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-indicato r dye 7-hydroxycoumarin-4-acetic acid (7-HCA) is covalently bound to b ovine serum albumin (BSA) at the positions of reactive amino groups. A highly stable and water-soluble complex (BSA-HCA) with a 1:4 molar st oichiometry is synthesized. Appearance of a strong absorption band at lambda(max) = 372 nm is associated to ionization of the 7-HCA chromoph ore when it is transferred from water into a basic microenvironment on the BSA surface. This particular surface site is related to the regio n(s) for high-affinity binding of long-chain fatty acids (FA). BSA-HCA responds to binding of FA (14-20 carbons) with immediate spectral cha nges and a decrease in 372 nm absorption. BSA-HCA provides an indicato r-protein having a range of practical applications for the quantitativ e determination of long-chain FA in biochemical studies. The lower det ection limit in a spectrophotometric method is approximate to 1 mu M F A. BSA-HCA is usable both in various buffers and in the presence of de tergents such as n-octylglucoside, Triton X-100 and CHAPS. A novel met hod for continuous assay of phospholipase A(2) activity with BSA-HCA a nd a mixed phosphatidylcholine/CHAPS micellar substrate is reported.