Lars Lindqvist

The pH dependence of fluorescein fluorescence

Abstract Fluorescence quantum yields Φf and corrected fluorescence spectra were determined for fluorescein over a wide pH range (10 M H2SO4 to 0.01 M NaOH) in aqueous solution. The results were interpreted by comparison with those obtained for the very similar dye 6-hydroxy-9-phenyl-fluoron (HPF) which lacks the fluorescein carboxyl group. We find for the fluorescein cation Φf = 0.9−1, for the neutral molecule Φf = 0.20−0.25, and for the monoanion Φf = 0.25−0.35. The neutral molecule and monoanion have identical fluorescence spectra: an emission with maximum at 515 nm, extending to about 700 nm. (The dianion also has a maximum at 515 nm but has a more narrow emission band.) The fluorescein cation dissociates in the excited state at an acidity corresponding to that of ≈3 M H2SO4. A detailed scheme of the protolytic reactions of fluorescein in the excited state is presented.

Transient effects in the nanosecond laser photolysis of carboxyhemoglobin: “CAGE” recombination annd spectral evolution of the protein

Abstract The transient light absorption changes at 400-600 nm observed following photodissociation of human adult carboxy-hemoglobin in aqueous solution (pH 7.0) by pulsed laser irradiation (7 ns fwhm, λ = 529 nm) reveal that geminate recombination is occurring during ≈ 100 ns after the dissociation. They also show that the spectrum of the photodeligated hemoglobin initially is slightly different from that of stable deoxyhemoglobin but develops into the latter in ≈5 μs. The recombination kinetics vary with the excitation condiitions (duration, intensity).

One-and two-photon ionization of aqueous tryptophan by the harmonics of the Nd laser

Abstract Study of the photoionization of aqueous tryptophan by the 265 nm harmonic of the Nd-glass laser at different laser pulse durations and energies established that this reaction is due to one-photon as well as two-photon absorption, the latter occurring mainly via the excited singlet slate Addition of the 353, or, alternatively, the 530 nm harmonic increased the photoelectron yield appreciably Double-pulse experiments (lower harmonic delayed with respect to the 265 nm pulse) were used to determine the relative importance of the excited singlet and triplet states in the two-photon processes responsible for this increase.

Intraproteic ligand diffusion in laser-photodissociated carboxyhemoglobin and subunits

Abstract Carboxyhemoglobin in aqueous solution was photodissociated by laser pulses of 1–7 ns at 530 nm. Kinetic spectrophotometry showed that geminate recombination of CO to the heme, occurring during the first 100–200 ns after dissociation, has the square-root-of-tune dependence expected for ligation during random-walk diffusion of the ligand in the protein. Isolated α and β subunits gave similar results.

Direct measurement of excited singlet-state lifetime in the homologous sequence adenine, adenosine, adenosine 5′-monophosphate and in calf thymus DNA

Abstract The excited singlet-state lifetimes of the nucleic acid components adenine, adenosine and adenosine 5′-monophosphate, and also that of calf thymus DNA were determined from transient absorption measurements using a UV-laser flash-photolysis apparatus with subpicosecond time resolution. Excitation at λ = 280 mm gave for adenine the value 1.6 ± 0.3 ps. The addition of ribose and the further addition of a phosphate group do not affect the lifetime significantly: the values 1.9 ± 0.3 and 2.1 ± 0.3 ps, respectively, were obtained. For calf thymus DNA the lifetime was shorter than the experimental limit of resolution, 0.4 ps.

Photosensitized generation of hydroxyl radical by eight new sulfur and selenium analogs of psoralen

Abstract Using spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) combined with electron spin resonance spectroscopy (ESR), irradiation at the PUVA wavelength (365 nm) of eight new sulfur and selenium analogs of psoralen was shown to generate hydroxyl radicals (OH ∗ ), in air-saturated aqueous solution. For each compound, the DMPO-OH spin adduct signal was detected and could be abolished when the photosensitization reaction was carried out in the presence of OH ∗ scavengers such as sodium formate or sodium benzoate. Others inhibitors were selected to investigate the mechanism of OH ∗ generation. A comparison between the different psoralen analogs, psoralen and 8-methoxypsoralen (8-MOP) indicated that the rate of hydroxyl radical production is in the same order or lower than for 8-MOP, except for 7H-thieno (3,2-g) [1] benzothiopyran-7-one which produces 2.7 times more OH ∗ than 8-MOP. Moreover, the presence of superoxide anion (O 2 ∗- as an intermediate, revealed by the effect of superoxide dismutase (SOD) addition on the ESR spectra, was confirmed by following the spectrophotometric reduction of cytochrome c . Laser flash photolysis experiments allowed us to visualize the triplet state of thio- and seleno-psoralens. Because these new psoralen analogs only display a low yield of OH ∗ production, they should be considered as potentially important new drugs to be used in photochemotherapy.

Stepwise Two‐Photon Excitation of 1,5‐Dihydroflavin Mononucleotide: Study of Flavosemiquinone Properties

Abstract— Photoionization of 1,5‐dihydroflavin mononucleotide in aqueous solution, leading to formation of hydrated electrons and flavosemiquinone radicals, was studied by nanosecond laser photolysis (excitation at LD = 354.7 nm). Analysis of the laser fluence dependence of the photoionization yield indicates that the reaction is a stepwise two‐photon reaction with the fluorescent state of the flavin as intermediate step. Extinction coefficients of the neutral and anionic flavosemiquinone were obtained from transient absorbance measurements at 310‐660 nm. The pro‐tolytic dissociation constant of the neutral radical was determined from a study at pH 7.3‐10.3 of the spectral evolution in the submicrosecond time range due to de‐protonation of this radical. The value pKa= 8.7 0.1 was obtained. The formation of tautomeric species of the neutral flavosemiquinone was suggested based on the existence of a spectral evolution of this species during the first microseconds after excitation.

Excited-state dynamics of fully reduced flavins and flavoenzymes studied at subpicosecond time resolution.

The photophysics of the fully reduced states of a number of flavins (flavin mononucleotide, flavin adenine dinucleotide and 3‐N‐methyllumiflavin) and flavoenzymes (glucose oxidase from Aspergillus niger and the flavodehydrogenase component isolated from flavocytochrome b2) was studied using subpicosecond laser excitation at Λ. = 312 nm. The prompt transient absorption spectra (measured from 400 to 850 nm) were all closely similar in the case of the free flavins in aqueous solution. The decay of the transient absorbance obeyed biexponential kinetics with a fast component of lifetime ranging from 4 to 130 ps and a slower phase with a lifetime above 1 ns. The spectral structure changed appreciably during the rapid decay phase. In contrast, in the case of the enzymes only a very slight decay was apparent over the probed time interval (1 ns) and the shape of the spectrum remained unchanged. It is proposed that the two transient spectra appearing in the free flavins correspond to two conformations differing by their degree of nonplanarity, whereas in the flavoenzymes only one conformation is possible.

FLUORESCENCE PROPERTIES OF HUMAN ADULT APOHEMOGLOBIN AND ITS SUBUNITS

Abstract. Fluorescence excitation, emission and quantum yields for human A apohemoglobin and its subunits have been examined and compared to those of horse heart apomyoglobin, a single subunit protein.

PHOTOPHYSICS OF METHYLENE BLUE-POLYNUCLEOTIDE COMPLEXES IN THE PICOSECOND TIME RANGE: INFLUENCE OF THE LOCAL STRUCTURE OF THE POLYNUCLEOTIDE

Abstract—