Browsing by Author "Michler, Andre"

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    PVPylation of catalase
    (Stellenbosch : Stellenbosch University, 2017-03) Michler, Andre; Klumperman, Bert; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.
    ENGLISH ABSTRACT: Hair greying has been attributed to the loss of pigment-forming melanocytes from the hair follicle. Melanocytes are lost due to a high concentration of hydrogen peroxide, a reactive oxidative species found in the hair follicle, reacting with the melanocytes. Hydrogen peroxide is formed through the melanogenesis process, where melanocytes are formed, which takes place in the growth phase of the hair. Repair mechanisms and enzymes exist to combat this problem, the results of these natural processes are insufficient. One of the enzymes present on hair is catalase. This enzyme catalyzes the decomposition of hydrogen peroxide into water and oxygen. Bioconjugation is a method of attaching polymers as carriers to active molecules for therapeutics and nanomedicine. In this method a polymer is conjugated to an enzyme, protein or biological active molecule with a specific end function. It has been shown that bioconjugation increases the half-life and stability of biological active molecules during their usage. In this thesis we are conjugating poly(N-vinylpyrrolidone) (PVP) to catalase to determine whether the PVP has an effect on the enzymatic activity and structure of catalase. PVP is a polymer synthesized through the polymerization of N-vinylpyrrolidone (NVP). The polymerizations described in this thesis are conducted through Reversible Addition Fragmentation chain Transfer (RAFT) mediated polymerization. In this polymerization a chain transfer agent (CTA) is used. This CTA is used due to its ease of use and control it has over the polymerization and the end functional groups on the CTA that can be used for post polymerization reactions. In this thesis we are functionalizing the one end group to a desired aldehyde functionalized end group, where this end group will be used in the conjugation with catalase. This conjugation to catalase will take place on the lysine amino acid groups present on catalase. RAFT-mediated polymerization was used successfully to polymerize four different molecular weight polymers, each with the desired end group. This end group was then successfully converted to the aldehyde end group needed for the conjugation reaction. Conjugation of PVP through the aldehyde end group was successful and an increase in the molecular weight of the enzyme is seen in SDS-PAGE. This is followed by enzymatic assay analyses, where it was found that conjugation of PVP to catalase does not have a negative effect on the catalytic activity of the enzyme. It was further found that there is actually an increase in the enzymatic activity of catalase if a small amount of polymer is conjugated to catalase. A change in the structure of catalase after conjugation was found. It was also determined that conjugation of a constant number of PVP chains with different molecular weight (below 40 x 103 g/mol), does not have a significant effect on the enzymatic activity of the catalase-PVP conjugate. Thus the conjugation of catalase was successful and catalase can be used in the presence of PVP for the decomposition of hydrogen peroxide.