Interactions between canine plasminogen and 8-anilino-1-naphthalene sulfonate: structural insights from a fluorescent probe.
Canine plasminogen (DPgn) binds the fluorescent molecule, 8-anilino-1-naphthalene sulfonate (ANS). Binding is favored by low pH as shown by steady-state fluorescence, native PAGE and isothermal titration calorimetry. Binding occurs at multiple sites. A strong site binds ANS with a Ka of 3.3 x 10(4) M(-1). Two weaker sites were observed with association constants of 6.2 x 10(3) M(-1) and 360 M(-1), respectively. ANS binds equally well to both the closed and opened conformations of DPgn at pH 3.0. At low pH there are changes in protein tertiary structure as monitored by sedimentation velocity analysis and circular dichroism. Binding sites for ANS were located in the proteolytic domain as well as the kringle domains. However, it is unlikely that binding occurs within the lysine binding sites of each respective kringle. Taken together, the data suggest that ANS may be binding to a pH induced molten globule state of DPgn.
Carter
D M
DM
Department of Biology, Concordia University, Montreal, Quebec, H4B 1R6, Canada. krnbltt@vax2.concordia.ca
Kornblatt
J A
JA
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018082:Kringles; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D010958:Plasminogen; D010196:Pancreatic Elastase