Isolation and characterization of carotenoproteins in higher plant systems
Dietz, Jane Marie
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https://hdl.handle.net/2142/19012
Description
Title
Isolation and characterization of carotenoproteins in higher plant systems
Author(s)
Dietz, Jane Marie
Issue Date
1990
Doctoral Committee Chair(s)
Erdman, John W.
Department of Study
Food Science and Human Nutrition
Discipline
Food Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture
Health Sciences
Food Science
Technology
Nutrition
Language
eng
Abstract
For the first time, a procedure has been developed for the isolation of intact $\alpha$- and $\beta$-carotene binding carotenoprotein from carrot chromoplasts. Initial attempts to isolate and purify this protein from spinach chloroplasts was unsuccessful because of contaminating chlorophyll and chlorophyll proteins.
Though the attempts were unsuccessful for a carotene binding protein in spinach, a lutein isomer binding carotenoprotein was effectively isolated. The isolation method employed metal chelate affinity chromatography using immobilized copper ions. While suitable for this more polar carotenoid protein complex, this method was not utilizable for non-polar carotene binding proteins. Degradation of $\beta$-carotene occurred during chromatography, apparently catalyzed by the copper ions.
The developed procedure for the isolation of the pure carotenoprotein from carrot chromoplast was quick and straightforward. Only French pressing, high speed centrifugation and gel filtration were needed to purify the protein.
The major protein peak collected from the gel filtration column was also the major carotenoid peak. This peak corresponded to a molecular weight of approximately 2,000 kDal for the native protein. Isoelectric focusing indicated the presence of a single protein band with a pI of 3.6. Further substantiation that this was a single pure protein complex was demonstrated by SDS-PAGE where only a single 54 kDal protein band was detected. This indicates that the native complex of 2,000 kDal was comprised of seemingly identical 54 kDal subunits.
Analysis of the carotenoprotein complex by r-phase HPLC demonstrated the protein to bind 1 mole of $\alpha$-carotene and 2 moles of $\beta$-carotene for each mole of the subunit.
The amino acid composition of the protein suggests that the protein contains a high percentage of $\alpha$-helical conformation in combination with a probability of numerous reverse turns. Given this information and the high percentage of hydrophobic amino acids present, it may be hypothesized that the helical structure of the protein may form a hydrophobic cleft along the surface of the protein which would allow attachment of the carotenes. This hypothesis is consistent with the ease in which the carotene may be dissociated from the protein complex during the isolation procedures.
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