Svante Eriksson

Experimental correction for the inner-filter effect in fluorescence spectra

Recorded fluorescence intensity is in general not proportional to sample concentration owing to absorption of the incident and emitted light passing through the sample to and from the point inside the cell where the emission is detected. This well known inner-filter effect depends on sample absorption and on instrument geometry, and is usually significant even in samples with rather low absorption (the error is about 8% at an absorbance of 0.06 in a 1 cm square cell). In this work we show that a particular experimental set-up can be calibrated for the inner-filter effect from the absorption and fluorescence excitation spectra of a suitable standard. The calibration takes only a few minutes and provides correction with sufficient accuracy for most practical situations.

Exciton splitting in the spectra of covalently linked porphyrins

Abstract Absorption spectra are obtained for bis- and penta-porphyrin derivatives of meso-tetra-phenylporphyrins in which the porphyrin units are connected via 1,4 substituted benzene bridges. The results are interpreted with the help of the CNDO/S model and the Forster model using transition charges or dipoles of the monomer. An energy splitting of about 10 nm occurs in the Soret band. The polarization directions for the dimer were examined using the method of stretched polymer films. The energy splitting, intensities and polarization of the Soret band are explained by the transition dipole model. Dual fluorescence, indicating the existence of an emitting charge transfer state, was not found.

The electronically excited states of 2-phenylindole

The light absorption of 2-phenylindole (2PI) in the UV region (210-350 nm) is investigated by means of linear dichroism in stretched polyethylene film and fluorescence polarization anisotropy in a propylene glycol glass. Experimentally, 2PI is found to have five distinct electronic transitions located above 200 nm for which the transition moments are determined. The conclusions are supported by quantum chemical calculations and the origin of the so-called composite band is discussed. Comparison with the absorption spectrum of the fluorescent DNA binding probe DAPI is also made and 2PI is found to be an appropriate model system for the electronic transitions of DAPI.

DNA Drug Interactions Studied with Polarized Light Spectroscopy: The DAPI Case

Attention is focused on flow linear dichroism (FLD) measured with phase—modulation technique as a simplistic tool for detecting and characterizing DNA drug interactions. FLD combined with DNA—induced circular dichroism (CD), and in some instances fluorescence polarization anisotropy (FPA), has thus for a number of intercalators and non—intercalators provided important information about effective binding geometries and orientational dynamics under varying solution and binding conditions. The present study of a classical non—intercalator, DAPI (4‘,6—diamidino—2—phenylindole), offers several surprising features. FLD supports that DAPI binds in the minor groove of DNA and poly(dA—dT), but suggests that it may be intercalated in poly(dG—dC), albeit fluorescence quenching by iodide ion contradicts classical intercalation. The existence of two “binding sites” on DNA, one with an extremely low density as evidenced from CD, is surprisingly enough also found with the homopolymer poly(dA—dT). Three possible explanations for this observation are discussed: 1) the low—density site may be a “transient site” owing to DNA conformational dynamics, 2) the second site may reflect interacting DAPI monomers or 3) be an effect of a small allosteric conformational change in DNA. However, first at very high degrees of occupancy exciton DAPI—DAPI interaction indicates the presence of true dimers. Results from molecular mechanics calculations are in agreement with that different binding modes are preferred in poly(dG—dC) and poly(dA—dT) but give no indication as to the origin of two DAPI binding sites in the latter.

Dynamics of benzo[a]pyrene diol epoxide adducts in poly(dG-dC · (dG-dC studied by synchrotron excited fluorescence polarization anisotropy decay

Time-resolved fluorescence studies have been performed on (+)-anti-7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene adducts in double-stranded poly(dG-dC).(dG-dC). Part of the adduct population gives rise to excimer fluorescence. The heterogeneous fluorescence emission decay curves at 22 degrees C could be resolved into three components with lifetimes: 0.4 ns, 3 ns and 24 ns for the total fluorescence (monomer and excimer emission), and 0.5 ns, 5 ns and 24 ns, respectively, for excimer emission alone. The relative amplitudes for the longer lifetimes were larger for the pure excimer population than for the mixed population. The fluorescence polarization anisotropy decay curves were resolved into two components of rotational correlation times: 0.4 ns and 25 ns for the total fluorescence and 0.3 ns and 33 ns for the excimer fluorescence. We interpret the two rotational correlation times to correspond to local motion of the adduct and segmental motion of the polynucleotide, respectively.

Interaction of benz(a)pyrene diol epoxide with chromatin studied by flow linear dichroism

Chromatin isolated from Ehrlich ascites cells was incubated with the tumourigenic compound (+)‐7β,8α‐dihydroxy‐9α,10α‐epoxy‐7,8,9,10‐tetrahydrobenz[a]pyrene [(+)‐anti‐BPDE] at low ionic strength and the modified chromatin was analysed using flow linear dichroism (LD). The results confirm that (+)‐anti‐BPDE preferentially binds to the DNA in the linker regions, and furthermore show that the long axis of the bound pyrenyl chromophore is oriented parallel or close to parallel to the average orientation of the chromatin fiber axis. The data indicate that the binding geometry of (+)‐anti‐BPDE in chromatin is similar to that in pure DNA and deoxyguanosine‐containing double‐helical oligonucleotides.