| Autor: |
Demant EJ; Department of Medical Biochemistry and Genetics, Panum Institute, University of Copenhagen, Denmark. |
| Jazyk: |
angličtina |
| Zdroj: |
Biochimica et biophysica acta [Biochim Biophys Acta] 1996 Nov 11; Vol. 1304 (1), pp. 43-55. |
| DOI: |
10.1016/s0005-2760(96)00106-3 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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