Abstract

The canine variant of the serum protein, plasminogen (DPgn) binds the fluorescent molecule, 8-anilino-l-naphthalene sulfonate (ANS). Binding is pronounced at low pH, as witnessed by steady-state fluorescence, native PAGE and isothermal titration calorimetry. At pH 6.3 ANS binding occurs, but with a largely diminished magnitude. Binding occurs at multiple sites. A strong site binds ANS with a K a of 3.3 {604} 10 4 M -1 . Two weaker sites were observed with K a 's of 6.2 {604} 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. Enhanced binding at low pH is accompanied with changes in protein flexibility as monitored by sedimentation velocity analysis. Changes in frictional ratios were observed indicating an increased flexibility for both conformations of DPgn at pH 3.0 compared to pH 6.3. This finding was confirmed from the observation of altered near UV circular dichroism spectra of the protein at low pH. Taken together, the data indicate that ANS may be binding to a pH induced molten globule state of DPgn. Binding sites for ANS were localized within kringle domains as well as the proteolytic domain. However, it is unlikely that binding occurs within the lysine binding sites of each respective kringle.