Cytochrome c films formed on silane self-assembled monolayer derivatized surfaces

Persistent Link:
http://hdl.handle.net/10150/288871
Title:
Cytochrome c films formed on silane self-assembled monolayer derivatized surfaces
Author:
Wood, Laurie Lynne, 1969-
Issue Date:
1998
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Formation and characterization of well-organized protein film assemblies are of high interest due to potential applications in biomolecular devices. The hypothesis that a macroscopically ordered protein film can be formed by site-directed, covalent binding of a protein to an appropriately derivatized surface formed the basis for the reported studies. The molecular architecture chosen to address this hypothesis consisted of yeast cytochrome c, a heme protein containing a unique binding site, immobilized on a surface derivatized with a silane self-assembled monolayer, (SAM). A combination of two techniques: total internal reflectance fluorescence (TIRF), to measure fluorescence anisotropy, and integrated optical waveguide-attenuated total reflectance (IOW-ATR) spectroscopy, to measure absorbance linear dichroism, was used to probe the macroscopic order of the heme groups in the film assemblies. Epifluorescence microscopy and absorbance (in an IOW-ATR geometry) were used to probe the nature of the surface-protein interactions and to determine relative protein affinities for different SAM-derivatized surfaces. The molecular orientation distribution for yeast cytochrome c immobilized on a 100% thiol-terminated SAM was 67° ± 39°. Partial protein removal was observed under a variety of rinse conditions, indicating that multiple protein-surface interactions may have contributed to the wide distribution value. Relative binding affinity constants and protein-surface interactions are compared for yeast cytochrome c and horse heart cytochrome c on 100% thiol-capped, 100% hydroxyl-capped, and mixed SAM-modified surfaces. These studies were also extended to include a variant of yeast cytochrome c, Thr8Cys/Cys102Thr. Similar adsorption and removal trends were observed for all the protein-SAM combinations. The adsorption isotherms indicated that at least two binding processes occur during formation of each protein film, high and low affinity binding. Removal studies indicated that the adsorption process is only partially reversible. It was concluded that employing a site-directed immobilization strategy does not necessarily produce a well-ordered protein film.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Chemistry, Analytical.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry
Degree Grantor:
University of Arizona
Advisor:
Saavedra, S. Scott

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleCytochrome c films formed on silane self-assembled monolayer derivatized surfacesen_US
dc.creatorWood, Laurie Lynne, 1969-en_US
dc.contributor.authorWood, Laurie Lynne, 1969-en_US
dc.date.issued1998en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractFormation and characterization of well-organized protein film assemblies are of high interest due to potential applications in biomolecular devices. The hypothesis that a macroscopically ordered protein film can be formed by site-directed, covalent binding of a protein to an appropriately derivatized surface formed the basis for the reported studies. The molecular architecture chosen to address this hypothesis consisted of yeast cytochrome c, a heme protein containing a unique binding site, immobilized on a surface derivatized with a silane self-assembled monolayer, (SAM). A combination of two techniques: total internal reflectance fluorescence (TIRF), to measure fluorescence anisotropy, and integrated optical waveguide-attenuated total reflectance (IOW-ATR) spectroscopy, to measure absorbance linear dichroism, was used to probe the macroscopic order of the heme groups in the film assemblies. Epifluorescence microscopy and absorbance (in an IOW-ATR geometry) were used to probe the nature of the surface-protein interactions and to determine relative protein affinities for different SAM-derivatized surfaces. The molecular orientation distribution for yeast cytochrome c immobilized on a 100% thiol-terminated SAM was 67° ± 39°. Partial protein removal was observed under a variety of rinse conditions, indicating that multiple protein-surface interactions may have contributed to the wide distribution value. Relative binding affinity constants and protein-surface interactions are compared for yeast cytochrome c and horse heart cytochrome c on 100% thiol-capped, 100% hydroxyl-capped, and mixed SAM-modified surfaces. These studies were also extended to include a variant of yeast cytochrome c, Thr8Cys/Cys102Thr. Similar adsorption and removal trends were observed for all the protein-SAM combinations. The adsorption isotherms indicated that at least two binding processes occur during formation of each protein film, high and low affinity binding. Removal studies indicated that the adsorption process is only partially reversible. It was concluded that employing a site-directed immobilization strategy does not necessarily produce a well-ordered protein film.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectChemistry, Analytical.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorSaavedra, S. Scotten_US
dc.identifier.proquest9901694en_US
dc.identifier.bibrecord.b3882565xen_US
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