Specificity and site of action of a mammary gland thioesterase which releases acyl moieties from thioester linkage to the fatty acid synthetase

The lactone (I) of 2-hydroxy-1-naphthaleneacetic acid was developed as a reagent for novel, highly efficient, covalent attachment of an excited-state proton transfer fluorescence probe (i.e. 2-naphthol) to protein amino groups. The lactone (I) was shown to react with amines faster than it was hydrolyzed. Reaction of the lactone (I) with bovine serum albumin (BSA) was faster than its reaction with corresponding concentrations of small organic amines, which suggested that the lactone (I) first adsorbed to BSA and subsequently reacted covalently; data are presented which suggest that this covalent binding occurs at a unique, single site on BSA (i.e., affinity labeling). Equilibrium studies involving instantaneous and time-dependent fluorescence changes were interpreted in terms of a 1:1 lactone:BSA labeling ratio at neutral pH. Steady-state fluorescence spectroscopy of the 1:1 lactone:BSA conjugate and of the conjugate of the lactone with glycine ethyl ester were recorded, compared and interpreted in terms of the microenvironment of the protein-bound fluorescence probe. Applications are suggested for use of this new and powerful procedure for study of the conformational changes and molecular interactions in other proteins.