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Separation of native and latent forms of human antithrombin by hydrophobic interaction high-performance liquid chromatography.

Abstract：

:Hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) was utilized for the separation of native human antithrombin (AT) and a partially denaturated form of AT, known as the latent form (L-AT). The AT used in this study is commercially available (Atenativ, Pharmacia & Upjohn, Sweden) and contains albumin as the main stabilizer. The AT was reconstituted and 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 tumor 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 chloride, respectively. A reference sample gave 42% L-AT when analyzed by the HIC method and 41% L-AT when analyzed by the heparin affinity chromatography method. The resolution between AT and L-AT was higher with the HIC method than with the heparin affinity method. Incubation of Atenativ at 45 degrees C for 15 h gave about 18% L-AT and was shown by native polyacrylamide gel electrophoresis to contain only monomeric AT. A good resolution between AT 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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Karlsson G,Winge S

doi

10.1006/prep.2000.1349

keywords:

subject

Has Abstract

pub_date

2001-02-01 00:00:00

pages

149-55

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(00)91349-4

journal_volume

21

pub_type

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