Study of pepsin phosphorylation using immobilized metal affinity chromatography.
The interactions of pepsin with immobilized trivalent metal ions and the participation of the enzyme phosphate group in this process were investigated using high performance immobilized metal affinity chromatography. Two different sorbents were used: the newly prepared one, consisting of Ga(3+ )chelate of (6-amino-1-hydroxyhexane-1,1-diyl) bis(phosphonic acid) covalently bound to a methacrylate support (BP-Ga(3+)), and the commercial one, containing immobilized Fe(3+ )ions (POROS MC20-Fe(3+)). The comparison of the behavior of porcine pepsin A and its partially dephosphorylated form on both sorbents showed that both forms of pepsin were adsorbed under the same conditions. To eliminate the participation of free carboxyl groups in pepsin adsorption, both enzyme forms were modified by amidation or esterification. Native enzyme and its partially dephosphorylated form both with modified carboxyl groups differed in their interaction with immobilized Ga(3+ )and Fe(3+). Phosphorylated pepsin molecules with esterified carboxyl groups were adsorbed on both sorbents while nonphosphorylated ones with esterified carboxyl groups were not adsorbed.
Novotna
Lenka
L
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic. lenka.novotna@LF1.cuni.cz
Hruby
Martin
M
Benes
Milan J
MJ
Kucerova
Zdenka
Z
D000327:Adsorption; D000818:Animals; D002614:Chelating Agents / Q000737:chemistry; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D005682:Gadolinium / Q000737:chemistry; D006801:Humans; D007477:Ions; D007501:Iron / Q000737:chemistry; D008670:Metals / Q000032:analysis*; D010434:Pepsin A / Q000032:analysis* / Q000737:chemistry*; D010766:Phosphorylation; D011108:Polymers / Q000737:chemistry; D013552:Swine
D002614:Chelating Agents; D007477:Ions; D008670:Metals; D011108:Polymers; D005682:Gadolinium; D007501:Iron; D010434:Pepsin A