Fibrinogen-beta-estradiol binding studied by fluorescence spectroscopy: denaturation and pH effects.
Fibrinogen is a blood plasma protein that plays a crucial role in hemostasis. It is known that erythrocyte aggregation increases in the presence of fibrinogen, and that beta-estradiol decreases erythrocyte aggregation with a constant fibrinogen concentration. In this work, we have used intrinsic tryptophan fluorescence to obtain information on the conformational changes of fibrinogen upon the recently proposed interaction with beta-estradiol. To evaluate the effect on the conformational changes during fibrinogen-beta-estradiol binding, fluorescence experiments were performed using guanidine hydrochloride (0-6 M) as denaturant, at different pH values. The results obtained for pH 6.5 and 8.0 showed no effect during the binding. The main differences were observed between pH 4.2 and 7.4, in the absence and in the presence of two different denaturant concentrations (1 and 5 M). A red shift of the fluorescence emission from 344 to 354 nm is observed when denaturant concentration is above 3 M for all studied pH values. This phenomenon may be explained by the loss of compact structure of the protein in the presence of denaturant, with tryptophan residues exposure to the aqueous environment and alteration of fibrinogen-beta-estradiol binding. These results demonstrate that the binding sites of fibrinogen are strongly dependent on the conformational state of the protein.
Instituto de Biopatologia Química and Unidade de Biopatologia Vascular, Faculdade de Medicina de Lisboa, 1649-028, Lisboa, Portugal. sabreu@fm.ul.pt
Sónia
S
Gonçalves
Nuno C
NC
Santos
J
J
Martins-Silva
Carlota
C
Saldanha
D001665:Binding Sites; D004958:Estradiol / Q000737:chemistry*; D005340:Fibrinogen / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis
D004958:Estradiol; D014364:Tryptophan; D005340:Fibrinogen; D019791:Guanidine