Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme.
Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.
Frýdlová
Jana
J
Department of Pathophysiology, Institute of Pathophysiology, First Faculty of Medicine, Charles University, U nemocnice 5, 128 53 Praha 2, Czech Republic.
Kucerová
Zdenka
Z
Tichá
Marie
M
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002846:Chromatography, Affinity; D007202:Indicators and Reagents; D008024:Ligands; D010434:Pepsin A / Q000032:analysis*; D020116:Pepsinogen A / Q000032:analysis*; D012685:Sepharose; D013379:Substrate Specificity; D013450:Sulfones; D013552:Swine
D000596:Amino Acids; D007202:Indicators and Reagents; D008024:Ligands; D013450:Sulfones; C009873:divinyl sulfone; D020116:Pepsinogen A; D012685:Sepharose; D010434:Pepsin A