Interaction of catechol O-methyltransferase with gold and silver nanoparticles
- Authors: Usman, Aminu
- Date: 2018
- Subjects: Parkinson's disease , Methyltransferases , Catechol , Nanoparticles
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61818 , vital:28063 , DOI https://doi.org/10.21504/10962/61818
- Description: Catechol O-methyltransferase (S-adenosyl-Z-methionine: catechol O-methyltransferase; COMT; EC 2.1.1.6) is a ubiquitous enzyme that catalyses the transfer of a methyl group from the cofactor, S-adenosyl-Z-methionine (SAM) to a hydroxyl group of endogenous and exogenous catechol-containing moieties. The physiological role of this enzyme is the methylation and thereby inactivation of the catechol-containing bio-active and bio-toxic compounds, including catechol-neurotransmitters, catechol-estrogens and catechol-containing drugs. Activity of this enzyme is implicated in the treatment of Parkinson’s disease and is associated with other diseases including breast cancer and an array neuropsychological disorders, such as schizophrenia. This thesis explores the use of gold and silver nanoparticles (NPs) (AuNPs and AgNPs) to inhibit the catalytic activity of mammalian COMT. Because of its accessibility and availability, we initially investigated bovine soluble COMT (BSCOMT) from liver tissue. Bioinformatic analyses and structural modeling revealed high (>90%) sequence similarity between BSCOMT and human soluble COMT (HSCOMT). BSCOMT was partially purified to 7.78 fold, 1.65% yield and had a specific activity of 0.052 U/mg. It had pH and temperature optima of 8.5 and 40oC, respectively. The Km, Vmax, Kcat and Kcat/Km towards esculetin methylation were respectively 1.475±0.130 pM, 0.0353±0.001 pmol/ml/min, 1.748 x 10-2±5.0x10-4 min-1 and 1.18x10-2 M-1. min-1. HSCOMT was expressed in Escherichia coli BL21(DE3) which showed optimal activity for esculetin methylation at pH and temperature of 7.0 and 30°C, respectively. It was purified to 5.62 fold, 22.6% yield with a specific activity of 3.85 U/mg. HSCOMT kinetic plots, upon incubation of the reaction mixture at 30°C for 5 min before addition of SAM was hyperbolic with Km, Vmax, Kcat and Kcat/Km values of 1.79 pM, 0.412 pmol/ml/min, 2.08 min-1 and 1.165 M-1. min-1, respectively. AuNPs and AgNPs showed a concentration dependent inhibition of HSCOMT activity upon increasing the 5 min incubation time to 1 h. Interestingly, HSCOMT kinetics, with 1 h incubation at 30°C, showed a sigmoidal curve, as well as increased activity. Incubation of the reaction mixture in the presence of 60 pM AuNPs and/or AgNPs for 1 hreversed the observed sigmoidal to a hyperbolic curve, with kinetic parameters comparable to those of 5 min incubation. SDS-PAGE analyses of HSCOMT after the kinetic experiments showed the enzyme incubated for 5 min as a monomer, while that which was incubated for 1 h migrated substantially as dimer. However, the HSCOMT incubated for 1 h in the presence of 60 pM AuNPs and/or AgNPs migrated as a monomer. This indicated that the extension of the incubation period allowed the dimerization of HSCOMT, which exhibited sigmoidal kinetics and higher activity. The presence of NPs impeded the HSCOMT dimerization which decreased the activity. Varying the concentration of SAM suggested that SAM had an allosteric modulatory effect on HSCOMT. Absorption spectroscopy indicated adsorption of HSCOMT on the gold and silver NP surfaces and the formation of NPs-HSCOMT corona. Fluorescence spectroscopy showed that the interaction of HSCOMT with both gold and silver NPs was governed by a static quenching mechanism, implying the formation of a non-fluorescent fluorophore-NP complex at the ground state. Further fluorometric analyses indicated that both gold and silver NPs had contact with Trp143; that the interactions were spontaneous and were driven by electrostatic interactions. Fourier transform infrared spectroscopic studies showed the adsorption of HSCOMT of the NPs surfaces to cause relaxation of the enzyme’s B-sheet structures. Molecular docking studies indicated involvement of largely hydrophilic amino acids, with the interacting distances of less than 3.5A. These findings signify the potential of nanotechnology in the control of COMT catalytic activity for the management of the COMT-related disorders. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Authors: Usman, Aminu
- Date: 2018
- Subjects: Parkinson's disease , Methyltransferases , Catechol , Nanoparticles
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61818 , vital:28063 , DOI https://doi.org/10.21504/10962/61818
- Description: Catechol O-methyltransferase (S-adenosyl-Z-methionine: catechol O-methyltransferase; COMT; EC 2.1.1.6) is a ubiquitous enzyme that catalyses the transfer of a methyl group from the cofactor, S-adenosyl-Z-methionine (SAM) to a hydroxyl group of endogenous and exogenous catechol-containing moieties. The physiological role of this enzyme is the methylation and thereby inactivation of the catechol-containing bio-active and bio-toxic compounds, including catechol-neurotransmitters, catechol-estrogens and catechol-containing drugs. Activity of this enzyme is implicated in the treatment of Parkinson’s disease and is associated with other diseases including breast cancer and an array neuropsychological disorders, such as schizophrenia. This thesis explores the use of gold and silver nanoparticles (NPs) (AuNPs and AgNPs) to inhibit the catalytic activity of mammalian COMT. Because of its accessibility and availability, we initially investigated bovine soluble COMT (BSCOMT) from liver tissue. Bioinformatic analyses and structural modeling revealed high (>90%) sequence similarity between BSCOMT and human soluble COMT (HSCOMT). BSCOMT was partially purified to 7.78 fold, 1.65% yield and had a specific activity of 0.052 U/mg. It had pH and temperature optima of 8.5 and 40oC, respectively. The Km, Vmax, Kcat and Kcat/Km towards esculetin methylation were respectively 1.475±0.130 pM, 0.0353±0.001 pmol/ml/min, 1.748 x 10-2±5.0x10-4 min-1 and 1.18x10-2 M-1. min-1. HSCOMT was expressed in Escherichia coli BL21(DE3) which showed optimal activity for esculetin methylation at pH and temperature of 7.0 and 30°C, respectively. It was purified to 5.62 fold, 22.6% yield with a specific activity of 3.85 U/mg. HSCOMT kinetic plots, upon incubation of the reaction mixture at 30°C for 5 min before addition of SAM was hyperbolic with Km, Vmax, Kcat and Kcat/Km values of 1.79 pM, 0.412 pmol/ml/min, 2.08 min-1 and 1.165 M-1. min-1, respectively. AuNPs and AgNPs showed a concentration dependent inhibition of HSCOMT activity upon increasing the 5 min incubation time to 1 h. Interestingly, HSCOMT kinetics, with 1 h incubation at 30°C, showed a sigmoidal curve, as well as increased activity. Incubation of the reaction mixture in the presence of 60 pM AuNPs and/or AgNPs for 1 hreversed the observed sigmoidal to a hyperbolic curve, with kinetic parameters comparable to those of 5 min incubation. SDS-PAGE analyses of HSCOMT after the kinetic experiments showed the enzyme incubated for 5 min as a monomer, while that which was incubated for 1 h migrated substantially as dimer. However, the HSCOMT incubated for 1 h in the presence of 60 pM AuNPs and/or AgNPs migrated as a monomer. This indicated that the extension of the incubation period allowed the dimerization of HSCOMT, which exhibited sigmoidal kinetics and higher activity. The presence of NPs impeded the HSCOMT dimerization which decreased the activity. Varying the concentration of SAM suggested that SAM had an allosteric modulatory effect on HSCOMT. Absorption spectroscopy indicated adsorption of HSCOMT on the gold and silver NP surfaces and the formation of NPs-HSCOMT corona. Fluorescence spectroscopy showed that the interaction of HSCOMT with both gold and silver NPs was governed by a static quenching mechanism, implying the formation of a non-fluorescent fluorophore-NP complex at the ground state. Further fluorometric analyses indicated that both gold and silver NPs had contact with Trp143; that the interactions were spontaneous and were driven by electrostatic interactions. Fourier transform infrared spectroscopic studies showed the adsorption of HSCOMT of the NPs surfaces to cause relaxation of the enzyme’s B-sheet structures. Molecular docking studies indicated involvement of largely hydrophilic amino acids, with the interacting distances of less than 3.5A. These findings signify the potential of nanotechnology in the control of COMT catalytic activity for the management of the COMT-related disorders. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
The evaluation of potential dietary media, measurement parameters and storage techniques for use in forensic entomotoxicology
- Mbatha, Erica Isabel Tavares Da Silva
- Authors: Mbatha, Erica Isabel Tavares Da Silva
- Date: 2018
- Subjects: Blowflies -- Feeding and feeds , Blowflies -- Larvae , Blowflies -- Physiology , Blowflies -- Collection and preservation , Poisons -- Analysis , Death -- Causes , Forensic pathology , Forensic entomology , Forensic entomotoxicology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63323 , vital:28393
- Description: The term forensic entomotoxicology was coined by Pounder and is used to describe the process of using insects to determine the presence or absence of toxicants in decomposing corpses. Forensic entomotoxicology is most applicable when the orthodox sources of evidence (i.e. blood and urine) are no longer available for testing due to the degree of putrefaction as a result of the decomposition process. As the field is relatively new, various authors have conducted studies to determine the effects of different toxicants on different insects. These studies have all been conducted in the absence of a standardised protocol and we hypothesise that this has led to conflicting results (i.e. two different authors will conduct a study using the same toxicant and model insect and the effects on the insects will differ significantly). The aim of this thesis was to identify the areas which might have led to the artefacts in the results and identify ways in which to standardise them. The three areas selected were the feeding substrates and the measures taken to quantify growth rate, as well as the preservation techniques that should be used for preserving larval flies. The recommendation from the literature review was that artificial diets would be the most appropriate dietary media to use for entomotoxicological studies. An artificial diet was selected and modified for potential used in entomotoxicological studies. Four different diets (no meat treatment, fish, beef and pork artificial diets) were used to rear Chrysomya chloropyga larvae and their growth rates were measured using length and width. The fly larvae reared on the fish and no meat treatment diets did not reach pupation stage. The beef and pork diets produced the largest larvae and the flies in these treatments reached adult stage. The recommendation was that the beef and pork treatments be tested with various toxicants to establish their stability in the matrix and the diet that provides the toxicants with the most stability should be used for future entomotoxicological studies. The two other factors selected for standardisation were the parameters used to quantify growth rate, as well as the preservation techniques used to store empty Chrysomya chloropyga pupal casings and Calliphora croceipalpis third instar larvae. Previous authors have suggested that width be used as an alternative to length to quantify growth rate. The results from this thesis show that length should continue to be used as the standard parameter because the incremental change in length is much larger than the change in width, and these larger increments allow for greater resolution when estimating the age of the larvae. Various authors have also suggested that pupal casings should be stored without any preservative, whereas fly larvae should be stored in concentrations of ethanol >70%. The results in this thesis have shown that the concentration of ethanol does not make any significant difference to the proportional change of length and width of the empty pupal casings and the third instar larvae. The recommendation is that when selecting the preservation technique, the integrity of the specimen for examination of other evidence (i.e. DNA or toxicological extraction) should take precedence. Although this thesis has not completely standardised the protocol for forensic entomotoxicology, it has indicated the areas that need to be focused on in order for standardisation to occur. Future studies should focus on standardisation, as this makes studies more comparable and ultimately makes entomotoxicological evidence admissible in the court of law.
- Full Text:
- Authors: Mbatha, Erica Isabel Tavares Da Silva
- Date: 2018
- Subjects: Blowflies -- Feeding and feeds , Blowflies -- Larvae , Blowflies -- Physiology , Blowflies -- Collection and preservation , Poisons -- Analysis , Death -- Causes , Forensic pathology , Forensic entomology , Forensic entomotoxicology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63323 , vital:28393
- Description: The term forensic entomotoxicology was coined by Pounder and is used to describe the process of using insects to determine the presence or absence of toxicants in decomposing corpses. Forensic entomotoxicology is most applicable when the orthodox sources of evidence (i.e. blood and urine) are no longer available for testing due to the degree of putrefaction as a result of the decomposition process. As the field is relatively new, various authors have conducted studies to determine the effects of different toxicants on different insects. These studies have all been conducted in the absence of a standardised protocol and we hypothesise that this has led to conflicting results (i.e. two different authors will conduct a study using the same toxicant and model insect and the effects on the insects will differ significantly). The aim of this thesis was to identify the areas which might have led to the artefacts in the results and identify ways in which to standardise them. The three areas selected were the feeding substrates and the measures taken to quantify growth rate, as well as the preservation techniques that should be used for preserving larval flies. The recommendation from the literature review was that artificial diets would be the most appropriate dietary media to use for entomotoxicological studies. An artificial diet was selected and modified for potential used in entomotoxicological studies. Four different diets (no meat treatment, fish, beef and pork artificial diets) were used to rear Chrysomya chloropyga larvae and their growth rates were measured using length and width. The fly larvae reared on the fish and no meat treatment diets did not reach pupation stage. The beef and pork diets produced the largest larvae and the flies in these treatments reached adult stage. The recommendation was that the beef and pork treatments be tested with various toxicants to establish their stability in the matrix and the diet that provides the toxicants with the most stability should be used for future entomotoxicological studies. The two other factors selected for standardisation were the parameters used to quantify growth rate, as well as the preservation techniques used to store empty Chrysomya chloropyga pupal casings and Calliphora croceipalpis third instar larvae. Previous authors have suggested that width be used as an alternative to length to quantify growth rate. The results from this thesis show that length should continue to be used as the standard parameter because the incremental change in length is much larger than the change in width, and these larger increments allow for greater resolution when estimating the age of the larvae. Various authors have also suggested that pupal casings should be stored without any preservative, whereas fly larvae should be stored in concentrations of ethanol >70%. The results in this thesis have shown that the concentration of ethanol does not make any significant difference to the proportional change of length and width of the empty pupal casings and the third instar larvae. The recommendation is that when selecting the preservation technique, the integrity of the specimen for examination of other evidence (i.e. DNA or toxicological extraction) should take precedence. Although this thesis has not completely standardised the protocol for forensic entomotoxicology, it has indicated the areas that need to be focused on in order for standardisation to occur. Future studies should focus on standardisation, as this makes studies more comparable and ultimately makes entomotoxicological evidence admissible in the court of law.
- Full Text:
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