A procedure for human pregnancy zone protein (and human alpha 2-macroglobulin) purification using hydrophobic interaction chromatography on phenyl-sepharose CL-4B column.
In the present work we describe a procedure for the purification of human pregnancy zone protein (PZP) from pooled late pregnancy plasma by using hydrophobic interaction chromatography (HIC) on a phenyl-Sepharose column. The HIC step allowed the complete isolation of haptoglobins and the partial separation of human alpha 2-macroglobulin (alpha 2-M) from a protein fraction containing PZP previously obtained by a DEAE-Sephacel chromatography. Pure and native PZP, with a recovery of nearly 25% and biological activity of protease-binding, was obtained by two definitive final steps consisting of zinc-chelate and size-filtration chromatographies. Moreover, we further present an alternative procedure for the purification of alpha 2-M from the same pregnancy plasma, based on the differential elution of PZP and alpha 2-M from the HIC. This purification step gave rise to a highly purified product with a recovery of 10%. This differential elution could be explained by differences in surface hydrophobicity observed between both proteins. In addition, considering the different hydrophobic properties exhibited by native PZP and PZP-protease complexes, HIC on phenyl-Sepharose column could also be used for separating both conformational states of PZP.
Chiabrando
G
G
Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina. gchiabra@fcq.uncor.edu
Bonacci
G
G
Sanchez
C
C
Ramos
A
A
Zalazar
F
F
Vides
M A
MA
D002614:Chelating Agents; D002847:Chromatography, Agarose / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011487:Protein Conformation; D012685:Sepharose / Q000031:analogs & derivatives; D015032:Zinc; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000302:isolation & purification*
D002614:Chelating Agents; D011257:Pregnancy Proteins; D000511:alpha-Macroglobulins; C033452:Phenyl-Sepharose CL-4B; D012685:Sepharose; D015032:Zinc