No está claro cuán grande es el papel de los antibióticos https://antibioticos-wiki.es en las relaciones competitivas entre los microorganismos en condiciones naturales. Zelman Waxman creía que este papel era mínimo, los antibióticos no se forman sino en culturas limpias en entornos ricos. Posteriormente, sin embargo, se descubrió que en muchos productos, la actividad de síntesis de antibióticos aumenta en presencia de otros tipos o productos específicos de su metabolismo.
Vol-15_no-4a.pdf
Polish J. of Environ. Stud. Vol. 15 No. 4A (2006), 59-61About 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 1.5510-5 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
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