Protein annealing: Thermal treatment of met-hemoglobin bound to α-zirconium phosphate/phosphonates results in initial denaturation followed by recovery of activity and structure

Jagannadham, V.; Bhambhani, Akhilesh; Kumar, Challa V.

doi:10.1016/j.micromeso.2005.09.019

Cited by 24 publications

(24 citation statements)

References 37 publications

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“…[9,10] Even though Hb is not an enzyme in biological systems, Hb catalyzes the oxidation of o-methoxyphenol by H 2 O 2 (peroxidase-like activity). [7,11] These interesting properties of Hb/a-ZrP prompted the challenge of improving the activity of Hb bound to a-ZrP.In an independent study, heme proteins were shown to bind to the double-helical calf thymus DNA (referred to as DNA hereafter), [12] and DNA inhibited the thermally induced aggregation of Hb. This observation raised the interesting possibility of using DNA to improve the properties of intercalated Hb and also of testing if Hb binding to DNA would assist DNA intercalation in the negatively charged galleries of a-ZrP (Scheme 1).…”

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confidence: 99%

“…Hb is not an enzyme, but its peroxidaselike activity has long been known. [16,11] Binding of Hb to DNA (no a-ZrP) indicated some reduction in activity, and this has been shown to be due to the slow, competitive, reaction of DNA as a substrate. [12] Therefore, it was not clear if co-intercalation of DNA would enhance or diminish the activity of intercalated Hb, but current results show that Hb activity is indeed enhanced (Fig.…”

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confidence: 99%

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Protein/DNA/Inorganic Materials: DNA Binding to Layered α‐Zirconium Phosphate Enhances Bound Protein Structure and Activity

Bhambhani

Kumar

2006

Advanced Materials

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Enzymes are highly efficient and specific biocatalysts with superb chemo-, regio-, stereo-, and chiral selectivities, but the exciting possibility of using enzymes in the laboratory is severely limited.[1] This is because enzymes are generally expensive, sensitive to pH/temperature, and unstable in organic media. [2] Binding of enzymes on solid supports can partly overcome these limitations, [3] and in specific cases such binding improved enzyme properties. [4] However, binding of enzymes to solids often results in some loss in activity, and the extent of loss depends on the solid, the nature of the enzyme, and the method of binding. Therefore, developing novel methods to improve the bound-enzyme activity is of intense interest.[5] Here, we show that bound-protein behavior is improved to a significant extent by double-helical DNA. This is also the very first example of DNA binding to negatively charged layered inorganic materials, and this observation opens new possibilities for applications in gene or RNA delivery. Previously, successful intercalation of a number of enzymes and proteins into the galleries of layered a-zirconium phosphate (a-Zr(HPO 4 ) 2 · H 2 O, [6] abbreviated as a-ZrP) was reported. [7] Intercalated proteins retained their structure as well as activity to a significant extent.[8] Intercalation of met-hemoglobin (Hb) in the galleries of a-ZrP improved the thermal stability of Hb. [9,10] Because of this improved stability, Hb/a-ZrP was catalytically active at elevated temperatures (> 90°C), while the free Hb rapidly denatured at these temperatures. [9,10] Even though Hb is not an enzyme in biological systems, Hb catalyzes the oxidation of o-methoxyphenol by H 2 O 2 (peroxidase-like activity). [7,11] These interesting properties of Hb/a-ZrP prompted the challenge of improving the activity of Hb bound to a-ZrP.In an independent study, heme proteins were shown to bind to the double-helical calf thymus DNA (referred to as DNA hereafter), [12] and DNA inhibited the thermally induced aggregation of Hb. This observation raised the interesting possibility of using DNA to improve the properties of intercalated Hb and also of testing if Hb binding to DNA would assist DNA intercalation in the negatively charged galleries of a-ZrP (Scheme 1). Our results are described below.Suspensions of a-ZrP (0-10 mM) were contacted with solutions of Hb (0-32 lM) and DNA (0-80 lM), and binding was monitored by centrifugation studies. The amount of DNA bound to a-ZrP increased steadily as a function of a-ZrP concentration (Fig. 1A) at constant concentrations of Hb (8 lM) and DNA (120 lM). This is the first observation of DNA binding to a negatively charged layered inorganic solid, and no binding was noted in the absence of Hb. All Hb present in the solution was intercalated under these conditions. The slope of the linear fit to the data indicates that 9 lM of DNA binds to every mM of a-ZrP. The binding of DNA to the solid, therefore, is favored because of the formation of a Hb-DNA complex prior to intercalation.When t...

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confidence: 99%

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confidence: 99%

Protein/DNA/Inorganic Materials: DNA Binding to Layered α‐Zirconium Phosphate Enhances Bound Protein Structure and Activity

Bhambhani

Kumar

2006

Advanced Materials

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“…26, 36–39 However, many of the preintercalators used to overcome the intercalation energy barrier are toxic and hamper the biological viability of the system. Alternatively, we used a hydrated phase of α-ZrP, θ-ZrP, which can easily intercalate large molecules without any preintercalators.…”

Section: Resultsmentioning

confidence: 99%

Direct intercalation of cisplatin into zirconium phosphate nanoplatelets for potential cancer nanotherapy

et al. 2013

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We report the use of zirconium phosphate nanoplatelets (ZrP) for the encapsulation of the anticancer drug cisplatin and its delivery to tumor cells. Cisplatin was intercalated into ZrP by direct-ion exchange and was tested in-vitro for cytotoxicity in the human breast cancer (MCF-7) cell line. The structural characterization of the intercalated cisplatin in ZrP suggests that during the intercalation process, the chloride ligands of the cisplatin complex were substituted by phosphate groups within the layers. Consequently, a new phosphate phase with the platinum complex directly bound to ZrP (cisPt@ZrP) is produced with an interlayer distance of 9.3 Å. The in-vitro release profile of the intercalated drug by pH stimulus shows that at low pH under lysosomal conditions the platinum complex is released with simultaneous hydrolysis of the zirconium phosphate material, while at higher pH the complex is not released. Experiments with the MCF-7 cell line show that cisPt@ZrP reduced the cell viability up to 40%. The cisPt@ZrP intercalation product is envisioned as a future nanotherapy agent for cancer. Taking advantage of the shape and sizes of the ZrP particles and controlled release of the drug at low pH, it is intended to exploit the enhanced permeability and retention effect of tumors, as well as their intrinsic acidity, for the destruction of malignant cells.

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“…When Hb intercalated a-ZrP is heated over the denaturation temperature of the free enzyme a recovery of the enzyme activity is observed after an initial activity loss [139]. This unusual behavior for free protein is even enhanced for a-ZrCMP and aZrCEP.…”

Section: Biohybridmentioning

confidence: 97%

“…Simultaneously a recovery of the structure was observed, showing that structural changes (dehydration, unfolding of the peptide chains, formation of random coils) may be reversible. The authors [139] evidenced that recovery of the activity after thermal treatment of Hb immobilized in the three solids leads to a new conformation of the protein resulting in an improved bioactivity. Using ITC analysis, the authors demonstrated that immobilization of Hemoglobin in a-ZrP was exothermic and proceeds via a single site binding model.…”

Section: Biohybridmentioning

confidence: 99%

Enzyme‐Based Bioinorganic Materials

Forano

Prévot

2007

Bio‐inorganic Hybrid Nanomaterials

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Trends in research materials currently focus on innovation in the field of intelligent or smart materials [1]. Smart materials are materials that display functionalities able to detect a change in the near environment, answer to a stimulus, modify a property, store, transfer or convert a signal, in a sense behave like living cells. The design of smart materials focuses on the concept of biomimetic materials [2][3][4] in order to reproduce specific and selective properties not deliverable by standard chemical or physical processes. Hence, there has been much effort concentrated on the development of enzyme-based biohybrid materials. Enzyme-based biohybrid materials are under much investigation for the operation of physical or chemical functions. Enzymes display active and selective properties such as molecular recognition, complexation or carrier functions, and a wide range of catalytic reactions (hydrolysis, condensation, redox reaction, isomerization, reaction transfer). Enzymes are thus very attractive as an alternative to traditional catalysts when chemical routes are difficult to apply. Enzymes are able to catalyze regio-and stereo-selective reactions with rate accelerations up to 10-17 fold [17]. They are interesting materials for realization of sensing devices, recognition systems and nanoreactors with many potential industrial developments in biocatalysis [4] for the food industry or effluent treatment [5,6], in biomedical domains for medical implants or enzyme delivery [7], in bioanalysis and affinity chromatography [8,9], and in biosensor technology [10][11][12].Engineering the development of biohybrid enzyme-based materials requires solution of the limitations of the use of enzymes due to their intrinsic fragility against the various environmental stresses and their narrow range of working conditions (e.g. short pH and temperature range). Embedding enzymes in protective matrices in order to solve these problems has been envisaged for many years [13,14]. Immobilization of enzymes in solid supports is seen as a strategy to preserve the active site, to improve the performance of the enzyme, that is, its activity, reaction rate and long-term stability, and to allow enzyme reuse. Physical and chemical properties of the solid supports such as particle size, surface area, pore diameter, mechanical j443 Bio-inorganic Hybrid Nanomaterials. Edited

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Protein annealing: Thermal treatment of met-hemoglobin bound to α-zirconium phosphate/phosphonates results in initial denaturation followed by recovery of activity and structure

Cited by 24 publications

References 37 publications

Protein/DNA/Inorganic Materials: DNA Binding to Layered α‐Zirconium Phosphate Enhances Bound Protein Structure and Activity

Protein/DNA/Inorganic Materials: DNA Binding to Layered α‐Zirconium Phosphate Enhances Bound Protein Structure and Activity

Direct intercalation of cisplatin into zirconium phosphate nanoplatelets for potential cancer nanotherapy

Enzyme‐Based Bioinorganic Materials

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