homology modelling was performed to calculate the protein structures for characterisation. In CA-VIII, the substitution of Ser for Pro at position 100 (variant S100P) resulted in destruction of the β-sheet that the SNV was located on. Little is known about the mechanism of interaction between CA-VIII and ITPR1, and residues involved. SiteMap and CPORT were used to identify binding site amino for CA-VIII and results identified 38 potential residues. Traditional FFs are incapable of performing MD simulations of metalloproteins. The AMBER ff14SB FF was extended and Zn2+ FF parameters calculated to add support for metalloprotein MD simulations. In the protein, Zn2+ was noted to have a charge less than +1. Variant effects on protein structure were then investigated using MD simulations. Root mean square deviation (RMSD) and radius of gyration (Rg) results indicated subtle SNV effects to the variant global structure in CA-II and CA-IV. However, with regards to CA-VIII RMSD analysis highlighted that variant presence was associated with increases to the structural rigidity of the protein. Principal component analysis (PCA) in conjunction with free energy analysis was performed to observe variant effects on protein conformational sampling in 3D space. The binding of BCT to CA-II induced greater protein conformational sampling and was associated with higher free energy. In CA-IV and CA-VIII PCA analysis revealed key differences in the mechanism of action of pathogenic and benign SNVs. In CA-IV, wild-type (WT) and benign variant protein structures clustered into single low energy well hinting at the presence of more stable structures. Pathogenic variants were associated with higher free energy and proteins sampled more conformations without settling into a low energy well. PCA analysis of CA-VIII indicated the opposite to CA-IV. Pathogenic variants were clustered into low energy wells, while the WT and benign variants showed greater conformational sampling. Dynamic cross correlation (DCC) analysis was performed using the MD-TASK suite to determine variant effects on residue movement. CA-II WT protein revealed that BCT and CO2 were associated with anti-correlated and correlated residue movement, highlighting at opposite mechanisms. In CA-IV and CA-VIII variant presence resulted in a change to residue correlation compared to the WT proteins. DRN analysis was performed to investigate SNV effects of residue accessibility and communication. Results demonstrated that SNVs are associated with allosteric effects on the CA protein structures, and effects are located on the stability assisting residues of the aromatic clusters and the active site of the proteins. CA-II studies discovered that Glu117 is the most important residue for communication, and variant presence results in a decrease to the usage of the residue. This effect was greatest in the CA-II H107Y SNV, and suggests that variants could have an effect on Zn2+ dissociation from the active site. Decreases to the usage of Zn2+ coordinating residues were also noted. Where this occurred, compensatory increases to the usage of other primary and secondary coordination residues were observed, that could possibly assist with the maintenance of Zn2+ within the active site. The CA-IV variants R69H and R219C highlighted potentially similar pathogenic mechanisms, whereas N86K and N177K hinted at potentially similar benign mechanisms. Within CA-VIII, variant presence was associated with changes to the accessibility of the N-terminal binding site residues. The benign CA-VIII variants highlighted possible compensatory mechanisms, whereby as one group of N-terminal residues loses accessibility, there was an increase to the accessibility of other binding site residues to possibly balance the effect. Catalytically, the proton shuttle residue His64 in CA-II was found to occupy a novel conformation named the “faux in” that brought the imidazole group even closer to the Zn2+ compared to the “in” conformation. Overall, compared to traditional MD simulations the incorporation of DRN allowed more detailed investigations into the variant mechanisms of action. This highlights the importance of network analysis in the study of the effects of missense mutations on the structure and function of proteins. Investigations of diseases at the molecular level is essential in the identification of disease pathogenesis and assists with the development of specifically tailored and better treatment options especially in the cases of genetically associated rare diseases.
- Full Text:
- Date Issued: 2020
The toxic effect of heavy metals on algal biomass (Spirulina sp.) and carbonic anhydrase activity, an enzyme which is central to algal application in metal precipitation
- Authors: Nightingale, Leigh
- Date: 2004
- Subjects: Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4093 , http://hdl.handle.net/10962/d1007858 , Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Description: Acid rmne drainage (AMD) is a major pollution problem througbout the world, adversely affecting both surface and groundwaters. AMD is principally associated with the mining of sulphide ores. The most commonly associated minerals being sulphur, copper, zinc, silver, gold, lead and uranium. As conventional methods for removing heavy metals from wastewater are often prohibitively expensive, the implementation of biological processes for the removal of heavy metals has become a realistic practice. The objectives of this project was firstly to establish the effect of copper, lead and nickel, heavy metals commonly found in AMD waters, on the enzyme carbonic anhydrase, which is an integral part of the carbon concentrating mechanism (CCM) and secondly, to determine the feasibility of using the alkalinity generated by Spindina for the precipitation of heavy metals from solution. Initially, batch flask experiments were performed and it was found that the algae were able to utilise the bicarbonate supplied in the medium, under CO, limiting conditions, through the induction of their CCM, resulting in the generation of carbonate. The effect of the inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), were also investigated in order to determine the importance of carbonic anhydrase (CA) in inorganic carbon accumulation and photosynthesis. Results obtained were consistent with those observed in literature and it was found that at IOOf.LM AZ and EZ, complete inhibition of photosynthesis and carbonic anhydrase occurred, with no oxygen being evolved. The results obtained from the inhibitor experiments substantiate the findings that carbonic anhydrase is an important part of the CCM, and that the dehydration of bicarbonate to carbon dioxide and hydroxide ions, is in fact an enzymatic process regulated by the enzyme carbonic anhydrase and is essential for efficient photosynthesis. The effect of heavy metals on Spirulina was also investigated. Lead, copper and nickel were all found to cause a reduction in the synthesis of chlorophyll a, which resulted in a decrease in photosynthetic efficiency and eventually death of the culture. The morphology of the algae was also severely affected by heavy metals, with degradation and aJmost complete disintegration of the algal filaments occurring. Using the Wilbur-Anderson assay method, carbonic anhydrase activity was found to be lower in the experimental flasks containing heavy metals, than the control flasks, reducing the algae's ability to utilise the bicarbonate in solution for effective photosynthesis. The Wilbur-Anderson assay method did not prove to be a reliable method for measuring changes in enzyme activity as results were found to be erratic. Therefore attempts were made to use an oxygen electrode as an alternative method for determining the effects of various parameters on enzyme activity and photosynthesis, this proved to be more successful. Because of the toxic effects of heavy metals on Spirulina it was decided that the use of the biogenic alkalinity generated by the algae for the precipitation of heavy metals may be successfully employed as an alternative method for bioremediation and metal recovery. Carbonate reacts readily with metals, therefore the carbonate produced by this algal system was used for the precipitation of metals. It was possible to categorise the precipitation reactions observed into three groups, namely those metals which, a) precipitate as hydroxides, b) precipitate as carbonates generated from the dissociation of bicarbonate and c) metals which can only precipitate if there is free carbonate present in solution.
- Full Text:
- Date Issued: 2004
- Authors: Nightingale, Leigh
- Date: 2004
- Subjects: Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4093 , http://hdl.handle.net/10962/d1007858 , Heavy metals -- Toxicology , Spirulina , Carbonic anhydrase , Algae -- Metabolism , Photosynthesis
- Description: Acid rmne drainage (AMD) is a major pollution problem througbout the world, adversely affecting both surface and groundwaters. AMD is principally associated with the mining of sulphide ores. The most commonly associated minerals being sulphur, copper, zinc, silver, gold, lead and uranium. As conventional methods for removing heavy metals from wastewater are often prohibitively expensive, the implementation of biological processes for the removal of heavy metals has become a realistic practice. The objectives of this project was firstly to establish the effect of copper, lead and nickel, heavy metals commonly found in AMD waters, on the enzyme carbonic anhydrase, which is an integral part of the carbon concentrating mechanism (CCM) and secondly, to determine the feasibility of using the alkalinity generated by Spindina for the precipitation of heavy metals from solution. Initially, batch flask experiments were performed and it was found that the algae were able to utilise the bicarbonate supplied in the medium, under CO, limiting conditions, through the induction of their CCM, resulting in the generation of carbonate. The effect of the inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), were also investigated in order to determine the importance of carbonic anhydrase (CA) in inorganic carbon accumulation and photosynthesis. Results obtained were consistent with those observed in literature and it was found that at IOOf.LM AZ and EZ, complete inhibition of photosynthesis and carbonic anhydrase occurred, with no oxygen being evolved. The results obtained from the inhibitor experiments substantiate the findings that carbonic anhydrase is an important part of the CCM, and that the dehydration of bicarbonate to carbon dioxide and hydroxide ions, is in fact an enzymatic process regulated by the enzyme carbonic anhydrase and is essential for efficient photosynthesis. The effect of heavy metals on Spirulina was also investigated. Lead, copper and nickel were all found to cause a reduction in the synthesis of chlorophyll a, which resulted in a decrease in photosynthetic efficiency and eventually death of the culture. The morphology of the algae was also severely affected by heavy metals, with degradation and aJmost complete disintegration of the algal filaments occurring. Using the Wilbur-Anderson assay method, carbonic anhydrase activity was found to be lower in the experimental flasks containing heavy metals, than the control flasks, reducing the algae's ability to utilise the bicarbonate in solution for effective photosynthesis. The Wilbur-Anderson assay method did not prove to be a reliable method for measuring changes in enzyme activity as results were found to be erratic. Therefore attempts were made to use an oxygen electrode as an alternative method for determining the effects of various parameters on enzyme activity and photosynthesis, this proved to be more successful. Because of the toxic effects of heavy metals on Spirulina it was decided that the use of the biogenic alkalinity generated by the algae for the precipitation of heavy metals may be successfully employed as an alternative method for bioremediation and metal recovery. Carbonate reacts readily with metals, therefore the carbonate produced by this algal system was used for the precipitation of metals. It was possible to categorise the precipitation reactions observed into three groups, namely those metals which, a) precipitate as hydroxides, b) precipitate as carbonates generated from the dissociation of bicarbonate and c) metals which can only precipitate if there is free carbonate present in solution.
- Full Text:
- Date Issued: 2004
Spirulina as a bioremediation agent : interaction with metals and involvement of carbonic anhydrase
- Authors: Payne, Rosemary Anne
- Date: 2000
- Subjects: Spirulina , Bioremediation , Carbonic anhydrase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3909 , http://hdl.handle.net/10962/d1003968 , Spirulina , Bioremediation , Carbonic anhydrase
- Description: Heavy metal contamination from mining and other industrial operations is becoming an increasing problem with regards to the depleting water resources in South Africa. This study involved the investigation of the use of an algal biomass as a possible alternative to the traditional chemical means of removing these metals. When the toxic effects of metals were investigated, Spirulina was found to have a threshold level of about 30 μM for copper, zinc and lead. Copper and zinc appeared to have a direct effect on the photosynthetic pathway, thereby causing a rapid decline in cell growth. Lead on the other hand seemed to affect surface properties and hence took longer to cause deterioration in growth. Although relatively low concentrations of metal may have a toxic effect on the cyanobacterium, Spirulina may have potential as a precipitation agent. The role of Spirulina in the precipitation of heavy metals appears to be through its ability to maintain a high pH in the surrounding medium, possibly through the enzyme carbonic anhydrase. Subsequent studies therefore focused on the assay and isolation of this enzyme. Two different radiotracer assays, in which carbonic anhydrase converts radiolabelled bicarbonate to carbon dioxide, were investigated, but were found to have several problems. Results were insensitive and could not be reproduced. The standard Wilbur-Anderson method subsequently investigated also proved to be insensitive with a tremendous degree of variability. Although not quantitative, SDS-PAGE proved to be the most reliable method of detection, and was therefore used in subsequent procedures. Chlamydomonas reinhardtii was the subject of initial enzyme isolation studies as these procedures are well documented. Although the published protocols proved unsuccessful, affinity chromatography of a membrane stock solution from Chlamydomonas reinhardtii yielded two relatively pure protein bands. These bands were presumed to represent two subunits of carbonic anhydrase, although Western blot analysis would be required to confirm their identity. Purification of carbonic anhydrase from Spirulina, however, proved unsuccessful and results obtained were very inconclusive. Hence, further analysis of Spirulina is required. The possibility of cloning CA from a genomic library was also considered, but suitable primers could not be designed from the aligned sequences.
- Full Text:
- Date Issued: 2000
- Authors: Payne, Rosemary Anne
- Date: 2000
- Subjects: Spirulina , Bioremediation , Carbonic anhydrase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3909 , http://hdl.handle.net/10962/d1003968 , Spirulina , Bioremediation , Carbonic anhydrase
- Description: Heavy metal contamination from mining and other industrial operations is becoming an increasing problem with regards to the depleting water resources in South Africa. This study involved the investigation of the use of an algal biomass as a possible alternative to the traditional chemical means of removing these metals. When the toxic effects of metals were investigated, Spirulina was found to have a threshold level of about 30 μM for copper, zinc and lead. Copper and zinc appeared to have a direct effect on the photosynthetic pathway, thereby causing a rapid decline in cell growth. Lead on the other hand seemed to affect surface properties and hence took longer to cause deterioration in growth. Although relatively low concentrations of metal may have a toxic effect on the cyanobacterium, Spirulina may have potential as a precipitation agent. The role of Spirulina in the precipitation of heavy metals appears to be through its ability to maintain a high pH in the surrounding medium, possibly through the enzyme carbonic anhydrase. Subsequent studies therefore focused on the assay and isolation of this enzyme. Two different radiotracer assays, in which carbonic anhydrase converts radiolabelled bicarbonate to carbon dioxide, were investigated, but were found to have several problems. Results were insensitive and could not be reproduced. The standard Wilbur-Anderson method subsequently investigated also proved to be insensitive with a tremendous degree of variability. Although not quantitative, SDS-PAGE proved to be the most reliable method of detection, and was therefore used in subsequent procedures. Chlamydomonas reinhardtii was the subject of initial enzyme isolation studies as these procedures are well documented. Although the published protocols proved unsuccessful, affinity chromatography of a membrane stock solution from Chlamydomonas reinhardtii yielded two relatively pure protein bands. These bands were presumed to represent two subunits of carbonic anhydrase, although Western blot analysis would be required to confirm their identity. Purification of carbonic anhydrase from Spirulina, however, proved unsuccessful and results obtained were very inconclusive. Hence, further analysis of Spirulina is required. The possibility of cloning CA from a genomic library was also considered, but suitable primers could not be designed from the aligned sequences.
- Full Text:
- Date Issued: 2000
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