Deconvolution of the fluorescence emission spectrum of human antithrombin and identification of the tryptophan residues that are responsive to heparin binding.
Heparin causes an allosterically transmitted conformational change in the reactive center loop of antithrombin and a 40% enhancement of tryptophan fluorescence. We have expressed four human antithrombins containing single Trp --> Phe mutations and determined that the fluorescence of antithrombin is a linear combination of the four tryptophans. The contributions to the spectrum of native antithrombin at 340 nm were 8% for Trp-49, 10% for Trp-189, 19% for Trp-225, and 63% for Trp-307. Trp-225 and Trp-307 accounted for the majority of the heparin-induced fluorescence enhancement, contributing 37 and 36%, respectively. Trp-49 and Trp-225 underwent spectral shifts of 15 nm to blue and 5 nm to red, respectively, in the antithrombin-heparin complex. The blue shift for Trp-49 is consistent with partial burial by contact with heparin, whereas the red shift for Trp-225 and large enhancement probably result from increased solvent access upon heparin-induced displacement of the contact residue Ser-380. The enhancement for Trp-307 may result from the heparin-induced movement of helix H seen in the crystal structure. The time-resolved fluorescence properties of individual tryptophans of wild-type antithrombin were also determined using the four variants and showed that Trp-225 and Trp-307 experienced the largest change in lifetime upon heparin binding, providing support for the steady-state fluorescence deconvolution.
Department of Biochemistry and Molecular Biology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
J L
JL
Meagher
J M
JM
Beechem
S T
ST
Olson
P G
PG
Gettins
D000990:Antithrombin III / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics
D014364:Tryptophan; D000990:Antithrombin III; D006493:Heparin