Mass spectrometric studies of the formation and reactivity of trans-[PtCl2(Am)(piperidinopiperidine)] x HCl complexes with ubiquitin

trans-[PtCl2(Am)(pip-pip)] x HCl complexes, where Am = ammine, methylamine and dimethylamine, react with ubiquitin to form 1:1 covalent adducts. The platinum complexes bind exclusively to Met1 of ubiquitin forming trans-[PtCl(S-Met1-Ub)(Am)(pip-pip)] adducts. These adducts are reactive towards nucleophiles and react with deoxyguanosine (dGMP) to form the ternary trans-[Pt(dGMP)(S-Met1-Ub) (Am)(pip-pip)] complex which is stable in water and even in the presence of excess glutathione (GSH). Reaction of trans-[PtCl(S-Met1-Ub)(Am)(pip-pip)] with GSH resulted in the rapid formation of the ternary complex trans-[Pt(GS)(S-Met1-Ub)(Am)(pip-pip)] which was not stable and slowly lost the platinum moiety; after 7 days the platinum moiety was completely removed and the native ubiquitin was regenerated.     

DIRECT RECORDING OF THE INITIALLY EXCITED AND THE SOLVENT RELAXED FLUORESCENCE EMISSION SPECTRA OF TRYPTOPHAN BY PHASE SENSITIVE DETECTION OF FLUORESCENCE

Abstract Phase sensitive detection of fluorescence was used to directly record the initially excited and the solvent-relaxed emission spectra of N-acetyl-L-tryptophanamide in propylene glycol. Emission from the initially excited state was suppressed by adjusting the phase sensitive detector to be out of phase with the emission on the short wavelength side of the fluorescence spectrum. Then, the phase sensitive intensities revealed the emission spectrum of the solvent relaxed state. Similarly, the emission from the solvent relaxed state was suppressed by adjusting the detector to be out of phase with the emission on the long wavelength side of the spectrum, allowing the spectrum of the initially excited state to be directly recorded. Distinct emission spectra could be recorded when the solvent relaxation time was comparable to the fluorescence lifetime. At higher or lower temperatures, emission occurs predominantly from a single state, and suppression of the fluorescence signal at any arbitrary wavelength resulted in suppression of the entire emission. A simple theory is described which allows the spectral relaxation times to be estimated from the phase sensitive intensities. From this analysis we obtained an activation energy for spectral relaxation of 3 kcal/mol. This activation energy is smaller than that found for the temperature dependence of fluorescence depolarization, 7.8 kcal/mol. We attribute this difference to the smaller molecular motions required for spectral relaxation.
The method of phase sensitive detection of fluorescence shows excellent resolving power and sensitivity, and this method should facilitate measurement of spectral relaxation around tryptophan residues in proteins.     

Detection of the reversibility of an excited-state reaction by phase-modulation fluorometry

We used phase and modulation fluorometry to investigate the excited-state proton transfer from 2-naphthol. Phase-sensitive detection of fluorescence allows determination of the phase angles of the two-excited species, naphthol and naphtholate. If the steady-state spectra of the individual species are known and are not identical, then this general procedure yields the phase angles irrespective of the extent of the spectral overlap. The phase difference (Δφ) between the naphthol and naphtholate emission is given by tan Δφ = ω/(Γ_R + k₂ where ω is the circular modulation frequency, Γ_R the decay rate of naphtholate fluorescence and k₂ the rate of the reverse reaction. Hence, Δφ reflects both the decay rate of the reaction product and the rate of the reverse action. This back reaction was also detected by comparison of phase shift (φ) and demodulation (m) values for the initially excited state. Specifically, the reverse reaction results in a double exponential decay of naphthol fluorescence, which is revealed by m/cosφ < 1. The concepts described herein are generally applicable to determination of the reversibility of excited-state reactions.     

Fluorescence probes of viscosity: A comparative study of the fluorescence anisotropy decay of perylene and 3,9-dibromoperylene in glycerol

The authors compare the results of fluorescence anisotropy decay measurements for glycerol solutions of perylene with those of 3,9-dibromoperylene (DBP). For both molecules a good linear dependence is observed between the glycerol viscosity (varied by temperature) and the longer rotational correlation time obtained as a result of a global (using data obtained at 256- and 430-nm excitation wavelengths) biexponential analysis of the fluorescence anisotropy decay, at least in the range of 7–60 P for perylene and 4–60 P for DBP. This significantly extends the reported range of 0.5 to 150 cP investigated by Williams and Ben-Amotz [1] with the probe BTBP.     

First chemoenzymatic synthesis of (R)- and (S)-1-(9H-fluoren-9-yl)ethanol

A simple chemoenzymatic synthesis of 1-(9H-fluoren-9-yl)ethanol stereoisomers is described. The enantiomers were resolved by a kinetically controlled transesterification with vinyl acetate in the presence of commercially available lipases. High-throughput screening and subsequent exhaustive investigation of the utility of the lipases in a stereoselective process of introducing chirality have been carried out. Lipase A from Candida antarctica as a cross-linked aggregate (CAL-A-CLEA) was the most efficient enzyme for the resolution of the title compound providing (S)-1-(9H-fluoren-9-yl)ethanol and its (R)-acetate in enantiopure form (>99% ee). Under mild reaction conditions, excellent enantioselectivity (E = 407), and good isolated yields of the products were obtained.     

Thermal rotations and relaxation of vibrational energy of excited dye molecules in solution

Measurements of polarization and decay time of fluorescence of dyes in solution as a function of excitation wavelength were performed. The rate of thermal rotational depolarization was found to be independent of vibrational energy excess. To explain the observed influence of excitation wavelength on the emission anisotropy, a change in the magnitude of torsional vibrations, in addition to possible mixing of states, is proposed.     

FLUORESCENCE QUENCHING FOR FLAVINS INTERACTING WITH EGG WHITE RIBOFLAVIN-BINDING PROTEIN

Abstract— The effect of flavin structure variation upon the binding process of flavin to hen egg white riboflavin was studied using fluorescence methods for formylmethylflavin (FMF), riboflavin (RF) and flavomononucleotide (FMN).
Measurements of flavin fluorescence intensities (steady state and phase-sensitive) and lifetimes were performed in a variety of RBP concentrations and temperatures (4 to 40°C). No fluorescence of flavoproteins was detected, while the fluorescence of flavins was found to be quenched by RBP. The overall quenching process is dominated by the static quenching (about 88%) due to the flavoprotein complex formation in the ground state, presumably a charge transfer complex.