Abnormal microheterogeneity detected in one commercial alpha 1-acid glycoprotein preparation using chromatography on immobilized metal affinity adsorbent and on hydroxyapatite.
The study of one commercial preparation of human alpha 1-acid glycoprotein (AAG) by isoelectric focusing and by different chromatographic methods, previously developed to purify and fractionate the genetic variants of AAG, revealed an abnormal heterogeneity for this preparation. In addition to the three main variants (F1, S and A) of AAG normally present, this preparation contained five other AAG variants (called here sigma, alpha, beta, delta and gamma), accounting for ca. 40% of the total. As it is very unlikely that the latter variants are rare AAG variants, the abnormal heterogeneity of this AAG preparation is most probably due to structural alterations occurring during the large scale isolation. The alpha and the sigma, beta, delta and gamma variants could correspond to altered forms of the A and the F1 and S variants, respectively, because of their similar retention behaviour on immobilized copper(II) ions and their similar drug binding properties. However, the elution of the variants from the immobilized metal affinity column suggested that sigma, alpha, beta, delta and gamma were desialylated. Chromatography on hydroxyapatite enabled the separation of the F1, S and A variants from the sigma, alpha, beta, delta and gamma variants. The inability of the latter variants to bind to hydroxyapatite suggests that the structural alterations might involve acidic amino acid residues. This proposal agreed with the isoelectric focusing study of variants sigma, alpha, beta, delta and gamma. Since the different separation methods used were able to resolve the variants of this AAG, this protocol could be used for characterization of commercial AAG proteins.
Hervé
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F
Service Hospitalo-Universitaire de Pharmacologie de Paris XII, Centre Hospitalier Intercommunal de Créteil, France.
Fouache
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F
Marché
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C
Tillement
J P
JP
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D017886:Durapatite / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006041:Goats; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000276:immunology; D007525:Isoelectric Focusing / Q000379:methods; D009961:Orosomucoid / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011233:Precipitin Tests; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet
D007106:Immune Sera; D009961:Orosomucoid; D017886:Durapatite