Binding of cationic porphyrin to isolated and encapsidated viral DNA analyzed by comprehensive spectroscopic methods.
The complexation of tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) with free and encapsidated DNA of T7 bacteriophage was investigated. To identify binding modes and relative concentrations of bound TMPyP forms, the porphyrin absorption spectra at various base pair/porphyrin ratios were analyzed. Spectral decomposition, fluorescent lifetime, and circular dichroism measurements proved the presence of two main binding types of TMPyP, e.g., external binding and intercalation both in free and in encapsidated DNA. Optical melting studies revealed that TMPyP increases the strand separation temperature of both free and native phage DNA and does not change the phase transition temperature of phage capsid proteins. From these findings we concluded that TMPyP binding does not influence the protein structure and/or the protein-DNA interaction. A combined analysis of absorption spectra and fluorescence decay curves made possible the determination of concentrations of free, externally bound, and intercalated porphyrin. As a perspective, our results facilitate a qualitative analysis of the TMPyP binding process at various experimental conditions.
Zupán
Kristóf
K
Institute of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.
Herényi
Levente
L
Tóth
Katalin
K
Majer
Zsuzsa
Z
Csík
Gabriella
G
D017123:Bacteriophage T7 / Q000235:genetics; D001665:Binding Sites; D002412:Cations; D004279:DNA, Viral / Q000737:chemistry* / Q000187:drug effects; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D044367:Phase Transition; D011166:Porphyrins / Q000737:chemistry*; D013057:Spectrum Analysis; D013696:Temperature
D002412:Cations; D004279:DNA, Viral; D011166:Porphyrins