A QM-MD simulation approach to the analysis of FRET processes in (bio)molecular systems. A case study: complexes of E. coli purine nucleoside phosphorylase and its mutants with formycin A.

Predicting FRET pathways in proteins using computer simulation techniques is very important for reliable interpretation of experimental data. A novel and relatively simple methodology has been developed and applied to purine nucleoside phosphorylase (PNP) complexed with a fluorescent ligand - formycin A (FA). FRET occurs between an excited Tyr residue (D*) and FA (A). This study aims to interpret experimental data that, among others, suggests the absence of FRET for the PNPF159A mutant in complex with FA, based on novel theoretical methodology. MD simulations for the protein molecule containing D*, and complexed with A, are carried out. Interactions of D* with its molecular environment are accounted by including changes of the ESP charges in S1, compared to S0, and computed at the SCF-CI level. FRET probability W F depends on the inverse six-power of the D*-A distance, R da . The orientational factor 0 < k(2) < 4 between D* and A is computed and included in the analysis. Finally W F is time-averaged over the MD trajectories resulting in its mean value. The red-shift of the tyrosinate anion emission and thus lack of spectral overlap integral and thermal energy dissipation are the reasons for the FRET absence in the studied mutants at pH 7 and above. The presence of the tyrosinate anion results in a competitive energy dissipation channel and red-shifted emission, thus in consequence in the absence of FRET. These studies also indicate an important role of the phenyl ring of Phe159 for FRET in the wild-type PNP, which does not exist in the Ala159 mutant, and for the effective association of PNP with FA. In a more general context, our observations point out very interesting and biologically important properties of the tyrosine residue in its excited state, which may undergo spontaneous deprotonation in the biomolecular systems, resulting further in unexpected physical and/or biological phenomena. Until now, this observation has not been widely discussed in the literature.

Captopril: determination in blood and pharmacokinetics after single oral dose.

The study describes a specific, precise, sensitive and accurate method for determination of unchanged captopril, an angiotensin-converting enzyme inhibitor, in human plasma. Captopril was stabilized by forming an adduct with p-bromophenacyl bromide and this adduct was measured by high-performance liquid chromatography with UV detection. The standard curve was linear over a range of 30-800 ng ml-1. The average yield of derivatization of the unchanged captopril was 73.6% and the recovery of captopril-adduct reached 93.1%. The limit of detection was 15 ng ml-1, while the quantitative limit was 30 ng ml-1. Inter- and intra-assay RSD was below 9%, but inter- and intra-assay accuracy was below 8%. On the basis of elaborated method, a single-dose pharmacokinetics in 12 men, in two doses (25 and 50 mg of captopril) has been investigated. The comparison of the pharmacokinetic parameters obtained from both doses of the drug have been made.

Biopharmaceutical evaluation of ferrous salts in form of oral long-acting tablets.

We determined the relative bioavailability in men of two preparations containing ferrous salts in the form of long-action tablets in relation to analogous conventional preparations in the from of drops and tablets. The comparison of the bioavailability of preparations slowly and quickly releasing the active substance did not show any prolonged effect and greater absorption of drugs slowly releasing the active substance along the alimentary tract.