Separation of native and latent forms of human antithrombin by hydrophobicinteraction high-performance liquid chromatography

Hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) w as utilized for the separation of native human antithrombin (AT) and a part ially denaturated form of AT, known as the latent form (L-AT). The AT used in this study is commercially available (Atenativ, Pharmacia & Upjohn, Swed en) and contains albumin as the main stabilizer. The AT was reconstituted a nd heat treated in order to generate L-AT. This latent form of AT has been shown to exhibit a strong antiangiogenic activity and also to suppress tumo r growth. The HPLC system included a TSK Phenyl 5PW column and a segmented gradient, 4.5-0 mol/L sodium chloride. Antithrombin was eluted at about 13 min, and L-AT, at 30 min, corresponding to about 4.2 and 1.6 mol/L sodium c hloride, respectively. A reference sample gave 42% L-AT when analyzed by th e HIC method and 41% L-AT when analyzed by the heparin affinity chromatogra phy method. The resolution between AT and L-AT was higher with the HIC meth od than with the heparin affinity method. Incubation of Atenativ at 45 degr eesC for 15 h gave about 18% L-AT and was shown by native polyacrylamide ge l electrophoresis to contain only monomeric AT. A good resolution between A T and L-AT, but not between albumin and L-AT, was also achieved by a linear gradient of 2-0 mol/L ammonium sulfate, in 25 mmol/L Tris/HCl, pH 8.0. (C) 2001 Academic Press.