(A) Nondenaturing PAGE electrophoresis of isolated antithrombins Wibble and normal control incubated for 2 hours at a range of temperatures as indicated. Because of limited material staining was by immuno- (Western) blotting. The gels show the formation of polymers, commencing at 50°C with antithrombin Wibble and 60°C with the normal control. (B) Electrophoresis in nondenaturing PAGE of antithrombin from the abnormal (Wibble) peak IV (see Fig 2) and the normal peak III. The two samples were incubated at 50°C for 24 hours with removal of aliquots at times denoted on the figure. The appearance of the faster latent band begins at 6 hours and is almost complete at 24 hours. Note the latent form just appearing at 24 hours in the normal control. Traces of polymers are present in the peak IV sample after 6 hours of incubation. (Staining is with Coomassie blue, as compared with the more sensitive immuno-staining of [A]). (C) Nondenaturing PAGE electrophoresis of antithrombin Wibble (peak IV, Fig 2) before (W) and after (W41)° 24 hours of incubation13 at 41°C, pH 8.0; standards for latent (L) and normal () antithrombin are shown; and also β-antithrombin (β) to exclude contamination of peak IV. The gel is sensitively silver-stained; note the absence of polymers in the W41° band, which shows almost complete conversion to the latent form. (D) Electrophoresis in nondenaturing PAGE of samples (at two different concentrations) from peak II and peak IV of the heparin-Sepharose chromatography of a carrier of antithrombin Wibble (Fig 2). On left is a control (normal ) antithrombin from peak III, and on the right is a control latent antithrombin.