Covalent complexes between serum albumin and 7-hydroxycoumarin-4-acetic acid: synthesis and applications in the spectrophotometric detection of long-chain fatty acids.

Using a hydrophobic 8-aminooctanoic acid cross-linker, the pH-indicator dye 7-hydroxycoumarin-4-acetic acid (7-HCA) is covalently bound to bovine serum albumin (BSA) at the positions of reactive amino groups. A highly stable and water-soluble complex (BSA-HCA) with a 1:4 molar stoichiometry is synthesized. Appearance of a strong absorption band at gamma max = 372 nm is associated to ionization of the 7-HCA chromophore when it is transferred from water into a basic microenvironment on the BSA surface. This particular surface site is related to the region(s) for high-affinity binding of long-chain fatty acids (FA). BSA-HCA responds to binding of FA (14-20 carbons) with immediate spectral changes and a decrease in 372 nm absorption. BSA-HCA provides an indicator-protein having a range of practical applications for the quantitative determination of long-chain FA in biochemical studies. The lower detection limit in a spectrophotometric method is approximately 1 microM FA. BSA-HCA is usable both in various buffers and in the presence of detergents such as n-octylglucoside, Triton X-100 and CHAPS. A novel method for continuous assay of phospholipase A2 activity with BSA-HCA and a mixed phosphatidylcholine/CHAPS micellar substrate is reported.