Published on Brock University (http://www.brocku.ca)
Our interests in tocopherol bioorganic chemistry includes activity in organic synthesis as well as protein biochemistry, structuiral biology, and analytical biochemistry.
Strong collaborations with active groups in the US and Europe mean that we are direcetly invovled with aspects of tocopherol biokinetics, intracellular location, and mode of activity of the tocopherol transfer protein, TTP, and other lipid trnasfer proteins, as weel as proteins that interact with membranes at some point in their biochemistry.
We regularly prepare deuterated forms of tocopherol and tocotrienol as in vivo tracers for biokinetic studies in animals and humans. We have recently completed the preparation of 16 different fluorescent forms of tocopherol and have embarked on versions of tocotrienol as well.
The photoaffinity labels we made several years ago are getting a new life in the exploration of tocopherol location in lipids rafts and selective proximity to certain membrane proteins.
Our newest synthetic efforts include the design and synthesis of novel forms of tocopherols that will disrupt their normal oxidative metabolism and aid in the understanding of the fate of the different forms of vitamin E.
Tocopherol Transfer Protein (TTP)
Every hydrophobic ligand would have trouble moving about the aqueous portion of cells were it not for specific transfer proteins. Transfer proteins exist for retinoids, sterols, fatty acids, phospholipids, cholesterol, vitamin D and, of course, vitamin E (tocopherols). Each ligand has particular biochemistry to which the transfer protein must be attuned.
For instance, the sterol binding proteins (SBP) and ceramide transfer proteins (CERT) are coordinated so that the biosynthesis of both cholesterol and ceramides are controlled together. Since tocopherols are vitamins, there is no necessity for controling biosynthesis in mamalian tissue, but it is reasonable to assume that TTP has some role to play in transporting tocopherol to particular membranes involved in vesicular traffic (i.e. VLDL secretion from liver cells) or to those membranes that are prone to lipid peroxidation.
We are looking at the features that control the rates of tocopehrol delivery to membranes by the TTP. To this end we have developed FRET assays using our fluorescent tocopherol derivative NBD-alpha-Tocopherol. By varying the composition of membrane acceptor, as well as making mutations in the transfer protein, we (along with Dr. Danny Manor, at Case Western Reserve University) have begun to decipher those features necessary for membrane recognition by TTP, and phosphlipids that effect efficeint transfer.
We base our experiemnts on two majo techniques. One, mentioned above is fluorescnece energy resonance transfer (FRET), and the other is called Dual Polarization Interferometry (DPI) We are using Farfield Scientific's AnaLight200. In brief, this is a surface adsorption technique that allows one to adsorb phosphlipid vesicles onto a sensor chip and measure the thickness, density, and mass of this layer. Stable phospholipid layers can also have protein adsorbed to them. From similar measuremnts of mass and thickness, we can calculate affinities of proteins for particular phosphlipid membranes.
Synthesis of New Forms of Tocopherols and Tocotrienols
We remain active in the organic synthesis of tocols and other hydrophobic compounds as tools for biochemical research. This has included a number of efforts such as:
• photoaffinity labels based on alpha-tocopherol, and other immobilized affinity ligands for retrieving novel tocol bindiong proteins from tissues
• fluorescent tocols
• isotopically substituted tocols (2H and 14C)
• inhibitors of tocol metabolism