Studies on the purification of peroxidase from horseradish roots using reverse micelles.
Horseradish peroxidase (HRP) was successfully purified from horseradish roots by a two-stage reverse-micellar extraction from the dialyzed aqueous extract. The anionic surfactant AOT dissolved in isooctane was used to produce the reverse-micellar phases. The narrow pH range at which HRP solubilization occurred was exploited to remove most of the contaminant proteins in the first forward extraction. In the second extraction stage, HRP was selectively solubilized and concentrated by using a volume ratio of 10 between the aqueous and organic phases. The HRP final specific activity was 86 guaiacol U mg-1, obtained with a purification factor of 80 and yield of 46%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two overlapping bands, with HRP corresponding to that at 43.8 kDa. Image analysis on isoelectric focusing (IEF) gels showed that the HRP was 80% pure. Ion exchange liquid chromatography showed that most of the specific activity was due to the basic isoenzyme with pI 8.5, which comprises 33.5% of the product. There were high HRP losses as a precipitate at the interface when direct reverse-micellar extraction was attempted from the crude extract. It is believed that the hydrophobic environment near the haem group of the HRP basic isoenzyme favors complex formation with the surfactant, and that this is promoted at higher protein concentrations.
Regalado
C
C
Department of Food Science and Technology, University of Reading, Berks, United Kingdom.
Asenjo
J A
JA
Pyle
D L
DL
D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002457:Cell Extracts / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D019343:Citric Acid / Q000494:pharmacology; D003956:Dialysis; D004143:Dioctyl Sulfosuccinic Acid; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008823:Micelles; D009795:Octanes / Q000378:metabolism; D016622:Silver Staining; D013501:Surface-Active Agents / Q000378:metabolism
D002457:Cell Extracts; D008823:Micelles; D009795:Octanes; D013501:Surface-Active Agents; D004143:Dioctyl Sulfosuccinic Acid; D019343:Citric Acid; D004492:Edetic Acid; D006735:Horseradish Peroxidase; C045798:2,2,4-trimethylpentane; D002118:Calcium