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Drzazga, Z.; Michnik, Anna; Bartoszek, M.; Beck, Evelin

doi:10.1023/a:1017910009660

Cited by 10 publications

(2 citation statements)

References 11 publications

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“…79 Hb thermogram, similarly, indicated a transition at 69 • C with a corresponding DH of 136 ± 12 kcal mol −1 (Table 9). 80 The Hb/DNA mixture (5 lM Hb/100 lM DNA/Tris buffer) showed a distinct, single, transition at 69 • C, which suggested the formation of a discrete Hb/DNA complex. Next, we tested the stability of protein/DNA complex bound to the solid.…”

Section: Spectral Characterization Of the Dna-based Biocatalystsmentioning

confidence: 95%

Novel enzyme/DNA/inorganic nanomaterials: a new generation of biocatalysts

2007

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The design, synthesis and properties of a new class of enzyme/DNA/inorganic nanobiomaterials are described here. DNA has been used to stabilize the enzymes intercalated in the galleries of the inorganic solid, alpha-Zr(iv) phosphate (alpha-Zr(HPO(4))(2).H(2)O, abbreviated as alpha-ZrP). Interestingly, the presence of DNA improved the activity and stability of the bound enzymes. Key studies leading to the current strategy are presented initially, and these are followed by more recent developments. Several enzymes and proteins, including horseradish peroxidase, lysozyme, glucose oxidase, chymotrypsin, bovine serum albumin, cytochrome c, met-hemoglobin and met-myoglobin are successfully intercalated in the galleries of alpha-ZrP, under benign ambient conditions (aqueous buffered solutions, at room temperature and neutral pH). These novel materials are characterized by XRD, SEM and TEM as well as by biochemical, calorimetric and spectroscopic methods. Spectroscopic studies (circular dichroism, CD), for example, indicated that co-intercalation of DNA improved the retention of bound enzyme structure. The activity was enhanced markedly (five-fold) when DNA is co-intercalated, when compared to the activity in the absence of DNA. Addition of DNA to the sample, after enzyme intercalation, did not make any improvements. Our hypothesis is that enzyme-DNA supramolecular complex binds to the solid and the unfavorable interactions between the enzyme and the solid are minimized. These novel nanobiocomposite materials provide a simple method for packaging DNA and aid in engineering more effective synthetic materials for gene/RNA-delivery and drug delivery applications.

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Section: Spectral Characterization Of the Dna-based Biocatalystsmentioning

confidence: 95%

Novel enzyme/DNA/inorganic nanomaterials: a new generation of biocatalysts

2007

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“…The wavelength used to activate benzophenone is ∼300 nm, which should not have great influence on the attached enzyme, but standard mercury lamps also provide light below 300 nm (184.9, 253.6 nm) . Several authors described the possibility of destabilizing proteins with UV light, − causing inactivation or loss of aromatics. In our experiment, we used both the Nd:YAG (third harmonic line at 355 nm) laser and mercury UV lamp to activate benzophenone and found no difference in the activity of immobilized trypsin (Supporting Information, Figure ).…”

Section: Resultsmentioning

confidence: 99%

Photoimmobilization of Proteins for Affinity Capture Combined with MALDI TOF MS Analysis

2004

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Affinity capture surfaces can be prepared in a number of ways. A method of obtaining such surfaces through UV-activated immobilization of binding proteins using a benzophenone derivative is reported. Photoimmobilized protein G was used to selectively capture and preconcentrate bovine IgG from a mixture with BSA, and the affinity of photoattached concanavalin A toward ovalbumin was compared with that of commercially available concanavalin A on agarose beads. The results of the capture after tryptic digestion were analyzed by MALDI TOF MS. Immobilized trypsin was also prepared through photoimmobilization and later used to digest hemoglobin. Immobilized enzyme digestion resulted in more partial cleavages than solution-phase digestion. More methionine and tryptophan oxidation was also observed. Photoimmobilization was shown to be a quick and easy way of immobilizing ligands on surfaces.

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Endonuclease-like activity of heme proteins

et al. 2005

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Heme proteins, metmyoglobin, methemoglobin, and metcytochrome c showed unusual affinity for double-stranded DNA. Calorimetric studies show that binding of methemoglobin to calf thymus DNA (CTDNA) is weakly endothermic, and the binding constant is 4.9+/-0.7x10(5) M(-1). The Soret absorption bands of the heme proteins remained unchanged, in the presence of excess CTDNA, but a new circular dichroic band appeared at 210 nm. Helix melting studies indicated that the protein-DNA mixture denatures at a lower temperature than the individual components. Thermograms obtained by differential scanning calorimetry of the mixture indicated two distinct transitions, which are comparable to the thermograms obtained for individual components, but there was a reduction in the excess heat capacity. Activation of heme proteins by hydrogen peroxide resulted in the formation of high valent Fe(IV) oxo intermediates, and CTDNA reacted rapidly under these conditions. The rate was first-order in DNA concentration, and this reactivity resulted in DNA strand cleavage. Upon activation with hydrogen peroxide, for example, the heme proteins converted the supercoiled pUC18 DNA into nicked circular and linear DNA. No reaction occurred in the absence of the heme protein, or hydrogen peroxide. These data clearly indicate a novel property of several heme proteins, and this is first report of the endonuclease-like activity of the heme proteins.

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Untitled

Cited by 10 publications

References 11 publications

Novel enzyme/DNA/inorganic nanomaterials: a new generation of biocatalysts

Novel enzyme/DNA/inorganic nanomaterials: a new generation of biocatalysts

Photoimmobilization of Proteins for Affinity Capture Combined with MALDI TOF MS Analysis

Endonuclease-like activity of heme proteins

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