Organic Artificial Proteinase with Active Site Comprising Three Salicylate Residues

Abstract

The first organic artificial proteinase based on synthetic materials is obtained by building active sites on the backbone of poly(ethylenimine) (PEI). The active site comprising three salicylate residues is prepared by preassemblage of three molecules of a salicylate derivative with Fe(III) ion followed by cross-linkage of the salicylates with PEI and demetalation of the resulting polymer. Proteinase activity of the artificial enzyme is demonstrated with hydrolytic cleavage of γ-globulin (Gbn). Both the heavy (50 kDa) and the light (25 kDa) chains of Gbn are effectively cleaved into peptides smaller than 5 kDa. The optimum activity of the artificial proteinase is manifested at pH 5−7. The half-life for cleavage of the two chains of Gbn by the artificial proteinase is ∼1 h at pH 7 and 50 °C when the concentration of the artificial active site is 0.4−1 mM. The activity (at pH 7) of the artificial proteinase prepared with PEI is comparable to that (at pH 9) of a catalytic antibody elicited by a joint hybridoma and combinatorial antibody library approach and to that (at pH < 3) achieved by the intramolecular carboxyl group in the hydrolysis of N-methyl maleamic acid. Thus, the approach reported here is useful for designing active sites of artificial enzymes.