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Vol-15_no-4a.pdf

Polish J. of Environ. Stud. Vol. 15 No. 4A (2006), 59-61 About the Interaction of Human Serum
Albumin with Nevirapine and Azidothymidine
A. Kluczewska1, K. Michalik1, Z. Drzazga1, M. Kaszuba2
1University of Silesia, A. Cheákowski’ Institute of Physics, Department of Medical Physics, Uniwersytecka 4, 40-007 Katowice, Poland 2Medical University of Silesia, Department of Prosthetic Dentistry, Abstract
Fluorescence measurements were used to characterize the interaction of human serum albumin (HSA) with the antiretroviral drugs of: nevirapine (NVP) and azidothymidine (AZT) in aqueous solutions. Comparison of characteristic parameters for static quenching at different excitation wavelengths (280 and 295 nm) and wavelength shifts in synchronous fluorescence spectral measurements revealed the interaction of drugs with different fluorophores in the HSA molecule: NVP showed higher binding affinity to tryptophan residues located in the subdomain IIA, whereas for AZT tyrosine residues in the subdomain IIIA of the protein were affected. Synchronous fluorescence spectra revealed a tendency for AZT to interact with the denaturated form of HSA confirming its toxicity. Keywords: Nevirapine; Azidothymidine; Synchronous fluorescence spectra
Introduction
Materials and methods
Therapy for Human Immunodeficiency Virus (HIV) includes several drugs decreasing the growth of the virus lyophilized and globulin free), azidothymidine reference to an extent that the treatment prevents or markedly standard (r.s.) (Lot 112K3485) and thymine (Lot 123K0719) delays the development of viral resistance to the drugs from Sigma Chemical Co., nevirapine and azidothymidine [1]. In HIV therapy two main classes of drugs are used, from Boehringer Ingelheim Pharmaceuticals Inc. and i.e. nevirapine (NVP), which belongs to non-nucleoside GlaxoSmithKline, were used. The final concentration of the reverse transcriptase inhibitors (NNRTI), and azido- aqueous protein solution was 1.55˜10-5 M in all experiments. thymidine (AZT), which is a nucleoside reverse Adding drugs to HSA solutions produced molar ratios of 0, 6, 15, 30, 45, and 60 for the assays. (NRTI). Combination of these two drugs reduces Emission fluorescence spectra of protein in the presence mother to child transmission of HIV [2]. Antiretroviral of drugs were studied at room temperature using a Hitachi drugs show some ability to plasma proteins binding but F-2500 spectrofluorimeter (Japan). Excitation wavelength only the unbound fraction of drug in plasma can block virus of OEX = 280 nm for exciting tryptophan (Trp) and tyrosine HIV activity. Therefore AVR drugs should bind weakly to (Tyr) residues and of 295 nm for (excites only Trp human serum albumin. In this paper, investigation of AVR excitation were used. The value of binding parameters were drug – human albumin interaction using fluorescence calculated according to the modified Stern-Volmer equation -F) 0
/(F
F 0

Q -1 [10-3 M-1]
.u.] 1000
escence Intensi
uor
3000
Fig. 1. (a) Double reciprocal curves of protein fluorescence in presence of drugs: NVP (squares), AZT (filled triangles), AZT r.s.
(open triangles) and Thymine (open circles) at OEX = 280 nm and OEX = 295 nm; (b) the effect of drugs on the synchronousfluorescence spectra of HSA for 'O = 60 nm and 'O = 15 nm To analyse conformational changes of HSA after drug residues was also revealed in a fluorescence experiment for titration synchronous fluorescence experiments was done. thymine, being a major decomposition product in aqueous Two different scanning intervals 'O = OEM - OEX were used solution. Excitation by a wavelength of 280 nm that for OEX = 280 nm: 'O = 15 nm and 'O = 60 nm. Obtained involves tyrosine residues caused quenching of fluorescence spectra could give characteristic information on the unlike to excitation by 295 nm, proving a binding affinity of respective protein tyrosine and tryptophan residues. thymine to hydrophilic tyrosine residues. The value of the Photometry using a spectrophotometer UV/VIS Jasco V- binding parameter for thymine [(8.2 r 0.5)˜103 M-1] is 530 was used as an assistant technique to qualification kind comparable with AZT r.s. [(8.2 r 0.2)˜103 M-1] and AZT [(9.7 r 1.5)˜103 M-1]. The obtained values of parameters are in good agreement and compatible with the dissociation equilibrium constant for binding of HIV reverse trans- Results and discussion
criptase inhibitors to HSA as reported by Bocedi et al. [7]. Under NVP and AZT titration of the pure sample of HSA the fluorescence as well as absorption spectra of Conclusions
studied mixtures indicated on static quenching process. Double reciprocal plots of F0/(F0-F) at various quencher NVP showed high binding affinity to tryptophan concentration used for determination of Ka value are residues located in the subdomain IIA, whereas AZT affects tyrosine residues in the subdomain IIIA of the protein. To obtain better insight into the molecule conformation Synchronous fluorescence spectra revealed a tendency for under drug titration, the synchronous fluorescence spectra AZT to interact with the denaturated form of HSA, proving with 'O = 60 nm and 'O = 15 nm were recorded (Fig. 1C and 1D, respectively). It should be noted, that AZT titration of HSA caused a marked red shift for the tryptophan residue (| 18 nm) in the emission maximum unlike to NVP. Such a Acknowledgements
red shift indicates that the environment of the tryptophan residue in the albumin becomes less hydrophobic and The research is partly supported by the M.N.E.P. (Grant results in a much higher exposition of Trp to the solvent after AZT addition than found for NVP. According to Burstein et al. [4], the fluorescence spectral maximum for protein-AZT mixtures at molar ratios of 30, 45 and 60 References
belongs to a class of tryptophan on the surface of the protein, completely exposed to water (emission maximum 1. In De Vita V., Hellman S., Curran S.J., Essex M., 350 – 353 nm) and results in the denaturation of the protein Fauci A.,: AIDS: Biology, Diagnosis, Treatment and polypeptide chain. Protein denaturation by high AZT Prevention. Lippincott-Raven Publishers, 177-195,
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drug [5]. The maximum emission of the synchronous fluorescence spectra in the case of HSA-NVP mixtures spectroscopy, Kluwer Academic, Plenum Publisher, keeps its position, which can be explained as an effect of N.York 1999.
immobilization by bond formation. The emission from the 3. Taha T.E., Kumewenda N.I., Gibbson A., Broadhead tryptophan residue can be classified into a spectral class of R.L., Fiscus S., Lema V., Liomba G., Nkhoma Ch., tryptophan residing on the native protein surface with Miotti P.G., Hoover D.R.: Short postexposure prophylaxis in newborn babies to reduce mother-to- Our results indicate, that NVP is bound only to child transmission of HIV-1: NVAZ randomised trial. tryptophan residue Trp 214, located in the subdomain II A, Lancet, 362, 1171-1177, 2003.
where a large hydrophobic cavity in HSA is present, 4. Burstein E.A., Vedenkina N.S., Ivakova M.N.: because the values of the association constant Ka [(10 r Fluorescence and the location of tryptophan residues in 0.3)˜103 M-1] as well as the number of binding sites n (1.0 r protein molecules. Photochem. Photobiol., 18, 263-
0.1) are the same at both excitation wavelengths (280 and 279, 1973.
295 nm). For AZT and the AZT reference standard (r.s.), 5. Wang, W.: Protein aggregation and its inhibition in the number of binding sites n obtained by the fluorescence biopharmaceutics. Int. J. Pharm., 289, 1-30, 2005.
method are higher than one (1.2 and 1.5, respectively). It 6. He X., Carter D.: Atomic structure and chemistry of was reported from X-ray structural analysis [6] that AZT is human serum albumin. Nature, 358, 209-215, 1992.
located in the binding pocket within the IIIA region, where 7. Bocedi A., Notaril S., Narciso P., Bolli A., Fasano M., four tyrosine residues are found (Tyr 401, Tyr 411, Tyr 452 Ascenzi P.: Binding of Anti-HIV drugs to human and Tyr 497). Preference of AZT binding to tyrosine serum albumin, IUBMB LIFE, 56, 609-614, 2004.

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