Potentiometric and multi-NMR studies of aluminum(III) complex with L-glutamate in acidic aqueous solutions.
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Complex formation studies of L-glutamate with aluminum(III) ion were conducted in acidic aqueous solutions (pH 2.0 - 5.5) by means of pH-metric titration and multinuclear (1H, 13C and 27Al) NMR techniques. The following results were obtained: (1) Al could weakly coordinate with Glu to form various mononuclear 1:1 (AlLH2+, AlL+, AlLH(-1)) species and dinuclear 2:1 (Al2L4+) species in acidic aqueous solutions, which somewhat agreed with previous findings. (2) The multi-NMR spectra of Al-Glu and Al-Asp strongly suggest that, besides negatively charged carboxylate donors (-COO(-), -COO(-)), the amino group of Glu can participate in the binding of Al in the AlL+ and AlLH(-1) species in the case of its deprotonation, which rather agreed with the case of Al-Asp. (3) These tridentate five-+seven-membered joint chelate (-COO(-), -NH2, -COO(-)) complexes exhibit an enhanced stability, which can help to better understand the biological studies that Al-Glu could cross the erythrocyte membrane and the blood-brain barrier (BBB) and be deposited selectively in various brain regions, particularly in the cortex. It will also help to intrinsically understand the Al's role in the biological transamination system, which is a very important process in all living things. (+info)
Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1.
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Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH- and FMN-binding domains of NR1 have been expressed and purified and their redox properties studied by stopped-flow and steady-state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (-315 +/- 5 mV) and semiquinone/dihydroquinone (-365 +/- 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is approximately 200-fold slower. Hydride transfer is rate-limiting in steady-state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped-flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP+ is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is -340 mV, cf-320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are -146 +/- 5 mV (oxidized/semiquinone) and -305 +/- 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin-binding domains. (+info)
Photo-accumulation of the P+QB- radical pair state in purple bacterial reaction centres that lack the QA ubiquinone.
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Photo-excitation of membrane-bound Rhodobacter sphaeroides reaction centres containing the mutation Ala M260 to Trp (AM260W) resulted in the accumulation of a radical pair state involving the photo-oxidised primary electron donor (P). This state had a lifetime of hundreds of milliseconds and its formation was inhibited by stigmatellin. The absence of the Q(A) ubiquinone in the AM260W reaction centre suggests that this long-lived radical pair state is P(+)Q(B)(-), although the exact reduction/protonation state of the Q(B) quinone remains to be confirmed. The blockage of active branch (A-branch) electron transfer by the AM260W mutation implies that this P(+)Q(B)(-) state is formed by electron transfer along the so-called inactive branch (B-branch) of reaction centre cofactors. We discuss how further mutations may affect the yield of the P(+)Q(B)(-) state, including a double alanine mutation (EL212A/DL213A) that probably has a direct effect on the efficiency of the low yield electron transfer step from the anion of the B-branch bacteriopheophytin (H(B)(-)) to the Q(B) ubiquinone. (+info)
On the possibility of excimer state formation in homodimers of the pyrimidine bases.
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A detailed investigation of the ground state and the first excited state of the pyrimidine bases of uracil, cytosine, thymine and their stacked homodimers is carried out by the Extended Huckel method. The first exicted state is shown to be the (see article)-state. The excitation is localized mainly on the C5-C6 bond region. The energy dependence of the ground state and the first excited state of the homodimers on the mutual position of bases is considered. For every dimer a minimum on the exicted state potential curve is found. This fact creates the possibility for the formation of excimer. The obtained data give some reason for the suggestion about participation of the excimer state as a precursor of photodimer. (+info)
Novel ibuprofen potentiometric membrane sensors based on tetraphenylporphyrinato indium(III).
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Two novel potentiometric membrane sensors responsive to the ibuprofen drug have been developed. These incorporate poly(vinyl chloride) and polyurethane matrix membranes containing 5,10,15,20-tetraphenylporphrinato (TPP) indium(II) ionophore plasticized with dibutylsebacate. The sensors show a near-Nernstian response with anionic slopes of -53 and -55 mV decade(-1), over the concentration range of 4.2 x 10(-6)-1.0 x 10(-2) and 3.3 x 10(-6)-1.0 x 10(-2) M ibuprofen within pH ranges of 4-9 and 5-9 for PVC and PU matrix membranes, respectively. A sensor based on a polyurethane membrane displays a lower detection limit and a wider linear working range, and a sensor based on a PVC membrane exhibits a better overall selectivity, especially in the presence of lipophilic organic anions. Both sensors are used for the quantification and quality-control assessment of ibuprofen in pharmaceutical preparations. The average recoveries are 99.1+/-0.3% and 99.3+/-0.3% for TPP In(III)-PVC and TPP In(III)-PU based membrane sensors, respectively. High selectivities towards ibuprofen in the presence of many anions, drug excipients and diluents are offered by both sensors, which exhibit a non-Hofmeister selectivity pattern. (+info)
Determination of dipyrone in pharmaceutical products by flow injection analysis with potentiometric detection.
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This work describes an FIA potentiometric procedure for the quantification of dipyrone in pharmaceutical products. For the detector, a tubular electrode comprising a polymeric membrane containing tetraoctylammonium as an electroactive material (5% w/w), dibutylphtalate as a mediator solvent (65% w/w) and PVC (30% w/w) directly applied above a graphite conductor support was used. This unit was incorporated into a monochannel FI-system with a 0.1 mol/L phosphate buffer solution (pH = 5.2) as the carrier solution. The electrode showed a linear response from 8.0 x 10(-4) to 10(-1) mol/L dipyrone, a slope of 62.1 +/- 0.2 mV/dec in pH 5.2 units, an injection volume of 500 microL and a carrier flow-rate of 6 mL/min. This procedure was applied to the analysis of pharmaceutical formulations (oral and injectable) containing dipyrone; the obtained results gave a relative error of less than 3.9% and coefficients of variation less than 1% and 5%, respectively, for the FIA and classical iodometric methods. (+info)
Interflavin electron transfer in human cytochrome P450 reductase is enhanced by coenzyme binding. Relaxation kinetic studies with coenzyme analogues.
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The role of coenzyme binding in regulating interflavin electron transfer in human cytochrome P450 reductase (CPR) has been studied using temperature-jump spectroscopy. Previous studies [Gutierrez, A., Paine, M., Wolf, C.R., Scrutton, N.S., & Roberts, G.C.K. Biochemistry (2002) 41, 4626-4637] have shown that the observed rate, 1/tau, of interflavin electron transfer (FADsq - FMNsq-->FADox - FMNhq) in CPR reduced at the two-electron level with NADPH is 55 +/- 2 s-1, whereas with dithionite-reduced enzyme the observed rate is 11 +/- 0.5 s-1, suggesting that NADPH (or NADP+) binding has an important role in controlling the rate of internal electron transfer. In relaxation experiments performed with CPR reduced at the two-electron level with NADH, the observed rate of internal electron transfer (1/tau = 18 +/- 0.7 s-1) is intermediate in value between those seen with dithionite-reduced and NADPH-reduced enzyme, indicating that the presence of the 2'-phosphate is important for enhancing internal electron transfer. To investigate this further, temperature jump experiments were performed with dithionite-reduced enzyme in the presence of 2',5'-ADP and 2'-AMP. These two ligands increase the observed rate of interflavin electron transfer in two-electron reduced CPR from 1/tau = 11 s-1 to 35 +/- 0.2 s-1 and 32 +/- 0.6 s-1, respectively. Reduction of CPR at the two-electron level by NADPH, NADH or dithionite generates the same spectral species, consistent with an electron distribution that is equivalent regardless of reductant at the initiation of the temperature jump. Spectroelectrochemical experiments establish that the redox potentials of the flavins of CPR are unchanged on binding 2',5'-ADP, supporting the view that enhanced rates of interdomain electron transfer have their origin in a conformational change produced by binding NADPH or its fragments. Addition of 2',5'-ADP either to the isolated FAD-domain or to full-length CPR (in their oxidized and reduced forms) leads to perturbation of the optical spectra of both the flavins, consistent with a conformational change that alters the environment of these redox cofactors. The binding of 2',5'-ADP eliminates the unusual dependence of the observed flavin reduction rate on NADPH concentration (i.e. enhanced at low coenzyme concentration) observed in stopped-flow studies. The data are discussed in the context of previous kinetic studies and of the crystallographic structure of rat CPR. (+info)
NMR spectra and potentiometry studies of aluminum(III) binding with coenzyme NAD+ in acidic aqueous solutions.
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Complexation and conformational studies of coenzyme NAD+ with aluminum were conducted in acidic aqueous solutions (pH 2-5) by means of potentiometry as well as multinuclear (1H, 13C, 31P, 27Al) and two-dimensional (1H, 1H-NOESY) NMR spectroscopy. These led to the following results: (1) Al could coordinate with NAD+ through the following binding sites: N7' of adenine and pyrophosphate free oxygen (O(A)1, O(N)1,O(A)2) to form various mononuclear 1:1 (AlLH23+, AlLH2+) and 2:1 (AlL2-) species, and dinuclear 2:2 (Al2L22+) species. (2) The conformations of NAD+ and Al-NAD+ depended on the solvents and different species in the complexes. The results suggest the occurrence of an Al-linked complexation, which causes structural changes at the primary recognition sites and secondary conformational alterations for coenzymes. This finding will help us to understand role of Al in biological enzyme reaction systems. (+info)