Immobile Artificial Metalloproteinase Containing Both Catalytic and Binding Groups

Abstract

Poly(chloromethylstyrene-co-divinylbenzene) (PCD) with 2% cross-linkage is developed as a backbone of immobile artificial enzymes. As the first artificial enzyme built on PCD, an artificial metalloproteinase is prepared by attaching the Cu(II) complex of cyclen as the catalytic center and guanidinium ion as the binding unit. The PCD derivatives prepared were characterized by scanning electron microscopy, IR spectroscopy, elemental analysis, titration of chloromethylphenyl moiety with triethylamine, complexation of p-nitrobenzoate ion to the guanidinium moiety, quantification of the cyclen moieties retaining high affinity for the Cu(II) ion, and determination of log K f for the Cu(II) binding sites. The proteinase activity was measured with γ-globulin (Gbn) by following cleavage of the two chains of Gbn by electrophoresis. The catalytic activity of the Cu(II) complex of cyclen toward Gbn was enhanced by more than 104 times upon attachment to PCD. Gbn complexed to the PCD derivative containing both Cu(II)-cyclen and guanidinium moieties was cleaved by hydrolysis into many pieces with a half-life as short as 10−30 min at pH 4.5−7 and 4 °C. Kinetic data revealed that guanidinium ions attached to PCD acted as effective binding sites for the protein, contributing considerably to the overall catalytic power of the immobile artificial metalloproteinase.

Hydrolysis of plasmid DNA catalyzed by Co(III) complex of cyclen attached to polystyrene

“…As mentioned above, the protein-cleaving activity of Cu(II)Cyc was greatly improved when Cu(II)Cyc was attached to PCD. 15 Enhancement of DNA hydrolyzing activity of CoCyc upon attachment to PCD in the present study suggests that PCD can be used as the backbone for immobile artificial metalloenzymes with highly activated metallo-catalytic centers.…”

“…In a previous study, 15 we observed that the proteincleaving activity of the Cu(II) complex of Cyc was enhanced remarkably upon attachment to a derivative of poly(chloromethylstyrene-co-divinylbenzene) (PCD). PCD, an insoluble resin, is a cross-linked polystyrene in which the styrene monomer contains chloromethyl group.…”

“…9 The content of CoCyc was estimated through measuring the amount of Co(III) ion released from the resin upon treatment with concentrated nitric acid by inductively coupled plasmaatomic emission spectroscopy. The content of guanidinium was measured by the binding experiment 15 with pnitrobenzoate. The contents of CoCyc unit and guanidinium ion in [CoCyc][Gua]…”

“…23,24 When the DNA is complexed by the resin, several CoCyc moieties would be contained in the region covered by the DNA. When estimated by the method employed previously, 15 25 In 3, the stripes and the dark ellipses represent the phosphate linkages and the CoCyc moieties, respectively. Availability of several catalytic units for each complexed DNA is not, therefore, sufficient to account for the high reactivity of the Co(III)-containing resins.…”

DNA Hybridization by Peptide Nucleic Acids Attached to Controlled Pore Glass

“…This suggests that adsorption of DNA onto the PNA-containing CPG (CPG-PNA) would occur mainly through interaction with the PNA moieties. The data of Figure 1 were analyzed in terms of the scheme of eq 1 and the expression of eq 2 derived therefrom by assuming independent binding to each binding site, by analogy with Langmuir adsorption as described 4 previously. In eq 1 and 2, BS stands for the binding site on the resin available for DNA hybridization, BS o for the total amount of the host site on the resin (expressed as the concentration attainable when the resin is dissolved), and K f (= k ad /k de ) for the formation constant of the BS-DNA complex.…”