Polyethyleneimine phosphate and citrate systems act like pseudo polyampholytes as a starting method to isolate pepsin.
The aqueous solution behaviour of polyethyleneimine (a cationic synthetic polymer) in the presence of anions (such as citrate and phosphate) was studied by means of turbidimetry. The variation of the absorbance at 420 nm of dilute mixture with pH, the polymer concentration and the ionic strength were examined. The mixture of polyethyleneinine citrate or polyethyleneinine phosphate behaves as a pseudo polyampholyte with an isoelectric point of 5.5 and 6.2 for phosphate and citrate respectively and a precipitation pH range between 3.5 and 8.0. Pepsin was completely precipitated with the polymer anion complex within this pH interval. Citrate showed a better precipitation effect than phosphate did. The precipitate was reversibly dissolved in NaCl (for concentrations higher than 0.2 M) and pepsin kept its biological activity. Studies of pepsin thermal stability (by differential scanning calorimetry) revealed that the polyethyleneimine presence increased the enzyme denaturation temperature. The circular dichroism spectrum of pepsin showed a non-significant loss of secondary and tertiary enzyme structure by the polyethyleneimine. However, the polymer presence increased the biological activity of pepsin.
Manzur
Alejandra
A
Bioseparation Lab., Physical Chemistry Department, Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario, FonCyT, CIUNR and CONICET, Suipacha 570 (S2002RLK), Rosario, Argentina.
Spelzini
Darío
D
Farruggia
Beatriz
B
Romanini
Diana
D
Picó
Guillermo
G
D002021:Buffers; D002951:Citrates; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D009391:Nephelometry and Turbidimetry; D010755:Organophosphates; D010434:Pepsin A / Q000302:isolation & purification* / Q000378:metabolism; D011094:Polyethyleneimine
D002021:Buffers; D002951:Citrates; D010755:Organophosphates; D011094:Polyethyleneimine; D010434:Pepsin A