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1.
Indian J Biochem Biophys ; 2013 Feb; 50(1): 64-71
Article in English | IMSEAR | ID: sea-147288

ABSTRACT

The hydrolysis of p-nitrophenyl phosphate (pNPP) by calf intestinal alkaline phosphatase (CIAP) was investigated with respect to kinetic parameters such as Vmax, Km and Kcat under varying pH, buffers, substrate concentration, temperature and period of incubation. Highest activity was obtained with Tris-HCl at pH 11, while in the case of glycine-NaOH buffer the peak activity was recorded at pH 9.5. The enzyme showed the following kinetic characteristics with pNPP in 50 mM Tris-HCl at pH 11 and 100 mM glycine-NaOH at pH 9.5 at an incubation temperature of 37°C: Vmax, 3.12 and 1.6 µmoles min-1 unit-1; Km, 7.6 × 10-4 M and 4 × 10-4 M; and Kcat, 82.98 s-1 and 42.55 s-1, respectively. CIAP displayed a high temperature optimum of 45°C at pH 11. The kinetic behaviour of the enzyme under different parameters suggested that the enzyme might undergo subtle conformational changes in response to the buffers displaying unique characteristics. Bioprecipitation of Cu2+ from 50 ppm of CuCl2 solution was studied where 64.3% of precipitation was obtained. Pi generated from CIAP-mediated hydrolysis of pNPP was found to bind with copper and precipitated as copper-phosphate. Thus, CIAP could be used as a test candidate in bioremediation of heavy metals from industrial wastes through generation of metal-phosphate complexes.


Subject(s)
Alkaline Phosphatase/chemistry , Alkaline Phosphatase/metabolism , Animals , Cattle/metabolism , Enzyme Activation , Enzyme Stability , Hydrolysis , Kinetics , Nitrophenols/chemistry , Organophosphorus Compounds/chemistry
2.
Indian J Biochem Biophys ; 2011 Feb; 48(1): 29-34
Article in English | IMSEAR | ID: sea-135297

ABSTRACT

Bacterial organophosphate hydrolases (OPH) have been shown to hydrolyze structurally diverse group of organophosphate (OP) compounds and nerve agents. Due to broad substrate range and unusual catalytic properties, the OPH has successfully been used to develop eco-friendly strategies for detection and decontamination of OP compounds. However, their usage has failed to gain necessary acceptance, due to short half-life of the enzyme and loss of activity during process development. In the present study, we report a simple procedure for immobilization of OPH on biocompatible gelatin pads. The covalent coupling of OPH using glutaraldehyde spacer has been found to dramatically improve the enzyme stability. There is no apparent loss of OPH activity in OPH-gelatin pads stored at room temperature for more than six months. As revealed by a number of kinetic parameters, the catalytic properties of immobilized enzyme are found to be comparable to the free enzyme. Further, the OPH‑gelatin pads effectively eliminate OP insecticide methyl parathion and nerve agent sarin.


Subject(s)
Enzyme Stability , Enzymes, Immobilized/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Gelatin/chemistry , Hydrolysis , Insecticides/poisoning , Methyl Parathion/chemistry , Organophosphorus Compounds/chemistry , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/isolation & purification , Phosphoric Monoester Hydrolases/metabolism , Sarin/chemistry , Substrate Specificity
3.
Braz. j. med. biol. res ; 41(7): 589-595, July 2008. ilus, tab
Article in English | LILACS | ID: lil-489521

ABSTRACT

Efonidipine hydrochloride is an antihypertensive and antianginal agent with fewer side effects and is better tolerated in the treatment of hypertension with renal impairment. Its interaction with bovine serum albumin (BSA) is of great use for the understanding of the pharmacokinetic and pharmacodynamic mechanisms of the drug. The binding of efonidipine to BSA was investigated by fluorescence spectroscopy and circular dichroism. BSA fluorescence was quenched by efonidipine, due to the fact that efonidipine quenched the fluorescence of tryptophan residues mainly by the collision mode. The thermodynamic parameters ÄH0 and ÄS0 were 68.04 kJ/mol and 319.42 J·mol-1·K-1, respectively, indicating that the hydrophobic interactions played a major role. The results of circular dichroism and synchronous fluorescence measurements showed that the binding of efonidipine to BSA led to a conformational change of BSA. The fraction of occupied sites (è) for the 8-anilino-1-naphthalein-sulfonic acid (ANS)-BSA system is 85 percent, whereas for the NZ-105-BSA system, it is 53 percent, which suggests that the interaction of ANS with BSA is stronger than that of NZ-105 with BSA. Binding studies in the presence of ANS indicated that efonidipine competed with ANS for hydrophobic sites of BSA. The effects of metal ions on the binding constant of the efonidipine-BSA complex were also investigated. The presence of metal ions Zn2+, Mg2+, Al3+, K+, and Ca2+ increased the binding constant of efonidipine_BSA complex, which may prolong the storage period of NZ-105 in blood plasma and enhance its maximum effects.


Subject(s)
Animals , Cattle , Dihydropyridines/chemistry , Nitrophenols/chemistry , Serum Albumin, Bovine/chemistry , Circular Dichroism , Models, Chemical , Organophosphorus Compounds/chemistry , Spectrometry, Fluorescence , Thermodynamics
4.
Egyptian Journal of Chemistry. 2007; (Special Issue): 45-58
in English | IMEMR | ID: emr-82189

ABSTRACT

Dialkyl phosphites [4] and trialkyl phosphites [5] attack the aldehydic carbonyl carbon of 4-methoxy-5-oxo-5H-furo[3,2-g] benzopyran-6-carboxaldehyde [3a] and 4,9-dimethoxy-5-oxo-5H-furo [3,2-g] benzopyran-6- carboxaldehyde [3b] yielding the corresponding alpha-hydroxyphosphonates [7]. Compounds 7 regenerate the starting gamma-pyrone derivatives [3a or 3b] and the appropriate dialkyl phosphites [4] upon thermolysis under reduced pressure. On the other hand, the reaction of 3 with ylidenetriphenylphosphoranes [6] proceeds according to the Wittig mechanism to give the respective ethylenes [E 17]. The reaction of 3b with diethyl cyanomethylphosphonate [18] under Homer-Wittig conditions yields 6-[[alpha-cyano-alpha diethoxyphosphoryl] ethyliden-yl]-4,9-dimethoxy-5-oxo-5H-furo [3,2-g] benzopyran [20]. Structures of the isolated new products were elucidated by compatible, analytical, chemical and spectroscopic measurements


Subject(s)
Phosphites/chemistry , Indicators and Reagents , Organophosphorus Compounds/chemistry
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