Bernard Alpert

Tryptophan emission from human hemoglobin and its isolated subunits.

Abstract— The emission spectra of human adult hemoglobin A0 and its isolated α and ß subunits were obtained using a highly sensitive photon‐counting spectrofluorometer. The quantum yields of the emissions, relative to free tryptophan, were also measured as well as the excitation polarization spectra for hemoglobin A0 and apohemoglobin. The fluorophore bis‐ANS was utilized to probe for the presence of apoproteins in the hemoprotein preparations. The work suggests that tryptophan may be useful as an intrinsic probe to study dynamical processes in hemoglobin.

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).

Dynamic and structural properties of glucose oxidase enzyme

Abstract The catalytic oxidation of β-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I′ band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH2 does not change the amount of α-helix present in the protein outer shell, but reorganises a fraction of random coil to β-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism.

Conformational heterogeneity in hemoglobin as determined by picosecond fluorescence decay measurements of the tryptopran residues

Abstract The fluorescence decay of the tryptophan residues in different derivatives of human hemoglobin (HbO. HbCO, Hb(deoxy), Hb(Met)) were examined in aqueous solutions at 20°C. Each derivative exhibited triple-exponential decay, kinetics with decay times between 70 and 90 ps. 1.8 and 1.9 ns, and 4.9 and 5.4 ns. The fractional fluorescence contribution of each component was different for each derivative. The results suggest that the different hemoglobin derivatives exist in solution as a mixture of at least three average conformations having different tryptoplian-heme orientations.

A model of dynamic quenching of fluorescence in globular proteins

A model is presented for the quenching of a fluorophore in a protein interior. At low quencher concentration the quenching process is determined by the acquisition rate of quencher by the protein, the migration rate of quencher in the protein interior, and the exit rate of quencher from the protein. In cases where the fluorescence emission observed in the absence of quencher could be described by a single exponential decay, the presence of quencher led to doubly exponential decay times, and the aforementioned exit rates of the quencher could be determined from experimental data. At high quencher concentration, the processes became more complex, and the deterministic rate equations used at low quencher concentration had to be modified to take into account the Poisson distribution of quencher molecules throughout the protein ensemble and also by using a migration rate for quencher in the protein interior that is a function of the quencher concentration. Simulations performed for typical fluorescent probes in proteins showed good agreement with experiments.

Oxygen distribution and migration within Mbdes Fe and Hbdes Fe. Multifrequency phase and modulation fluorometry study.

Quenching of the intensity and lifetime of porphyrin fluorescence from Mbdes Fe and Hbdes Fe (iron-free myoglobin and hemoglobin) by oxygen was investigated using a multifrequency cross-correlation phase fluorometer. The single exponential decay characteristic of porphyrin emission of Mbdes Fe and Hbdes Fe became doubly exponential upon application of oxygen pressure. The results were interpreted in terms of a general model of dynamic quenching of fluorescence in globular proteins. The model accounted for the rate k+ of acquisition of quencher by the protein, the exit rate k- of quencher from the protein, and the migration rate chi of quencher in the protein interior. The values of k+, k-, and chi were different for Mbdes Fe and Hbdes Fe. The addition of 40% sucrose, which increased the bulk viscosity sixfold, modified these rates. These results are discussed and compared with previous quenching studies on proteins. The significance of these results and the model for the interpretation of protein quenching studies is emphasized.

Förster energy transfer from tryptophan to flavin in glucose oxidase enzyme

Abstract The difference in the properties of the tryptophan fluorescence of the hologlucose oxidase forms, as compared to those of the apoprotein, can unambiguously be interpreted as due to a Forster energy transfer from the tryptophan residues to the flavinic group. Indeed, the atomic absorption of a glucose oxidase solution shows that the enzyme is not associated to any metals which could be responsible for the tryptophan fluorescence quenching effect. The data show that only 7 Trp residues are partially coupled to the flavin group and that the strength of this coupling is dependent on the flavin redox state.

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.

Involvement of protein dynamics in enzyme stability. The case of glucose oxidase.

Dynamics of glucose oxidase immobilized and in solution were compared through their tryptophan fluorescence spectra, decay times and quenching by acrylamide. Energy barrier for thermal inactivation and melting temperature of both soluble and immobilized enzyme were also measured. Data show that the fluctuation amplitude is at the origin of protein instability.

A fluorescence decay time study of tryptophan in isolated hemoglobin subunits

The time‐resolved fluorescence behavior of tryptophan residues in isolated human hemoglobin subunits was determined using a sync‐pumped dye laser system and time‐correlated single photon counting detection. Two decay components having values near 80 ps and 2 ns were found in the fluorescence decay of the α‐subunit. The data for the β‐chains were best fitted with 3 decay components of 90 ps, 2.5 ns and 6.4 ns. We propose that the decay times correspond to conformations of the proteins in which the disposition of the tryptophan to the heme residue differs.