High-performance anion-exchange chromatographic study of desialylated human alpha 1-acid glycoprotein variants. Development of a fractionation method for the protein slow variants.
The three main desialylated variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG), a serum acute-phase reactant, were analysed by high-performance anion-exchange chromatography in order to determine their optimum separation conditions. The analysis consisted of three steps, as follows: (1) A desialylated commercial AAG was separated into one "fast"- and one "slow"-migrating fraction by preparative isoelectrofocusing. The "fast" and "slow" fractions were shown to contain the F1 variant and a mixture of the S and A variants, respectively. (2) The pH titration curves of these two fractions were then measured by strong anion-exchange chromatography with several buffer systems of increasing pH. From the data obtained, it was not possible to select the optimum conditions to separate the "fast" variant F1 from the "slow" variants A and S. However, the S and A variants were shown to ionize very differently. (3) The specific fractionation of the S and A variants was therefore carried out by anion-exchange chromatography under operating conditions based on the data obtained from the study of their pH titration curves. This was performed both with the "slow"-migrating fraction obtained by preparative isoelectrofocusing of commercial AAG and with an AAG (containing only variants S and A) purified from an individual serum on immobilized Cibacron Blue F3G-A. Identification of the fractionated proteins was achieved by analytical isoelectrofocusing.
Herve
F
F
Service Hospitalo-Universitaire de Pharmacologie de Paris XII, Centre Hospitalier Intercommunal de Créteil, France.
Duche
J C
JC
Sportes
N
N
Tillement
J P
JP
D002852:Chromatography, Ion Exchange / Q000379:methods*; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009961:Orosomucoid / Q000032:analysis* / Q000235:genetics; D010641:Phenotype; D011500:Protein Synthesis Inhibitors; D014227:Triazines
D009961:Orosomucoid; D011500:Protein Synthesis Inhibitors; D014227:Triazines; C012842:Cibacron Blue F 3GA