Hey guys I will soon be conducting enzyme assays with a series of fatty acids of varying solubility. Has anyone got any advice on how to solubilise these for kinetic studies without increasing the temperature?
Thanks again guys
We usually start with 10% ethanolic fatty acid solutions, then dissolve them in a small amount of basic solution (i.e. 1M KOH or NaOH). A small amount of these solutions are then diluted in the appropriate buffer for the assay. Depending on the concentration we need or the pH of buffer, we'll include either Tween-20 or Triton X-100 (reduced form) in the buffer. A sample protocol can be found in Fukushige et al. (2005) J. Agric. Food Chem. 53: 5691-5694.
A promising new vehicle for solubilization of FA is the family of cyclodextrins (CDs), which are cyclic oligosaccharides formed by bacterial degradation of starch. These molecules typically contain six (α), seven (β), or eight (γ) glucose residues linked by (1→ 4) glycosidic bonds. They have a polar surface and a hydrophobic cylindrical cavity that can bind and solubilize a wide variety of hydrophobic molecules, such as cholesterol and FA, while remaining soluble in water (8). The number of CD molecules required to solubilize one FA molecule increases with an increase in the hydrocarbon chain length of FA (9), although the exact stoichiometry is difficult to determine.
Thanks very much guys. I'll try both methods and get back to you!
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