PVPylation of catalase

Michler, Andre (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

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.

AFRIKAANSE OPSOMMING: Haarvergrysing word toegeskryf aan die verlies van die pigmentvormende melanosiete in die haarfollikel. Melanosiete gaan verlore as gevolg van 'n hoë konsentrasie waterstofperoksied, 'n reaktiewe oksideringspesie wat gevind word in die haarfollikel, wat reageer met die melanosiete. Waterstofperoksied word gevorm deur 'n proses wat melanogenesis genoem word, waar melanosiete ook gevorm word, wat plaasvind in die groeifase van die haar. Herstelmeganismes en ensieme bestaan om hierdie probleem te beveg, maar die resultate na 'n tyd is nie bevredegind nie. Katalase is 'n ensiem wat teenwoordig is in hare. Dit verhoog die tempo van waterstofperoksied ontbinding na water en suurstof. Bio-konjugasie is 'n metode waar polimere gebruik word as toevoermiddels vir terapie en nanogeneeskunde. In die metode word 'n polimeer gekonjugeer aan 'n ensiem, proteïen of biologiese aktiewe molekule met 'n spesifiek eindfunksie. Daar is bewys dat bio-konjugasie die halfleeftyd en stabiliteit van hierdie biologiese aktiewe molekules tydens gebruik verhoog. In hierdie tesis word die konjugasie van 'n eksterne katalase tot polyvinylpyrrolidone (PVP) gedoen, om te bepaal of die PVP 'n effek het op die ensiematiese aktiwiteit en struktuur van katalase. PVP is 'n polimeer wat gevorm word met die polimerisasie reaksie van N-vinylpyrrolidone (NVP). Dié polimerisasie reaksie in hierdie studie is gedoen deur omkeerbare addisie fragmentasie kettingoordrag (RAFT) bemiddelde polimerisasie. In hierdie polimerisasie word 'n kettingoordragmiddel (CTA) gebruik. Hierdie CTA word eerstens gebruik as gevolg van die gemak van gebruik en beheer wat dit gee oor die polimerisasie. Tweedens kan die eindfunksionele groepe op hierdie CTA gebruik word vir post-polimerisasie reaksies. In hierdie tesis word een van hierdie eindgroepe verander na 'n gewenste aldehied eindgroep, waar hierdie eindgroep gebruik gaan word in die konjugasie met katalase. RAFT-bemiddelde polimerisasie is suksesvol gebruik om vier verskillende molekulêre gewigspolimere te sintetiseer, elk met die gekose eindgroep. Hierdie eindgroep word dan suksesvol omgeskakel na 'n aldehied eindgroep wat benodig word vir die konjugasie reaksie. Konjugasie van PVP deur hierdie aldehied eindgroep was suksesvol en 'n verhoging in die molekulêre gewig van die ensiemkonjugaat word gesien. Hierdie is gevolg deur ensiematiese aktiwiteitsanalise, waar dit bevind is dat die konjugasie van PVP tot katalase nie 'n negatiewe effek op die ontbindingsproses van waterstofperoksied het nie. Daar is verder bevind dat daar eintlik 'n verhoging in die ensiematiese aktiwiteit van katalase is as 'n klein hoeveelheid van die polimeer tot katalase gekonjugeer is. 'n Verskil in die struktuur van katalase word gevind wat verwys na 'n verandering ná konjugasie. Daar is ook bevind dat met die konjugasie van verskillende molekulêre gewigspolimere onder 40 x 103 g/mol, nie 'n groot effek op die ensiematiese aktiwiteit in vergelyking tot mekaar het nie. Dus was die konjugasie van katalase suksesvol en katalase kan gebruik word in die teenwoordigheid van PVP met die ontbinding van waterstofperoksied.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100847
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