The use of phenyl-Sepharose for the affinity purification of proteinases

Abstract

Phenyl-Sepharose is most often used as an adsorbent for hydrophobic interaction interaction chromatography (HIC). We report on its effective use for the affinity purification of some extracellular thermostable proteinases from bacterial sources.Proteinases belonging to the serine, aspartate and metallo mechanistic classes were effectively retained by the media. Purification factors in the range of 2.9 -60 and enzyme activity yields in excess of 88% were obtained. In some cases homogeneous enzyme was obtained from culture supernatants in a single step. A number of other proteinases from mammalian sources were also retained. The specificity of the enzyme/support interaction was studied. Proteinases complexed with peptide inhibitors (pepstatin and chymostatin)showed reduced binding to phenyl Sepharose indicating interaction with the active site cleft whereas modification with low molecular weight active site directed inactivators such as PMSF and DAN did not, indicating that binding may not be dependent on sites immediately adjacent to the catalytic site. Pepsinogen and the pro-enzyme form of the serine proteinase from the thermophilic Bacillus sp., strain Ak.1 were not retained by the media and could be resolved in an efficient manner from their active counterparts. KeywordsProteinase, purification, phenyl-Sepharose

Interaction of pepsin with aromatic amino acids and their derivatives immobilized to Sepharose

“…Phenyl-Sepharose is very often used as a carrier in hydrophobic chromatography. The usefulness of this carrier has also been reported in the separation of aspartate proteinases from various sources [46][47][48].…”

“…The behavior of pepsin and its complex with pepstatin A on immobilized ligands that are based on derivatives of aromatic amino acids differs from that of the same enzyme and its complex on Phenyl-Sepharose (Table 3) [46]. The complex of porcine pepsin A with pepstatin A exhibited reduced binding to PhenylSepharose in comparison with the enzyme in the absence of the inhibitor [46].…”

“…The complex of porcine pepsin A with pepstatin A exhibited reduced binding to PhenylSepharose in comparison with the enzyme in the absence of the inhibitor [46]. Phenyl-Sepharose is very often used as a carrier in hydrophobic chromatography.…”

Preparation of affinity carriers for the study of binding properties of aspartic proteinases

“…For the isolation of proteinases, they might represent a suitable and simple affinity carrier lacking peptide bonds. Immobilized L-phenylalanine has been used for the isolation of fungal proteinases [15,16] and Phenyl-Sepharose for the affinity purification of proteinases from bacterial sources belonging to serine, aspartate, and metallo mechanistic classes [17]. The obtained results have shown that immobilized aromatic amino acids and their derivatives are suitable affinity matrices to study binding properties of pepsin and its zymogen.…”

The major extracellular proteinases of the silverleaf fungus, Chondrostereum purpureum, are metalloproteinases

“…A number of other purification procedures were attempted. These included S-sepharose, phenyl sepharose (Prescott et al, 1993), ion exchange on HiTrap Q and HiTrap SP (Pharmacia), rechromatography on Superdex G75 in the presence of EDTA or zinc ions and ammonium sulphate precipitation. None of these methods led to useful separations of activity and all resulted in large losses of proteinase activity.…”