Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor's Name

John T. Landrum

First Advisor's Committee Title

Committee chair

Second Advisor's Name

Watson Lees

Second Advisor's Committee Title

Committee member

Third Advisor's Name

Bruce McCord

Third Advisor's Committee Title

Committee member

Fourth Advisor's Name

Piero Gardinali

Fourth Advisor's Committee Title

Committee member

Fifth Advisor's Name

Manuel Barbieri

Fifth Advisor's Committee Title

Committee member

Keywords

Carotenoid, carotenoid binding protein, lutein, fluorescence, surface plasmon resonance

Date of Defense

6-17-2016

Abstract

This dissertation describes the purification and partial characterization of CBP from the epidermis of the monarch butterfly larvae (Danaus plexippus). A yellow protein-carotenoid complex was extracted from the yellow pigmented epidermal tissue from monarch butterfly larvae by homogenization. Additional steps in the purification process included differential precipitation with ammonium sulfate, cation and anion chromatography, and lastly size exclusion chromatography. Polyacrylamide gel electrophoresis demonstrates that a single protein was isolated (M-LBP) having a ~60 kDa molecular weight, the value has subsequently been confirmed by HR-tandem MS. Lutein is the sole carotenoid bound by M-LBP with a stoichiometry of the binding of 2: 1. Immunohistochemistry results show that M-LBP has no cross-reactivity to antibodies for silk worm CBP (Bombix mori) but does have cross-reactivity with antibodies for horn worm epidermal CBP (Agrius convolvuli). Binding affinities were determined using surface plasmon resonance for the carotenoids lutein (KD = 18.6 ± 0.7), R,R-zeaxanthin (KD = 990 ± 60), R,S-zeaxanthin (KD = 60 ± 2). Tryptophyphan fluorescence lifetimes were determined for the apoprotein and compared to those of the native M-LBP. Tryptophan fluorescence lifetimes were found to be 3.9 ns and 3.0 ns, respectively for these two forms of the protein, indicating that upon dissociation of the carotenoid from the protein the tryptophan fluorophore adopts a position where it has less interaction with the polar surface environment.

Identifier

FIDC000724

Available for download on Saturday, July 21, 2018

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