Binding of Hoechst 33258 and its derivatives to DNA.

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)].poly[d(AT)], poly(dA).poly(dT), and DNA dodecamer with the sequence 5'-CGTATATATACG-3'. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)].poly[d(AT)] and poly(dA).poly(dT).

DNA sequence recognition by bis-linked netropsin and distamycin derivatives.

We studied the interaction of cis-diammine Pt(II)-bridged bis-netropsin, cis-diammine Pt(II)-bridged bis-distamycin and oligomethylene-bridged bis-netropsin with synthetic DNA fragments containing pseudosymmetrical AT-rich nucleotide sequences and compared it with the interaction of the parent compounds netropsin and distamycin A. For fragments containing multiple blocks of (AIT)4 and (T/A)4 separated by zero, one, two and three GC-base pairs, DNase I footprinting and CD spectroscopy studies reveal that 5'-TTTTAAAA-3' is the strongest affinity binding site for cis-diammine Pt(II)-bridged bis-netropsin and bis-distamycin. They both bind less strongly to a DNA region containing the sequence 5'-AAAATTTT-3'. Netropsin, distamycin A and oligomethylene-bridged bis-netropsin exhibit far less sequence discrimination.

Contribution of protein conformation to heme stereochemistry and reactivity. Low-temperature magnetic circular dichroism data.

Visible and near infrared magnetic circular dichroism (MCD) spectra of heme proteins and enzymes as well as those of a protein-free heme bound to 2-methylimidazole were recorded and compared at 4.2 K in unrelaxed metastable and relaxed equilibrium heme stereochemistry. The relaxed and unrelaxed stereochemistries of a 5-coordinate ferrous heme were generated by chemical reduction of iron at room temperature before freezing the sample and by photolysis of CO or O2 complexes at 4.2 K, respectively. The results are discussed in terms of a protein contribution into energies of the Fe-N epsilon(His) and Fe-N(pyrrols) bonds and their change on a ligand binding. We observed and analyzed cases of weak (myoglobin, hemoglobin) and strong (leghemoglobin, peroxidases) constraints imposed by the protein conformation on the proximal heme stereochemistry by comparing the bond energies in proteins with those in the protoheme-(2-methylimidazole) model compound. The role of a protein moiety in modulating the ligand binding properties of leghemoglobin and the heme reactivity of horseradish peroxidase is discussed.

Heme-linked ionization and ligand binding produce identical changes of proximal heme stereochemistry in reduced horseradish peroxidase. Evidence for existence of two protein conformations.

The visible and near infrared magnetic circular dichroism spectra of chemically reduced horseradish peroxidase at neutral and alkaline pH values and 5-coordinate protoheme-(2-methylimidazole) at pH 9.1 were compared at 4.2 K with those of photolysis products of their carbon monoxide complexes. From the results obtained we concluded that: (i) there are two protein conformations of HRP which determine the geometry of the Fe-N(His) bond; (ii) the transition from one conformation (heme stereochemistry) to another can be induced by either heme-linked ionization or ligand binding; (iii) a trigger mechanism for switching between two conformations has to exist.

Low- and ultralow-temperature magnetic circular dichroism studies of reduced cytochromes P-450-LM2 and P-420-LM2 and of photo-products of their co-complexes. The spin-state and axial ligation of heme iron.

MCD spectra of reduced cytochromes P-450 and P-420 have been recorded in the spectral region 350-800 nm at temperatures 4.2-290 K and were compared with the respective low-temperature photolysed CO-complexes at 4.2 K. The MCD data are consistent with the suggestions that: the heme iron is high-spin in the reduced proteins and in the photolysed species; mercaptide is the protein-derived ligand of the heme iron in the reduced cytochrome P-450, as well as in its CO-complex; imidazole of histidine is the fifth ligand of the heme iron both in the reduced P-420 and its CO-complex; structural changes in the heme iron coordination sphere occur at CO-binding.