Interaction of pepsin with aromatic amino acids and their derivatives immobilized to Sepharose.
The interaction of porcine pepsin A with immobilized derivatives of aromatic amino acids was investigated. Divinyl sulfone-activated Sepharose was used to immobilize N-acetyl-l-phenylalanine and 3,5-diiodo-l-tyrosine via their free carboxyl groups and l-tyrosine via its amino group. Immobilized l-tyrosine was iodinated after coupling. The optimum conditions for the separation of porcine pepsin A using the prepared affinity carriers were studied and the following parameters were established: enzyme recovery, reproducibility of analyses, capacity and dependence of the elution peak area on the concentration of the loaded enzyme. The ability of the prepared affinity carriers to retain various types of proteins was compared under optimum conditions for porcine pepsin A separation. While immobilized 3,5-diiodo-l-tyrosine and iodinated l-tyrosine-Sepharose adsorbed relatively high amounts of bovine serum albumin and ovalbumin, only negligible amounts of these proteins were adsorbed to immobilized N-acetyl-l-phenylalanine. The behavior of porcine pepsin A was the same as its complex with pepstatin A on the prepared affinity carriers, indicating that the enzyme active site is not involved in the studied interaction.
Frýdlová
Jana
J
Institute of Pathophysiology and Center of Experimental Hematology, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53 Praha 2, Czech Republic. jana.frydlova@LF1.cuni.cz
Kucerová
Zdenka
Z
Tichá
Marie
M
D024322:Amino Acids, Aromatic / Q000737:chemistry*; D000818:Animals; D002846:Chromatography, Affinity; D005029:Ethylenediamines / Q000737:chemistry; D010434:Pepsin A / Q000737:chemistry*; D010649:Phenylalanine / Q000737:chemistry; D012685:Sepharose; D013450:Sulfones / Q000737:chemistry; D013552:Swine; D014443:Tyrosine / Q000737:chemistry
D024322:Amino Acids, Aromatic; D005029:Ethylenediamines; D013450:Sulfones; D014443:Tyrosine; D010649:Phenylalanine; C009873:divinyl sulfone; D012685:Sepharose; D010434:Pepsin A