Nondenaturing PAGE electrophoreses. (A) Normal () antithrombin incubated at 50°C at pH7.4 (buffer A) and in 20% glycerol showing initial appearance at 12 hours of slow dimer band with complete conversion to latent form by 96 hours. A similar result but with more rapid change occurred under the same conditions in the absence of glycerol. L, latent control;  + L, 1:1 mixture of  and latent antithrombin. (B) A time sequence of incubation of the unstable antithrombin Wibble variant (50°C, pH 7.4, buffer B).10 Careful alignment shows how the transition to the latent form is preceded, in reciprocal proportions, by the formation of the slow dimeric component. Trace polymer bands are also present with the position of the loop-sheet dimer and trimer arrowed. (C) Isoelectric focusing of the stable slow electrophoretic component (lane 3), prepared as in (A), confirms its heterodimeric composition, with separation into equal bands of -antithrombin and latent antithrombin. Lanes 1 and 4, -antithrombin; lane 2, latent standard. (D) Mixing of antithrombins  (left) and β (right) 1:1 with latent antithrombin immediately gives the single slow dimeric component that retains its electrophoretic integrity even after 7 days at room temperature. (E) Incubation of normal -antithrombin in buffer B (pH 7.4, 50 mmol/L KCl) showing conversion over 54 hours at 37°C to the slow dimeric form, with acceleration at 41°C to give the appearance of the free latent band at 48 hours.