Binding of heparin to human antithrombin III as studied by measurements of tryptophan fluorescence.

Corrected fluorescence excitation and emission spectra of human antithrombin III have been determined. The fluorescence observed originates almost entirely from tryptophan residues. Reduction of the disulfide bonds followed by carboxymethylation did not change the fluorometric properties of the protein. The binding of heparin to antithrombin III caused a marked fluorescence enhancement by about 30% of the intrinsic protein emission intensity. Various samples of heparin yielded different binding curves. Heparin fractionated by gel filtration seemed to be bound to two sites on antithrombin III with association constants of 0.6-10(6)m-1 and 0.2-10(6)M-1 respectively. Heparin, prepared by affinity chromatography on matrix-bound antithrombin III appeared to be bound to only one site with an association constant of 2.3-10(6)M-1. Under similar conditions heparin caused no increase of the intrinsic protein emission intensity when added to reduced and carboxymethylated antithrombin III. The implications of these findings are discussed.